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  1. Evaluation of new biomarker genes for differentiating Haemophilus influenzae from Haemophilus haemolyticus.

    Science.gov (United States)

    Theodore, M Jordan; Anderson, Raydel D; Wang, Xin; Katz, Lee S; Vuong, Jeni T; Bell, Melissa E; Juni, Billie A; Lowther, Sara A; Lynfield, Ruth; MacNeil, Jessica R; Mayer, Leonard W

    2012-04-01

    PCR detecting the protein D (hpd) and fuculose kinase (fucK) genes showed high sensitivity and specificity for identifying Haemophilus influenzae and differentiating it from H. haemolyticus. Phylogenetic analysis using the 16S rRNA gene demonstrated two distinct groups for H. influenzae and H. haemolyticus.

  2. Plasmid containing a DNA ligase gene from Haemophilus influenzae

    International Nuclear Information System (INIS)

    McCarthy, D.; Griffin, K.; Setlow, J.K.

    1984-01-01

    A ligase gene from Haemophilus influenzae was cloned into the shuttle vector pDM2. Although the plasmid did not affect X-ray sensitivity, it caused an increase in UV sensitivity of the wild-type but not excision-defective H. influenzae and a decrease in UV sensitivity of the rec-1 mutant. 14 references, 2 figures

  3. Delineation of the species Haemophilus influenzae by phenotype, multilocus sequence phylogeny, and detection of marker genes

    DEFF Research Database (Denmark)

    Nørskov-Lauritsen, Niels; Overballe, MD; Kilian, Mogens

    2009-01-01

    To obtain more information on the much-debated definition of prokaryotic species, we investigated the borders of Haemophilus influenzae by comparative analysis of H. influenzae reference strains with closely related bacteria including strains assigned to Haemophilus haemolyticus, cryptic genospec......To obtain more information on the much-debated definition of prokaryotic species, we investigated the borders of Haemophilus influenzae by comparative analysis of H. influenzae reference strains with closely related bacteria including strains assigned to Haemophilus haemolyticus, cryptic...... genospecies biotype IV, and the never formally validated species "Haemophilus intermedius". Multilocus sequence phylogeny based on six housekeeping genes separated a cluster encompassing the type and the reference strains of H. influenzae from 31 more distantly related strains. Comparison of 16S rRNA gene...

  4. Haemophilus ducreyi Hfq contributes to virulence gene regulation as cells enter stationary phase.

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    Gangaiah, Dharanesh; Labandeira-Rey, Maria; Zhang, Xinjun; Fortney, Kate R; Ellinger, Sheila; Zwickl, Beth; Baker, Beth; Liu, Yunlong; Janowicz, Diane M; Katz, Barry P; Brautigam, Chad A; Munson, Robert S; Hansen, Eric J; Spinola, Stanley M

    2014-02-11

    factor RpoS to adapt to stresses and stationary phase. However, homologs of RpoS are absent in some bacterial pathogens, including Haemophilus ducreyi, which causes chancroid and facilitates the acquisition and transmission of HIV-1. Here, we provide evidence that, in the absence of an RpoS homolog, Hfq serves as a major contributor of stationary-phase gene regulation and that Hfq is required for H. ducreyi to infect humans. To our knowledge, this is the first study describing Hfq as a major contributor of stationary-phase gene regulation in bacteria and the requirement of Hfq for the virulence of a bacterial pathogen in humans.

  5. Detection of cryptic genospecies misidentified as Haemophilus influenzae in routine clinical samples by assessment of marker genes fucK, hap, and sodC.

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    Nørskov-Lauritsen, Niels

    2009-08-01

    Clinical isolates of Haemophilus influenzae were assessed for the presence of fucK, hap, and sodC by hybridization with gene-specific probes, and isolates diverging from the expected H. influenzae genotype were characterized by phenotype and 16S rRNA gene sequencing. Two of 480 isolates were finally classified as variant strains ("nonhemolytic Haemophilus haemolyticus").

  6. Detection of Cryptic Genospecies Misidentified as Haemophilus influenzae in Routine Clinical Samples by Assessment of Marker Genes fucK, hap, and sodC▿

    Science.gov (United States)

    Nørskov-Lauritsen, Niels

    2009-01-01

    Clinical isolates of Haemophilus influenzae were assessed for the presence of fucK, hap, and sodC by hybridization with gene-specific probes, and isolates diverging from the expected H. influenzae genotype were characterized by phenotype and 16S rRNA gene sequencing. Two of 480 isolates were finally classified as variant strains (“nonhemolytic Haemophilus haemolyticus”). PMID:19535530

  7. Identification, distribution, and expression of novel genes in 10 clinical isolates of nontypeable Haemophilus influenzae.

    Science.gov (United States)

    Shen, Kai; Antalis, Patricia; Gladitz, John; Sayeed, Sameera; Ahmed, Azad; Yu, Shujun; Hayes, Jay; Johnson, Sandra; Dice, Bethany; Dopico, Richard; Keefe, Randy; Janto, Benjamin; Chong, William; Goodwin, Joseph; Wadowsky, Robert M; Erdos, Geza; Post, J Christopher; Ehrlich, Garth D; Hu, Fen Z

    2005-06-01

    We hypothesize that Haemophilus influenzae, as a species, possesses a much greater number of genes than that found in any single H. influenzae genome. This supragenome is distributed throughout naturally occurring infectious populations, and new strains arise through autocompetence and autotransformation systems. The effect is that H. influenzae populations can readily adapt to environmental stressors. The supragenome hypothesis predicts that significant differences exist between and among the genomes of individual infectious strains of nontypeable H. influenzae (NTHi). To test this prediction, we obtained 10 low-passage NTHi clinical isolates from the middle ear effusions of patients with chronic otitis media. DNA sequencing was performed with 771 clones chosen at random from a pooled genomic library. Homology searching demonstrated that approximately 10% of these clones were novel compared to the H. influenzae Rd KW20 genome, and most of them did not match any DNA sequence in GenBank. Amino acid homology searches using hypothetical translations of the open reading frames revealed homologies to a variety of proteins, including bacterial virulence factors not previously identified in the NTHi isolates. The distribution and expression of 53 of these genes among the 10 strains were determined by PCR- and reverse transcription PCR-based analyses. These unique genes were nonuniformly distributed among the 10 isolates, and transcription of these genes in planktonic cultures was detected in 50% (177 of 352) of the occurrences. All of the novel sequences were transcribed in one or more of the NTHi isolates. Seventeen percent (9 of 53) of the novel genes were identified in all 10 NTHi strains, with each of the remaining 44 being present in only a subset of the strains. These genic distribution analyses were more effective as a strain discrimination tool than either multilocus sequence typing or 23S ribosomal gene typing methods.

  8. Characterization of antimicrobial resistance genes in Haemophilus parasuis isolated from pigs in China

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    Yongda Zhao

    2018-04-01

    Full Text Available Background Haemophilus parasuis is a common porcine respiratory pathogen that causes high rates of morbidity and mortality in farmed swine. We performed a molecular characterization of antimicrobial resistance genes harbored by H. parasuis from pig farms in China. Methods We screened 143 H. parasuis isolates for antimicrobial susceptibility against six fluoroquinolone antibiotics testing by the broth microdilution method, and the presence of 64 antimicrobial resistance genes by PCR amplification and DNA sequence analysis. We determined quinolone resistance determining region mutations of DNA gyrase (gyrA and gyrB and topoisomerase IV (parC and parE. The genetic relatedness among the strains was analyzed by pulsed-field gel electrophoresis. Results Susceptibility test showed that all isolates were low resistance to lomefloxacin (28.67%, levofloxacin (20.28%, norfloxacin (22.38%, ciprofloxacin (23.78%, however, high resistance levels were found to nalidixic acid (82.52% and enrofloxacin (55.94%. In addition, we found 14 antimicrobial resistance genes were present in these isolates, including blaTEM-1, blaROB-1, ermB, ermA, flor, catl, tetB, tetC, rmtB, rmtD, aadA1, aac(3′-llc, sul1, and sul2 genes. Interestingly, one isolate carried five antibiotic resistance genes (tetB, tetC, flor, rmtB, sul1. The genes tetB, rmtB, and flor were the most prevalent resistance genes in H. parasuis in China. Alterations in the gyrA gene (S83F/Y, D87Y/N/H/G were detected in 81% of the strains and parC mutations were often accompanied by a gyrA mutation. Pulsed-field gel electrophoresis typing revealed 51 unique patterns in the isolates carrying high-level antibiotic resistance genes, indicating considerable genetic diversity and suggesting that the genes were spread horizontally. Discussion The current study demonstrated that the high antibiotic resistance of H. parasuis in piglets is a combination of transferable antibiotic resistance genes and multiple target

  9. Characterization of antimicrobial resistance genes in Haemophilus parasuis isolated from pigs in China.

    Science.gov (United States)

    Zhao, Yongda; Guo, Lili; Li, Jie; Huang, Xianhui; Fang, Binghu

    2018-01-01

    Haemophilus parasuis is a common porcine respiratory pathogen that causes high rates of morbidity and mortality in farmed swine. We performed a molecular characterization of antimicrobial resistance genes harbored by H. parasuis from pig farms in China. We screened 143 H. parasuis isolates for antimicrobial susceptibility against six fluoroquinolone antibiotics testing by the broth microdilution method, and the presence of 64 antimicrobial resistance genes by PCR amplification and DNA sequence analysis. We determined quinolone resistance determining region mutations of DNA gyrase ( gyrA and gyrB ) and topoisomerase IV ( parC and parE ). The genetic relatedness among the strains was analyzed by pulsed-field gel electrophoresis. Susceptibility test showed that all isolates were low resistance to lomefloxacin (28.67%), levofloxacin (20.28%), norfloxacin (22.38%), ciprofloxacin (23.78%), however, high resistance levels were found to nalidixic acid (82.52%) and enrofloxacin (55.94%). In addition, we found 14 antimicrobial resistance genes were present in these isolates, including bla TEM-1 , bla ROB-1 , ermB, ermA, flor, catl, tetB, tetC, rmtB, rmtD, aadA1, aac(3')-llc, sul1, and sul2 genes. Interestingly, one isolate carried five antibiotic resistance genes ( tetB, tetC, flor, rmtB, sul1 ). The genes tetB , rmtB, and flor were the most prevalent resistance genes in H. parasuis in China. Alterations in the gyrA gene (S83F/Y, D87Y/N/H/G) were detected in 81% of the strains and parC mutations were often accompanied by a gyrA mutation. Pulsed-field gel electrophoresis typing revealed 51 unique patterns in the isolates carrying high-level antibiotic resistance genes, indicating considerable genetic diversity and suggesting that the genes were spread horizontally. The current study demonstrated that the high antibiotic resistance of H. parasuis in piglets is a combination of transferable antibiotic resistance genes and multiple target gene mutations. These data provide novel

  10. Characterization of the rec-1 gene of Haemophilus influenzae and behavior of the gene in Escherichia coli

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    Setlow, J.K.; Spikes, D.; Griffin, K.

    1988-09-01

    The rec-1 gene of Haemophilus influenzae was cloned into a shuttle vector that replicates in Escherichia coli as well as in H. influenzae. The plasmid, called pRec1, complemented the defects of a rec-1 mutant in repair of UV damage, transformation, and ability of prophage to be induced by UV radiation. Although UV resistance and recombination were caused by pRec1 in E. coli recA mutants, UV induction of lambda and UV mutagenesis were not. We suggest that the ability of the H. influenzae Rec-1 protein to cause cleavage of repressors but not the recombinase function differs from that of the E. coli RecA protein.

  11. Characterization of the rec-1 gene of Haemophilus influenzae and behavior of the gene in Escherichia coli

    International Nuclear Information System (INIS)

    Setlow, J.K.; Spikes, D.; Griffin, K.

    1988-01-01

    The rec-1 gene of Haemophilus influenzae was cloned into a shuttle vector that replicates in Escherichia coli as well as in H. influenzae. The plasmid, called pRec1, complemented the defects of a rec-1 mutant in repair of UV damage, transformation, and ability of prophage to be induced by UV radiation. Although UV resistance and recombination were caused by pRec1 in E. coli recA mutants, UV induction of lambda and UV mutagenesis were not. We suggest that the ability of the H. influenzae Rec-1 protein to cause cleavage of repressors but not the recombinase function differs from that of the E. coli RecA protein

  12. Detection of a Bacteriophage Gene Encoding a Mu-like Portal Protein in Haemophilus parasuis Reference Strains and Field Isolates by Nested Polymerase Chain Reaction

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    A nested PCR assay was developed to determine the presence of a gene encoding a bacteriophage Mu-like portal protein, gp29, in 15 reference strains and 31 field isolates of Haemophilus parasuis. Specific primers, based on the gene’s sequence, were utilized. A majority of the virulent reference strai...

  13. Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus.

    Science.gov (United States)

    Lubys, A; Lubienè, J; Kulakauskas, S; Stankevicius, K; Timinskas, A; Janulaitis, A

    1996-07-15

    The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.

  14. The capsule biosynthesis locus of Haemophilus influenzae show conspicuous similarity to the corresponding locus in Haemophilus sputorum and may have been recruited from this species by horizontal gene transfer

    DEFF Research Database (Denmark)

    Nielsen, Signe Maria; de Gier, Camilla; Dimopoulou, Chrysoula

    2015-01-01

    in export and processing of the capsular material, show high similarity to the corresponding genes in capsulate lineages of the pathogenic species Haemophilus influenzae; indeed, standard bexA and bexB PCRs for detection of capsulated strains of H. influenzae give positive results with strains of H....... sputorum was only distantly related to H. influenzae. In contrast to H. influenzae, the capsule locus in H. sputorum is not associated with transposases or other transposable elements. Our data suggest that the capsule locus of capsulate lineages of H. influenzae may relatively recently have been recruited...

  15. Inactivation of the Haemophilus ducreyi luxS gene affects the virulence of this pathogen in human subjects.

    Science.gov (United States)

    Labandeira-Rey, Maria; Janowicz, Diane M; Blick, Robert J; Fortney, Kate R; Zwickl, Beth; Katz, Barry P; Spinola, Stanley M; Hansen, Eric J

    2009-08-01

    Haemophilus ducreyi 35000HP contains a homologue of the luxS gene, which encodes an enzyme that synthesizes autoinducer 2 (AI-2) in other gram-negative bacteria. H. ducreyi 35000HP produced AI-2 that functioned in a Vibrio harveyi-based reporter system. A H. ducreyi luxS mutant was constructed by insertional inactivation of the luxS gene and lost the ability to produce AI-2. Provision of the H. ducreyi luxS gene in trans partially restored AI-2 production by the mutant. The luxS mutant was compared with its parent for virulence in the human challenge model of experimental chancroid. The pustule-formation rate in 5 volunteers was 93.3% (95% confidence interval, 81.7%-99.9%) at 15 parent sites and 60.0% (95% confidence interval, 48.3%-71.7%) at 15 mutant sites (1-tailed P < .001). Thus, the luxS mutant was partially attenuated for virulence. This is the first report of AI-2 production contributing to the pathogenesis of a genital ulcer disease.

  16. Physical size of the donor locus and transmission of Haemophilus influenzae ampicillin resistance genes by deoxyribonucleic acid-mediated transformation

    International Nuclear Information System (INIS)

    Bendler, J.W. III

    1976-01-01

    The properties of donor deoxyribonucleic acid (DNA) from three clinical isolates and its ability to mediate the transformation of competent Rd strains to ampicillin resistance were examined. A quantitative technique for determining the resistance of individual Haemophilus influenzae cells to ampicillin was developed. When this technique was used, sensitive cells failed to tolerate levels of ampicillin greater than 0.1 to 0.2 μg/ml, whereas three resistant type b β-lactamase-producing strains could form colonies 1- to 3-μg/ml levels of the antibiotic. DNA extracted from the resistant strains elicited transformation of the auxotrophic genes in a multiply auxotrophic Rd strain. For two of the donors, transformation to ampicillin resistance occurred after the uptake of a single DNA molecule approximately 10 4 -fold less frequently than transformation of auxotrophic loci and was not observed to occur at all with the third. The frequency of transformation to ampicillin resistance was two- to fivefold higher in strain BC200 (Okinaka and Barnhart, 1974), which was cured of a defective prophage. All three clinical ampicillin-resistant strains were poor recipients, but the presence of the ampicillin resistant genes in strain BC200 did not reduce its competence

  17. Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia.

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    Abdeldaim, Guma M K; Strålin, Kristoffer; Olcén, Per; Blomberg, Jonas; Mölling, Paula; Herrmann, Björn

    2013-06-01

    A quantitative polymerase chain reaction (PCR) for the fucK gene was developed for specific detection of Haemophilus influenzae. The method was tested on sputum and nasopharyngeal aspirate (NPA) from 78 patients with community-acquired pneumonia (CAP). With a reference standard of sputum culture and/or serology against the patient's own nasopharyngeal isolate, H. influenzae etiology was detected in 20 patients. Compared with the reference standard, fucK PCR (using the detection limit 10(5) DNA copies/mL) on sputum and NPA showed a sensitivity of 95.0% (19/20) in both cases, and specificities of 87.9% (51/58) and 89.5% (52/58), respectively. In a receiver operating characteristic curve analysis, sputum fucK PCR was found to be significantly superior to sputum P6 PCR for detection of H. influenzae CAP. NPA fucK PCR was positive in 3 of 54 adult controls without respiratory symptoms. In conclusion, quantitative fucK real-time PCR provides a sensitive and specific identification of H. influenzae in respiratory secretions. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Chancroid and Haemophilus ducreyi.

    Science.gov (United States)

    Morse, S A

    1989-01-01

    Haemophilus ducreyi is the causative agent of chancroid, one of the genital ulcerative diseases. H. ducreyi is the major cause of genital ulcer disease in Africa and Southeast Asia and is of increasing concern in the United States. Definitive diagnosis of chancroid requires the isolation and identification of H. ducreyi, but isolation of this organism is difficult and the available medium is not optimal for all strains. Fluorescent antibody and serologic tests are of limited value. In general, our knowledge of this organism is rather limited, and indeed, recent studies have questioned the placement of H. ducreyi in the genus Haemophilus. H. ducreyi has relatively few biochemical activities, and epidemiologic studies are limited because there are limited phenotypic markers available for strain typing. Specific virulence factors of H. ducreyi have yet to be identified. Antimicrobial resistance in H. ducreyi is of special concern, as this organism has acquired both gram-negative and gram-positive resistance determinants. In addition, some of these determinants can be mobilized and transferred to other Haemophilus species or to Neisseria gonorrhoeae. Images PMID:2650859

  19. Expression of Haemophilus ducreyi collagen binding outer membrane protein NcaA is required for virulence in swine and human challenge models of chancroid.

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    Fulcher, Robert A; Cole, Leah E; Janowicz, Diane M; Toffer, Kristen L; Fortney, Kate R; Katz, Barry P; Orndorff, Paul E; Spinola, Stanley M; Kawula, Thomas H

    2006-05-01

    Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection.

  20. Fastidious Gram-Negatives: Identification by the Vitek 2 Neisseria-Haemophilus Card and by Partial 16S rRNA Gene Sequencing Analysis

    DEFF Research Database (Denmark)

    Wolff Sönksen, Ute; Christensen, Jens Jørgen; Nielsen, Lisbeth

    2010-01-01

    Taxonomy and identification of fastidious Gram negatives are evolving and challenging. We compared identifications achieved with the Vitek 2 Neisseria-Haemophilus (NH) card and partial 16S rRNA gene sequence (526 bp stretch) analysis with identifications obtained with extensive phenotypic...... characterization using 100 fastidious Gram negative bacteria. Seventy-five strains represented 21 of the 26 taxa included in the Vitek 2 NH database and 25 strains represented related species not included in the database. Of the 100 strains, 31 were the type strains of the species. Vitek 2 NH identification...

  1. Fastidious Gram-Negatives: Identification by the Vitek 2 Neisseria-Haemophilus Card and by Partial 16S rRNA Gene Sequencing Analysis

    DEFF Research Database (Denmark)

    Wolff Sönksen, Ute; Christensen, Jens Jørgen; Nielsen, Lisbeth

    2010-01-01

    Taxonomy and identification of fastidious Gram negatives are evolving and challenging. We compared identifications achieved with the Vitek 2 Neisseria-Haemophilus (NH) card and partial 16S rRNA gene sequence (526 bp stretch) analysis with identifications obtained with extensive phenotypic...... characterization using 100 fastidious Gram negative bacteria. Seventy-five strains represented 21 of the 26 taxa included in the Vitek 2 NH database and 25 strains represented related species not included in the database. Of the 100 strains, 31 were the type strains of the species. Vitek 2 NH identification...... results: 48 of 75 database strains were correctly identified, 11 strains gave `low discrimination´, seven strains were unidentified, and nine strains were misidentified. Identification of 25 non-database strains resulted in 14 strains incorrectly identified as belonging to species in the database. Partial...

  2. Origin of the diversity in DNA recognition domains in phasevarion associated modA genes of pathogenic Neisseria and Haemophilus influenzae.

    Science.gov (United States)

    Gawthorne, Jayde A; Beatson, Scott A; Srikhanta, Yogitha N; Fox, Kate L; Jennings, Michael P

    2012-01-01

    Phase variable restriction-modification (R-M) systems have been identified in a range of pathogenic bacteria. In some it has been demonstrated that the random switching of the mod (DNA methyltransferase) gene mediates the coordinated expression of multiple genes and constitutes a phasevarion (phase variable regulon). ModA of Neisseria and Haemophilus influenzae contain a highly variable, DNA recognition domain (DRD) that defines the target sequence that is modified by methylation and is used to define modA alleles. 18 distinct modA alleles have been identified in H. influenzae and the pathogenic Neisseria. To determine the origin of DRD variability, the 18 modA DRDs were used to search the available databases for similar sequences. Significant matches were identified between several modA alleles and mod gene from distinct bacterial species, indicating one source of the DRD variability was via horizontal gene transfer. Comparison of DRD sequences revealed significant mosaicism, indicating exchange between the Neisseria and H. influenzae modA alleles. Regions of high inter- and intra-allele similarity indicate that some modA alleles had undergone recombination more frequently than others, generating further diversity. Furthermore, the DRD from some modA alleles, such as modA12, have been transferred en bloc to replace the DRD from different modA alleles.

  3. Outer membrane protein P4 is not required for virulence in the human challenge model of Haemophilus ducreyi infection.

    Science.gov (United States)

    Janowicz, Diane M; Zwickl, Beth W; Fortney, Kate R; Katz, Barry P; Bauer, Margaret E

    2014-06-24

    Bacterial lipoproteins often play important roles in pathogenesis and can stimulate protective immune responses. Such lipoproteins are viable vaccine candidates. Haemophilus ducreyi, which causes the sexually transmitted disease chancroid, expresses a number of lipoproteins during human infection. One such lipoprotein, OmpP4, is homologous to the outer membrane lipoprotein e (P4) of H. influenzae. In H. influenzae, e (P4) stimulates production of bactericidal and protective antibodies and contributes to pathogenesis by facilitating acquisition of the essential nutrients heme and nicotinamide adenine dinucleotide (NAD). Here, we tested the hypothesis that, like its homolog, H. ducreyi OmpP4 contributes to virulence and stimulates production of bactericidal antibodies. We determined that OmpP4 is broadly conserved among clinical isolates of H. ducreyi. We next constructed and characterized an isogenic ompP4 mutant, designated 35000HPompP4, in H. ducreyi strain 35000HP. To test whether OmpP4 was necessary for virulence in humans, eight healthy adults were experimentally infected. Each subject was inoculated with a fixed dose of 35000HP on one arm and three doses of 35000HPompP4 on the other arm. The overall parent and mutant pustule formation rates were 52.4% and 47.6%, respectively (P = 0.74). These results indicate that expression of OmpP4 in not necessary for H. ducreyi to initiate disease or progress to pustule formation in humans. Hyperimmune mouse serum raised against purified, recombinant OmpP4 did not promote bactericidal killing of 35000HP or phagocytosis by J774A.1 mouse macrophages in serum bactericidal and phagocytosis assays, respectively. Our data suggest that, unlike e (P4), H. ducreyi OmpP4 is not a suitable vaccine candidate. OmpP4 may be dispensable for virulence because of redundant mechanisms in H. ducreyi for heme acquisition and NAD utilization.

  4. Fastidious Gram-Negatives: Identification by the Vitek 2 Neisseria-Haemophilus Card and by Partial 16S rRNA Gene Sequencing Analysis.

    Science.gov (United States)

    Sönksen, Ute Wolff; Christensen, Jens Jørgen; Nielsen, Lisbeth; Hesselbjerg, Annemarie; Hansen, Dennis Schrøder; Bruun, Brita

    2010-12-31

    Taxonomy and identification of fastidious Gram negatives are evolving and challenging. We compared identifications achieved with the Vitek 2 Neisseria-Haemophilus (NH) card and partial 16S rRNA gene sequence (526 bp stretch) analysis with identifications obtained with extensive phenotypic characterization using 100 fastidious Gram negative bacteria. Seventy-five strains represented 21 of the 26 taxa included in the Vitek 2 NH database and 25 strains represented related species not included in the database. Of the 100 strains, 31 were the type strains of the species. Vitek 2 NH identification results: 48 of 75 database strains were correctly identified, 11 strains gave `low discrimination´, seven strains were unidentified, and nine strains were misidentified. Identification of 25 non-database strains resulted in 14 strains incorrectly identified as belonging to species in the database. Partial 16S rRNA gene sequence analysis results: For 76 strains phenotypic and sequencing identifications were identical, for 23 strains the sequencing identifications were either probable or possible, and for one strain only the genus was confirmed. Thus, the Vitek 2 NH system identifies most of the commonly occurring species included in the database. Some strains of rarely occurring species and strains of non-database species closely related to database species cause problems. Partial 16S rRNA gene sequence analysis performs well, but does not always suffice, additional phenotypical characterization being useful for final identification.

  5. Genes from plasmid pKM101 in Haemophilus influenzae: separation of functions of mucA and mucB

    International Nuclear Information System (INIS)

    Balganesh, M.; Setlow, J.K.

    1985-01-01

    Haemophilus influenzae, normally not mutable by UV, became UV mutable with a recombinant plasmid insertion. A 7.8-kilobase-pair (kbp) fragment of the plasmid pKM101 containing the mucA and mucB genes was ligated to the shuttle vector pDM2, and a Rec- strain of H. influenzae was transformed with the ligated mixture. All of the transformants, unlike the parent Rec- strain, were resistant to UV, could carry out postreplication repair and Weigle reactivation, showed greatly increased spontaneous mutation, and contained a plasmid carrying an insert of only 1.2 rather than 7.8 kbp. This plasmid in a umuC mutant strain of Escherichia coli complemented a pKM101 derivative lacking mucA function but with an intact mucB gene, although there was no complementation with a mucA+ mucB- plasmid, suggesting that the newly constructed plasmid coded for the mucA protein; this is in accord with the restriction analysis and hybridization between the plasmid and a probe containing all of the mucA gene but only a small fraction of mucB. When one of the H. influenzae Rec- transformants lost the plasmid, the resistance to UV was retained but the high spontaneous mutation and UV mutability were not. The fact that there was hybridization between the chromosome of the cured strain and a probe containing both muc genes but none when almost no mucB was present suggested that at least part of the mucB gene had been integrated into the Rec- chromosome. Five different postreplication repair-proficient strains became UV mutable and had high spontaneous mutation rates caused by the putative mucA plasmid, indicating that these strains already possessed a chromosomal equivalent of the mucB gene

  6. Haemophilus segnis endocarditis

    DEFF Research Database (Denmark)

    Bangsborg, Jette Marie; Tvede, M; Skinhøj, P

    1988-01-01

    Haemophilus segnis is a rarely recognised commensal in the oropharynx. We wish to report the first published case of endocarditis caused by H. segnis. The patient, a 76-year-old female did not recover until after 2 courses of ampicillin given for a total of 57 days. In the second course of treatm......Haemophilus segnis is a rarely recognised commensal in the oropharynx. We wish to report the first published case of endocarditis caused by H. segnis. The patient, a 76-year-old female did not recover until after 2 courses of ampicillin given for a total of 57 days. In the second course...

  7. Reclassification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nvo., comb. nov., Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V factor-independent isolates

    DEFF Research Database (Denmark)

    Nørskov-Lauritsen, N.; Kilian, Mogens

    2006-01-01

    -independent growth was identified in Haemophilus aphrophilus. The gene encodes a polypeptide of 462 amino acids that shows 74.5 % amino acid sequence identity to the corresponding enzyme from Actinobacillus actinomycetemcomitans. Ten isolates of Haemophilus paraphrophilus all carried a nadV pseudogene. DNA from...... Haemophilus aphrophilus was able to transform Haemophilus paraphrophilus into the NAD-independent phenotype. The transformants carried a full-length nadV inserted in the former locus of the pseudogene. The DNA-DNA relatedness between the type strains of Haemophilus aphrophilus and Haemophilus paraphrophilus...

  8. A plasmid carrying mucA and mucB genes from pKM101 in Haemophilus influenzae and Escherichia coli

    International Nuclear Information System (INIS)

    Spikes, D.; Setlow, J.K.

    1989-01-01

    The plasmid pMucAMucB, constructed from the Haemophilus influenzae vector pDM2, and a similar plasmid, constructed from pBR322, increased the survival after UV irradiation of Escherichia coli AB1157 with the umu-36 mutation and also caused UV-induced mutation in the E. coli strain. In H. influenzae, pMucAMucB caused a small but reproducible increase in survival after UV irradiation in wild-type cells and in a rec-1 mutant, but there was no increase in spontaneous mutation in the wild type or in the rec-1 mutant and no UV-induced mutation

  9. Evolution of the paralogous hap and iga genes in Haemophilus influenzae: evidence for a conserved hap pseudogene associated with microcolony formation in the recently diverged Haemophilus aegyptius and H. influenzae biogroup aegyptius

    DEFF Research Database (Denmark)

    Kilian, Mogens; Poulsen, Knud; Lomholt, Hans Bredsted

    2002-01-01

    genetic polymorphism and pronounced mosaic-like patterns in both genes, but no evidence of intrastrain recombination between the two genes. A conserved hap pseudogene was present in all strains of H. aegyptius and H. influenzae biogroup aegyptius, each of which constituted distinct subpopulations...... on conjunctival cells, previously termed microcolony formation. The fact that individual hap pseudogenes differed from the ancestral sequence by zero to two positions within a 1.5 kb stretch suggests that the silencing event happened approximately 2000-11,000 years ago. Divergence of H. aegyptius and H...

  10. Haemophilus haemolyticus: A Human Respiratory Tract Commensal to Be Distinguished from Haemophilus influenzae

    DEFF Research Database (Denmark)

    Murphy, T.F.; Brauer, A.L.; Sethi, S.

    2007-01-01

    Background. Haemophilus influenzae is a common pathogen in adults with chronic obstructive pulmonary disease (COPD). In a prospective study, selected isolates of apparent H. influenzae had an altered phenotype. We tested the hypothesis that these variant strains were genetically different from...... typical H. influenzae.Methods. A prospective study of adults with COPD was conducted. Strains of apparent H. influenzae obtained from a range of clinical sources were evaluated by ribosomal DNA sequence analysis, multilocus sequence analysis, DNA-DNA hybridization, and sequencing of the conserved P6 gene.......Results. Variant strains were determined to be Haemophilus haemolyticus by means of 4 independent methods. Analysis of 490 apparent H. influenzae strains, identified by standard methods, revealed that 39.5% of sputum isolates and 27.3% of nasopharyngeal isolates were H. haemolyticus. Isolates obtained from...

  11. Haemophilus parainfluenzae urethritis among homosexual men.

    Science.gov (United States)

    Hsu, Meng-Shiuan; Wu, Mei-Yu; Lin, Tsui-Hsien; Liao, Chun-Hsing

    2015-08-01

    Haemophilus parainfluenzae is a common inhabitant of the human upper respiratory tract of the normal oral microflora. We report three men who had been having unprotected sex with men (MSM) and subsequently acquired H. parainfluenzae urethritis, which was confirmed by 16S rRNA gene sequencing analysis. Two men were treated with ceftriaxone and doxycycline, and the third man was treated with clarithromycin. All three patients responded to treatment. This case series highlights the potential role of H. parainfluenzae as a sexually transmitted genitourinary pathogen. Copyright © 2012. Published by Elsevier B.V.

  12. Hib Disease (Haemophilus Influenzae Type b)

    Science.gov (United States)

    ... Fitness Diseases & Conditions Infections Drugs & Alcohol School & Jobs Sports Expert Answers (Q&A) Staying Safe Videos for Educators Search English Español Hib Disease (Haemophilus Influenzae Type b) KidsHealth / For Teens / Hib Disease (Haemophilus Influenzae ...

  13. NNDSS - Table II. Giardiasis to Haemophilus influenza

    Data.gov (United States)

    U.S. Department of Health & Human Services — NNDSS - Table II. Giardiasis to Haemophilus influenza - 2017. In this Table, provisional cases of selected notifiable diseases (≥1,000 cases reported during the...

  14. NNDSS - Table II. Giardiasis to Haemophilus influenza

    Data.gov (United States)

    U.S. Department of Health & Human Services — NNDSS - Table II. Giardiasis to Haemophilus influenza - 2014. In this Table, all conditions with a 5-year average annual national total of more than or equals 1,000...

  15. NNDSS - Table II. Giardiasis to Haemophilus influenza

    Data.gov (United States)

    U.S. Department of Health & Human Services — NNDSS - Table II. Giardiasis to Haemophilus influenza - 2018. In this Table, provisional cases of selected notifiable diseases (≥1,000 cases reported during the...

  16. NNDSS - Table II. Giardiasis to Haemophilus influenza

    Data.gov (United States)

    U.S. Department of Health & Human Services — NNDSS - Table II. Giardiasis to Haemophilus influenza - 2015.In this Table, provisional cases of selected notifiable diseases (≥1,000 cases reported during the...

  17. NNDSS - Table II. Giardiasis to Haemophilus influenza

    Data.gov (United States)

    U.S. Department of Health & Human Services — NNDSS - Table II. Giardiasis to Haemophilus influenza - 2016. In this Table, provisional* cases of selected† notifiable diseases (≥1,000 cases reported during the...

  18. An isolate of Haemophilus haemolyticus produces a bacteriocin-like substance that inhibits the growth of nontypeable Haemophilus influenzae.

    Science.gov (United States)

    Latham, Roger D; Gell, David A; Fairbairn, Rory L; Lyons, A Bruce; Shukla, Shakti D; Cho, Kum Yin; Jones, David A; Harkness, Nick M; Tristram, Stephen G

    2017-04-01

    Nontypeable Haemophilus influenzae (NTHi) frequently colonises the upper respiratory tract and is an important cause of respiratory infections. Resistance to antibiotics is an emerging trend in NTHi and alternative prevention or treatment strategies are required. Haemophilus haemolyticus is a common commensal occupying the same niche as NTHi and, if able to produce substances that inhibit NTHi growth, may have a role as a probiotic. In this study, ammonium sulphate extracts from broth culture of 100 H. haemolyticus isolates were tested for the presence of substances inhibitory to NTHi using a well diffusion assay. One isolate produced a substance that consistently inhibited the growth of NTHi. The substance was inactivated by protease enzymes and had a molecular size of ca. 30 kDa as determined by size exclusion chromatography. When the substance was tested against bacteria from eight Gram-negative and three Gram-positive genera, only Haemophilus spp. were inhibited. Quantitative PCR testing showed the substance to be different to 'haemocin', the previously described bacteriocin of H. influenzae type b. These molecular characteristics, together with narrow-spectrum activity, suggest the substance may be a novel bacteriocin, and there is potential for this H. haemolyticus isolate to function as a probiotic for reduction of colonisation and subsequent infection with NTHi. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  19. Phylogeny of the genus Haemophilus as determined by comparison of partial infB sequences

    DEFF Research Database (Denmark)

    Hedegaard, J; Okkels, H; Bruun, B

    2001-01-01

    A 453 bp fragment of infB, the gene encoding translation initiation factor 2, was sequenced and compared from 66 clinical isolates and type strains of Haemophilus species and related bacteria. Analysis of the partial infB sequences obtained suggested that the human isolates dependent on X and V...... factor, H. influenzae, H. haemolyticus, H. aegyptius and some cryptic genospecies of H. influenzae, were closely related to each other. H. parainfluenzae constituted a heterogeneous group within the boundaries of the genus, whereas H. aphrophilus/paraphrophilus and Actinobacillus actinomycetemcomitans...... were only remotely related to the type species of the genus Haemophilus H. parahaemolyticus and H. paraphrohaemolyticus took up an intermediary position and may not belong in the genus Haemophilus sensu stricto. Ambiguous results were obtained with seven isolates tentatively identified as H. segnis...

  20. Epidemiology of Haemophilus ducreyi Infections.

    Science.gov (United States)

    González-Beiras, Camila; Marks, Michael; Chen, Cheng Y; Roberts, Sally; Mitjà, Oriol

    2016-01-01

    The global epidemiology of Haemophilus ducreyi infections is poorly documented because of difficulties in confirming microbiological diagnoses. We evaluated published data on the proportion of genital and nongenital skin ulcers caused by H. ducreyi before and after introduction of syndromic management for genital ulcer disease (GUD). Before 2000, the proportion of GUD caused by H. ducreyi ranged from 0.0% to 69.0% (35 studies in 25 countries). After 2000, the proportion ranged from 0.0% to 15.0% (14 studies in 13 countries). In contrast, H. ducreyi has been recently identified as a causative agent of skin ulcers in children in the tropical regions; proportions ranged from 9.0% to 60.0% (6 studies in 4 countries). We conclude that, although there has been a sustained reduction in the proportion of GUD caused by H. ducreyi, this bacterium is increasingly recognized as a major cause of nongenital cutaneous ulcers.

  1. Host-pathogen interplay of Haemophilus ducreyi.

    Science.gov (United States)

    Janowicz, Diane M; Li, Wei; Bauer, Margaret E

    2010-02-01

    Haemophilus ducreyi, the causative agent of the sexually transmitted infection chancroid, is primarily a pathogen of human skin. During infection, H. ducreyi thrives extracellularly in a milieu of professional phagocytes and other antibacterial components of the innate and adaptive immune responses. This review summarizes our understanding of the interplay between this pathogen and its host that leads to development and persistence of disease. H. ducreyi expresses key virulence mechanisms to resist host defenses. The secreted LspA proteins are tyrosine-phosphorylated by host kinases, which may contribute to their antiphagocytic effector function. The serum resistance and adherence functions of DsrA map to separate domains of this multifunctional virulence factor. An influx transporter protects H. ducreyi from killing by the antimicrobial peptide LL37. Regulatory genes have been identified that may coordinate virulence factor expression during disease. Dendritic cells and natural killer cells respond to H. ducreyi and may be involved in determining the differential outcomes of infection observed in humans. A human model of H. ducreyi infection has provided insights into virulence mechanisms that allow this human-specific pathogen to survive immune pressures. Components of the human innate immune system may also determine the ultimate fate of H. ducreyi infection by driving either clearance of the organism or an ineffective response that allows disease progression.

  2. Drosophila Myc is required for normal DREF gene expression

    International Nuclear Information System (INIS)

    Dang Thi Phuong Thao; Seto, Hirokazu; Yamaguchi, Masamitsu

    2008-01-01

    The Drosophila DNA replication-related element-binding factor (dDREF) is required for the expression of many proliferation-related genes carrying the DRE sequence, 5'-TATCGATA. Finding a canonical E-box, 5'-CACGTG, in the dDREF gene promoter prompted us to explore the possibility that the dDREF gene is a target of Drosophila Myc (dMyc). Luciferase transient expression assays combined with RNA interference in Drosophila S2 cells revealed that knockdown of dmyc reduced dDREF gene promoter activity by 35% to 82%, an effect at least partly mediated by the E-box in the promoter. dm 4 /Y hemizygous mutant larvae demonstrated no maternal dMyc and severe impairment of dDREF mRNA transcription. dMyc loss of function in dm 2 /dm 2 homozygous mutant follicle cell clones also resulted in loss of anti-dDREF immunostaining in nuclei. In contrast, co-expression of dMyc-dMax up-regulated dDREF promoter activity in S2 cells. Furthermore, dMyc over-expressing clones exhibited a high level of dDREF gene expression in wing and eye discs. These results taken together indicate that dMyc is indeed required for dDREF gene expression

  3. First Necrotizing Fasciitis Caused by Haemophilus influenza Serotype a

    Science.gov (United States)

    Quach, Giang T.; Frisby, Jared; Kralovich, Kurt; Bohra, Mustafa

    2017-01-01

    Necrotizing fasciitis (NF) is an infrequently encountered skin infection that has high morbidity and mortality, even with prompt medical and surgical intervention. We describe the case of a 67-year-old male presenting with significant NF in his left lower extremity, despite aggressive surgical intervention, and included multiple surgical debridements, ACell Matrix, split-thickness, and negative wound VAC therapy. Ultimately, this patient required a below the knee amputation. This is the first documented case of Haemophilus influenza type a causing NF. PMID:29124073

  4. Invasive Disease Caused by Nontypeable Haemophilus Influenzae

    Centers for Disease Control (CDC) Podcasts

    2015-11-12

    Dr. Elizabeth Briere discusses Nontypeable Haemophilus influenzae which causes a variety of infections in children and adults.  Created: 11/12/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 11/17/2015.

  5. Low occurrence of 'non-haemolytic Haemophilus haemolyticus' misidentified as Haemophilus influenzae in cystic fibrosis respiratory specimens, and frequent recurrence of persistent H. influenzae clones despite antimicrobial treatment.

    Science.gov (United States)

    Fenger, Mette G; Ridderberg, Winnie; Olesen, Hanne V; Nørskov-Lauritsen, Niels

    2012-12-01

    Non-influenzae commensal Haemophilus species of low pathogenicity may be difficult to discriminate from Haemophilus influenzae. We investigated the level of misidentifications in respiratory specimens from cystic fibrosis patients and evaluated the colonisation dynamics of genuine H. influenzae isolates. One hundred and ninety-two presumptive H. influenzae isolates were re-examined by assessment of marker genes sodC and fucK, and isolates with aberrant genotypes were subjected to multilocus sequence typing. Misidentifications (3%) were mainly caused by failure to identify porphyrin-synthesising strains, and only a single strain (0.5%) could be classified as 'non-haemolytic Haemophilus haemolyticus'. Sequential isolates of confirmed H. influenzae isolates from individual patients were typed by pulsed-field gel electrophoresis. Despite the routine prescription of antimicrobial therapy, the majority of H. influenzae isolates were identical with at least one of the strains cultured from the two preceding positive samples from the same patient. Copyright © 2012 Elsevier GmbH. All rights reserved.

  6. Search for missing schizophrenia genes will require a new ...

    Indian Academy of Sciences (India)

    2013-08-06

    Aug 6, 2013 ... causal gene(s)?. The successful search for disease genes is based on a ..... 2010 Mobile interspersed repeats are major structural variants in ... Petronis A., Paterson A. D. and Kennedy J. L. 1999 Schizophrenia: an epigenetic ...

  7. Nociceptor-Enriched Genes Required for Normal Thermal Nociception

    Directory of Open Access Journals (Sweden)

    Ken Honjo

    2016-07-01

    Full Text Available Here, we describe a targeted reverse genetic screen for thermal nociception genes in Drosophila larvae. Using laser capture microdissection and microarray analyses of nociceptive and non-nociceptive neurons, we identified 275 nociceptor-enriched genes. We then tested the function of the enriched genes with nociceptor-specific RNAi and thermal nociception assays. Tissue-specific RNAi targeted against 14 genes caused insensitive thermal nociception while targeting of 22 genes caused hypersensitive thermal nociception. Previously uncategorized genes were named for heat resistance (i.e., boilerman, fire dancer, oven mitt, trivet, thawb, and bunker gear or heat sensitivity (firelighter, black match, eucalyptus, primacord, jet fuel, detonator, gasoline, smoke alarm, and jetboil. Insensitive nociception phenotypes were often associated with severely reduced branching of nociceptor neurites and hyperbranched dendrites were seen in two of the hypersensitive cases. Many genes that we identified are conserved in mammals.

  8. 21 CFR 866.3300 - Haemophilus spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... serological tests to identify Haemophilus spp. directly from clinical specimens or tissue culture isolates derived from clinical specimens. The identification aids in the diagnosis of diseases caused by bacteria belonging to the genus Haemophilus and provides epidemiological information on diseases cause by these...

  9. Functional requirements driving the gene duplication in 12 Drosophila species.

    Science.gov (United States)

    Zhong, Yan; Jia, Yanxiao; Gao, Yang; Tian, Dacheng; Yang, Sihai; Zhang, Xiaohui

    2013-08-15

    Gene duplication supplies the raw materials for novel gene functions and many gene families arisen from duplication experience adaptive evolution. Most studies of young duplicates have focused on mammals, especially humans, whereas reports describing their genome-wide evolutionary patterns across the closely related Drosophila species are rare. The sequenced 12 Drosophila genomes provide the opportunity to address this issue. In our study, 3,647 young duplicate gene families were identified across the 12 Drosophila species and three types of expansions, species-specific, lineage-specific and complex expansions, were detected in these gene families. Our data showed that the species-specific young duplicate genes predominated (86.6%) over the other two types. Interestingly, many independent species-specific expansions in the same gene family have been observed in many species, even including 11 or 12 Drosophila species. Our data also showed that the functional bias observed in these young duplicate genes was mainly related to responses to environmental stimuli and biotic stresses. This study reveals the evolutionary patterns of young duplicates across 12 Drosophila species on a genomic scale. Our results suggest that convergent evolution acts on young duplicate genes after the species differentiation and adaptive evolution may play an important role in duplicate genes for adaption to ecological factors and environmental changes in Drosophila.

  10. Molecular surveillance of true nontypeable Haemophilus influenzae: an evaluation of PCR screening assays.

    Science.gov (United States)

    Binks, Michael J; Temple, Beth; Kirkham, Lea-Ann; Wiertsema, Selma P; Dunne, Eileen M; Richmond, Peter C; Marsh, Robyn L; Leach, Amanda J; Smith-Vaughan, Heidi C

    2012-01-01

    Unambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination. Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22), Hh (n = 27) or equivocal (n = 11), were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3) and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction. Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease.

  11. Organization of Genes Required for the Oxidation of Methanol to Formaldehyde in Three Type II Methylotrophs

    Science.gov (United States)

    Bastien, C.; Machlin, S.; Zhang, Y.; Donaldson, K.; Hanson, R. S.

    1989-01-01

    Restriction maps of genes required for the synthesis of active methanol dehydrogenase in Methylobacterium organophilum XX and Methylobacterium sp. strain AM1 have been completed and compared. In these two species of pink-pigmented, type II methylotrophs, 15 genes were identified that were required for the expression of methanol dehydrogenase activity. None of these genes were required for the synthesis of the prosthetic group of methanol dehydrogenase, pyrroloquinoline quinone. The structural gene required for the synthesis of cytochrome cL, an electron acceptor uniquely required for methanol dehydrogenase, and the genes encoding small basic peptides that copurified with methanol dehydrogenases were closely linked to the methanol dehydrogenase structural genes. A cloned 22-kilobase DNA insert from Methylsporovibrio methanica 81Z, an obligate type II methanotroph, complemented mutants that contained lesions in four genes closely linked to the methanol dehydrogenase structural genes. The methanol dehydrogenase and cytochrome cL structural genes were found to be transcribed independently in M. organophilum XX. Only two of the genes required for methanol dehydrogenase synthesis in this bacterium were found to be cotranscribed. PMID:16348074

  12. Identifying Haemophilus haemolyticus and Haemophilus influenzae by SYBR Green real-time PCR.

    Science.gov (United States)

    Latham, Roger; Zhang, Bowen; Tristram, Stephen

    2015-05-01

    SYBR Green real time PCR assays for protein D (hpd), fuculose kinase (fucK) and [Cu, Zn]-superoxide dismutase (sodC) were designed for use in an algorithm for the identification of Haemophilus influenzae and H. haemolyticus. When tested on 127 H. influenzae and 60 H. haemolyticus all isolates were identified correctly. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

  13. Difficult identification of Haemophilus influenzae, a typical cause of upper respiratory tract infections, in the microbiological diagnostic routine.

    Science.gov (United States)

    Hinz, Rebecca; Zautner, Andreas Erich; Hagen, Ralf Matthias; Frickmann, Hagen

    2015-03-01

    Haemophilus influenzae is a key pathogen of upper respiratory tract infections. Its reliable discrimination from nonpathogenic Haemophilus spp. is necessary because merely colonizing bacteria are frequent at primarily unsterile sites. Due to close phylogenetic relationship, it is not easy to discriminate H. influenzae from the colonizer Haemophilus haemolyticus. The frequency of H. haemolyticus isolations depends on factors like sampling site, patient condition, and geographic region. Biochemical discrimination has been shown to be nonreliable. Multiplex PCR including marker genes like sodC, fucK, and hpd or sequencing of the 16S rRNA gene, the P6 gene, or multilocus-sequence-typing is more promising. For the diagnostic routine, such techniques are too expensive and laborious. If available, matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry is a routine-compatible option and should be used in the first line. However, the used database should contain well-defined reference spectra, and the spectral difference between H. influenzae and H. haemolyticus is small. Fluorescence in-situ hybridization is an option for less well-equipped laboratories, but the available protocol will not lead to conclusive results in all instances. It can be used as a second line approach. Occasional ambiguous results have to be resolved by alternative molecular methods like 16S rRNA gene sequencing.

  14. Virtual screening of phytochemicals to novel targets in Haemophilus ducreyi towards the treatment of Chancroid.

    Science.gov (United States)

    Tripathi, Pranav; Chaudhary, Ritu; Singh, Ajeet

    2014-01-01

    Conventionally, drugs are discovered by testing chemically synthesized compounds against a battery of in vivo biological screens. Information technology and Omic science enabled us for high throughput screening of compound libraries against biological targets and hits are then tested for efficacy in cells or animals. Chancroid, caused by Haemophilus ducreyi is a public health problem and has been recognized as a cofactor for Human Immunodeficiency Virus (HIV) transmission. It facilitates HIV transmission by providing an accessible portal entry, promoting viral shedding, and recruiting macrophages as well as CD4 cells to the skin. So, there is a requirement to develop an efficient drug to combat Chancroid that can also diminish HIV infection. In-silico screening of potential inhibitors against the target may facilitate in detection of the novel lead compounds for developing an effective chemo preventive strategy against Haemophilus ducreyi. The present study has investigated the effects of approximately 1100 natural compounds that inhibit three vital enzymes viz. Phosphoenolpyruvate phosphotransferase, Acetyl-coenzyme A carboxylase and Fructose 1, 6-bisphosphatase of Haemophilus ducreyi in reference to a commercial drug Rifabutin. Results reveal that the lead compound uses less energy to bind to target. The lead compound parillin has also been predicted as less immunogenic in comparison to Rifabutin. Further, better molecular dynamics, pharmacokinetics, pharmacodynamics and ADME-T properties establish it as an efficient chancroid preventer.

  15. Haemophilus ducreyi Cutaneous Ulcer Strains Are Nearly Identical to Class I Genital Ulcer Strains.

    Directory of Open Access Journals (Sweden)

    Dharanesh Gangaiah

    Full Text Available Although cutaneous ulcers (CU in the tropics is frequently attributed to Treponema pallidum subspecies pertenue, the causative agent of yaws, Haemophilus ducreyi has emerged as a major cause of CU in yaws-endemic regions of the South Pacific islands and Africa. H. ducreyi is generally susceptible to macrolides, but CU strains persist after mass drug administration of azithromycin for yaws or trachoma. H. ducreyi also causes genital ulcers (GU and was thought to be exclusively transmitted by microabrasions that occur during sex. In human volunteers, the GU strain 35000HP does not infect intact skin; wounds are required to initiate infection. These data led to several questions: Are CU strains a new variant of H. ducreyi or did they evolve from GU strains? Do CU strains contain additional genes that could allow them to infect intact skin? Are CU strains susceptible to azithromycin?To address these questions, we performed whole-genome sequencing and antibiotic susceptibility testing of 5 CU strains obtained from Samoa and Vanuatu and 9 archived class I and class II GU strains. Except for single nucleotide polymorphisms, the CU strains were genetically almost identical to the class I strain 35000HP and had no additional genetic content. Phylogenetic analysis showed that class I and class II strains formed two separate clusters and CU strains evolved from class I strains. Class I strains diverged from class II strains ~1.95 million years ago (mya and CU strains diverged from the class I strain 35000HP ~0.18 mya. CU and GU strains evolved under similar selection pressures. Like 35000HP, the CU strains were highly susceptible to antibiotics, including azithromycin.These data suggest that CU strains are derivatives of class I strains that were not recognized until recently. These findings require confirmation by analysis of CU strains from other regions.

  16. Haemophilus influenzae type b pneumonia in Egyptian children ...

    African Journals Online (AJOL)

    Haemophilus influenzae (Hi) causes more than 3 million cases of serious disease, mainly meningitis and ... One hundred patients with community-acquired pneumonia were investigated for Hib by both real-time PCR and bacterial culture.

  17. Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpB Real-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections.

    Science.gov (United States)

    Reddington, Kate; Schwenk, Stefan; Tuite, Nina; Platt, Gareth; Davar, Danesh; Coughlan, Helena; Personne, Yoann; Gant, Vanya; Enne, Virve I; Zumla, Alimuddin; Barry, Thomas

    2015-09-01

    Haemophilus influenzae is a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzae diagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzae in RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpB gene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilus spp. Subsequently, 44 Haemophilus clinical isolates were collected, and 36 isolates were identified as H. influenzae using a gold standard methodology that combined the results of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fucK diagnostic assay. Using the novel smpB diagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n = 98) were blindly tested with the gold standard and smpB diagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpB assay when used directly on respiratory specimens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Chancroid and Haemophilus ducreyi: an update.

    Science.gov (United States)

    Trees, D L; Morse, S A

    1995-07-01

    Haemophilus ducreyi is a fastidious gram-negative bacillus that causes the sexually transmitted infection chancroid. Chancroid is a major genital ulcerative disease in Africa, Southeast Asia, the Caribbean, and Latin America and is of increasing concern in the United States. Genital ulcerative disease and chancroid in particular have been associated with facilitating the transmission of human immunodeficiency virus. The diagnosis of chancroid based on the clinical appearance of the genital lesion or on the isolation of H. ducreyi on selective medium is relatively insensitive. However, recent advances in nonculture diagnostic tests have enhanced our ability to diagnose chancroid. There has been renewed interest in understanding the pathogenesis of H. ducreyi. In vitro and in vivo models have been developed to help identify important virulence determinants. Through the use of biochemical and molecular techniques, macromolecular components that may be important in virulence have been identified.

  19. The Laboratory Diagnosis of Haemophilus ducreyi

    Directory of Open Access Journals (Sweden)

    Michelle Alfa

    2005-01-01

    Full Text Available Chancroid is a sexually transmitted infection caused by Haemophilus ducreyi. This fastidious, Gram-negative coccobacilli dies rapidly outside the human host, making diagnostic testing using culture methods difficult. This genital ulcer infection is not common in Canada and, therefore, can often be misdiagnosed. The objective of the present paper is to provide practical approaches for the diagnosis of chancroid in Canadian patients where the prevalence of this infection is low. Issues related to sample collection, sample transport and available diagnostic tests are reviewed, and several alternative approaches are outlined. Although antigen detection, serology and genetic amplification methods have all been reported for H ducreyi, none are commercially available. Culture is still the primary method available to most laboratories. However, the special media necessary for direct bedside inoculation is often not available; therefore, communication with the diagnostic laboratory and rapid specimen transport are essential when chancroid is suspected

  20. Genes required for Lactococcus garvieae survival in a fish host.

    Science.gov (United States)

    Menéndez, Aurora; Fernández, Lucia; Reimundo, Pilar; Guijarro, José A

    2007-10-01

    Lactococcus garvieae is considered an emergent pathogen in aquaculture and it is also associated with mastitis in domestic animals as well as human endocarditis and septicaemia. In spite of this, the pathogenic mechanisms of this bacterium are poorly understood. Signature-tagged mutagenesis was used to identify virulence factors and to establish the basis of pathogen-host interactions. A library of 1250 L. garvieae UNIUD074-tagged Tn917 mutants in 25 pools was screened for the ability to grow in fish. Among them, 29 mutants (approx. 2.4 %) were identified which could not be recovered from rainbow trout following infection. Sequence analysis of the tagged Tn917-interrupted genes in these mutants indicated the participation in pathogenesis of the transcriptional regulatory proteins homologous to GidA and MerR; the metabolic enzymes asparagine synthetase A and alpha-acetolactate synthase; the ABC transport system of glutamine and a calcium-transporting ATPase; the dltA locus involved in alanylation of teichoic acids; and hypothetical proteins containing EAL and Eis domains, among others. Competence index experiments in several of the selected mutants confirmed the relevance of the Tn917-interrupted genes in the development of the infection process. The results suggested some of the metabolic routes and enzymic systems necessary for the complete virulence of this bacterium. This work is believed to represent the first report of a genome-wide scan for virulence factors in L. garvieae. The identified genes will further our understanding of the pathogenesis of L. garvieae infections and may provide targets for intervention or lead to the development of novel therapies.

  1. Basic Characterization of Natural Transformation in a Highly Transformable Haemophilus parasuis Strain SC1401

    Science.gov (United States)

    Dai, Ke; He, Lvqin; Chang, Yung-Fu; Cao, Sanjie; Zhao, Qin; Huang, Xiaobo; Wu, Rui; Huang, Yong; Yan, Qigui; Han, Xinfeng; Ma, Xiaoping; Wen, Xintian; Wen, Yiping

    2018-01-01

    Haemophilus parasuis causes Glässer's disease and pneumonia, incurring serious economic losses in the porcine industry. In this study, natural competence was investigated in H. parasuis. We found competence genes in H. parasuis homologous to ones in Haemophilus influenzae and a high consensus battery of Sxy-dependent cyclic AMP (cAMP) receptor protein (CRP-S) regulons using bioinformatics. High rates of natural competence were found from the onset of stationary-phase growth condition to mid-stationary phase (OD600 from 0.29 to 1.735); this rapidly dropped off as cells reached mid-stationary phase (OD600 from 1.735 to 1.625). As a whole, bacteria cultured in liquid media were observed to have lower competence levels than those grown on solid media plates. We also revealed that natural transformation in this species is stable after 200 passages and is largely dependent on DNA concentration. Transformation competition experiments showed that heterogeneous DNA cannot outcompete intraspecific natural transformation, suggesting an endogenous uptake sequence or other molecular markers may be important in differentiating heterogeneous DNA. We performed qRT-PCR targeting multiple putative competence genes in an effort to compare bacteria pre-cultured in TSB++ vs. TSA++ and SC1401 vs. SH0165 to determine expression profiles of the homologs of competence-genes in H. influenzae. Taken together, this study is the first to investigate natural transformation in H. parasuis based on a highly naturally transformable strain SC1401. PMID:29473023

  2. Metabolic versatility in Haemophilus influenzae: a metabolomic and genomic analysis

    Directory of Open Access Journals (Sweden)

    Dk Seti Maimonah Pg eOthman

    2014-03-01

    Full Text Available Haemophilus influenzae is a host adapted human pathogen known to contribute to a variety of acute and chronic diseases of the upper and lower respiratory tract as well as the middle ear. At the sites of infection as well as during growth as a commensal the environmental conditions encountered by H. influenzae will vary significantly, especially in terms of oxygen availability, however, the mechanisms by which the bacteria can adapt their metabolism to cope with such changes have not been studied in detail. Using targeted metabolomics the spectrum of metabolites produced during growth of H. influenzae on glucose in RPMI-based medium was found to change from acetate as the main product during aerobic growth to formate as the major product during anaerobic growth. This is likely caused by a switch in the major pyruvate degrading route. Neither lactate nor succinate or fumarate were major products of H. influenzae growth under any condition studied Gene expression studies and enzyme activity data revealed that despite an identical genetic makeup and very similar metabolite production profiles, H. influenzae strain Rd appeared to favour glucose degradation via the PPP, while strain 2019, a clinical isolate, showed higher expression of enzymes involved in glycolysis. Components of the respiratory chain were most highly expressed during microaerophilic and anaerobic growth in both strains, but again clear differences existed in the expression of genes associated e.g. with NADH oxidation, nitrate and nitrite reduction in the two strains studied.Together our results indicate that H. influenzae uses a specialized type of metabolism that could be termed ‘respiration assisted fermentation’ where the respiratory chain likely serves to alleviate redox imbalances caused by incomplete glucose oxidation, and at the same time provides a means of converting a variety of compounds including nitrite and nitrate that arise as part of the host defence mechanisms.

  3. Haemophilus ducreyi: from sexually transmitted infection to skin ulcer pathogen.

    Science.gov (United States)

    Lewis, David A; Mitjà, Oriol

    2016-02-01

    This article provides an overview of the biology, epidemiology, clinical features, diagnostic tests, and treatment of Haemophilus ducreyi infection, with special reference to the decline of chancroid and the recent emergence of H. ducreyi as a pathogen responsible for chronic limb ulceration clinically similar to yaws. Chancroid has declined in importance as a sexually transmitted infection in most countries where it was previously endemic. Chancroid may be caused by either class I or class II H. ducreyi isolates; these two classes diverged from each other approximately 1.95 million years ago. H. ducreyi has recently emerged as a cause of chronic skin ulceration in the Pacific region and Africa. Based on sequencing of whole genomes and defined genetic loci, it appears that the cutaneous H. ducreyi strains diverged from the class I genital strains relatively recently. H. ducreyi should be considered as a major cause of chronic limb ulceration in both adults and children and appropriate molecular diagnostic assays are required to determine ulcer aetiology. The high prevalence of H. ducreyi-related cutaneous ulceration in yaws-endemic countries has challenged the validity of observational surveys to monitor the effectiveness of the WHO's yaws eradication campaign.

  4. Functional requirements for bacteriophage growth: gene essentiality and expression in mycobacteriophage Giles.

    Science.gov (United States)

    Dedrick, Rebekah M; Marinelli, Laura J; Newton, Gerald L; Pogliano, Kit; Pogliano, Joseph; Hatfull, Graham F

    2013-05-01

    Bacteriophages represent a majority of all life forms, and the vast, dynamic population with early origins is reflected in their enormous genetic diversity. A large number of bacteriophage genomes have been sequenced. They are replete with novel genes without known relatives. We know little about their functions, which genes are required for lytic growth, and how they are expressed. Furthermore, the diversity is such that even genes with required functions - such as virion proteins and repressors - cannot always be recognized. Here we describe a functional genomic dissection of mycobacteriophage Giles, in which the virion proteins are identified, genes required for lytic growth are determined, the repressor is identified, and the transcription patterns determined. We find that although all of the predicted phage genes are expressed either in lysogeny or in lytic growth, 45% of the predicted genes are non-essential for lytic growth. We also describe genes required for DNA replication, show that recombination is required for lytic growth, and that Giles encodes a novel repressor. RNAseq analysis reveals abundant expression of a small non-coding RNA in a lysogen and in late lytic growth, although it is non-essential for lytic growth and does not alter lysogeny. © 2013 Blackwell Publishing Ltd.

  5. Molecular cloning and characterization of genes required for nucleotide excision repair in yeast

    International Nuclear Information System (INIS)

    Friedberg, E.C.

    1987-01-01

    Nucleotide excision repair in the yeast S. cerevisiae is a complex process which involves a large number of genes. At least five of these genes (RAD1, RAD2, RAD3, RAD4 and RAD10) are absolutely required for this process and mutations in any of these genes result in no detectable excision repair in vivo. In order to understand the function of these genes in DNA repair, the authors isolated a number of them by screening a yeast genomic library for recombinant plasmids which complement the phentoype of sensitivity to ultraviolet (UV) radiation imparted to mutant strains. A plasmid containing the RAD4 gene was isolated by an alternative strategy which will be discussed. The cloned genes have been extensively characterized. It has been determined that the RAD3 gene is essential for the viability of haploid yeast cells in the absence of DNA damage. The RAD2 gene is inducible by treatment of cells with a variety of DNA-damaging agents, including UV radiation and ionizing radiation. The RAD10 gene shares considerable amino acid sequence homology with a cloned gene involved in nucleotide excision repair in human cells. Yeast is a particularly versatile organism for studying gene function by molecular and genetic approaches and emphasis is placed on many of the techniques used in the present studies

  6. Haemophilus somnus (Histophilus somni) in bighorn sheep

    Science.gov (United States)

    2006-01-01

    Abstract Respiratory disease and poor lamb recruitment have been identified as limiting factors for bighorn-sheep populations. Haemophilus somnus (recently reclassified as Histophilus somni) is associated with respiratory disease in American bison, domestic sheep, and cattle. It is also harbored in their reproductive tracts and has been associated with reproductive failure in domestic sheep and cattle. Therefore, reproductive tract and lung samples from bighorn sheep were evaluated for the presence of this organism. Organisms identified as H. somnus were isolated from 6 of 62 vaginal but none of 12 preputial swab samples. Antigen specific to H. somnus was detected by immunohistochemical study in 4 of 12 formalin-fixed lung tissue samples of bighorn sheep that died with evidence of pneumonia. Notably, H. somnus was found in alveolar debris in areas of inflammation. The 6 vaginal isolates and 2 H. somnus isolates previously cultured from pneumonic lungs of bighorn sheep were compared with 3 representative isolates from domestic sheep and 2 from cattle. The profiles of major outer membrane proteins and antigens for all of the isolates were predominantly similar, although differences that may be associated with the host–parasite relationship and virulence were detected. The DNA restriction fragment length profiles of the bighorn-sheep isolates had similarities not shared with the other isolates, suggesting distinct phylogenetic lines. All of the isolates had similar antimicrobial profiles, but the isolates from the bighorn sheep produced less pigment than those from the domestic livestock, and growth of the former was not enhanced by CO2. Wildlife biologists and diagnosticians should be aware of the potential of these organisms to cause disease in bighorn sheep and of growth characteristics that may hinder laboratory detection. PMID:16548330

  7. Haemophilus somnus (Histophilus somni) in bighorn sheep.

    Science.gov (United States)

    Ward, Alton C S; Weiser, Glen C; Anderson, Bruce C; Cummings, Patrick J; Arnold, Karen F; Corbeil, Lynette B

    2006-01-01

    Respiratory disease and poor lamb recruitment have been identified as limiting factors for bighorn-sheep populations. Haemophilus somnus (recently reclassified as Histophilus somni) is associated with respiratory disease in American bison, domestic sheep, and cattle. It is also harbored in their reproductive tracts and has been associated with reproductive failure in domestic sheep and cattle. Therefore, reproductive tract and lung samples from bighorn sheep were evaluated for the presence of this organism. Organisms identified as H. somnus were isolated from 6 of 62 vaginal but none of 12 preputial swab samples. Antigen specific to H. somnus was detected by immunohistochemical study in 4 of 12 formalin-fixed lung tissue samples of bighorn sheep that died with evidence of pneumonia. Notably, H. somnus was found in alveolar debris in areas of inflammation. The 6 vaginal isolates and 2 H. somnus isolates previously cultured from pneumonic lungs of bighorn sheep were compared with 3 representative isolates from domestic sheep and 2 from cattle. The profiles of major outer membrane proteins and antigens for all of the isolates were predominantly similar, although differences that may be associated with the host-parasite relationship and virulence were detected. The DNA restriction fragment length profiles of the bighorn-sheep isolates had similarities not shared with the other isolates, suggesting distinct phylogenetic lines. All of the isolates had similar antimicrobial profiles, but the isolates from the bighorn sheep produced less pigment than those from the domestic livestock, and growth of the former was not enhanced by CO2. Wildlife biologists and diagnosticians should be aware of the potential of these organisms to cause disease in bighorn sheep and of growth characteristics that may hinder laboratory detection.

  8. Molecular surveillance of true nontypeable Haemophilus influenzae: an evaluation of PCR screening assays.

    Directory of Open Access Journals (Sweden)

    Michael J Binks

    Full Text Available BACKGROUND: Unambiguous identification of nontypeable Haemophilus influenzae (NTHi is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh; however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination. METHODOLOGY/PRINCIPAL FINDINGS: Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22, Hh (n = 27 or equivocal (n = 11, were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3 and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction. CONCLUSIONS/SIGNIFICANCE: Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease.

  9. The Aspergillus flavus Homeobox Gene, hbx1, Is Required for Development and Aflatoxin Production

    Directory of Open Access Journals (Sweden)

    Jeffrey W. Cary

    2017-10-01

    Full Text Available Homeobox proteins, a class of well conserved transcription factors, regulate the expression of targeted genes, especially those involved in development. In filamentous fungi, homeobox genes are required for normal conidiogenesis and fruiting body formation. In the present study, we identified eight homeobox (hbx genes in the aflatoxin-producing ascomycete, Aspergillus flavus, and determined their respective role in growth, conidiation and sclerotial production. Disruption of seven of the eight genes had little to no effect on fungal growth and development. However, disruption of the homeobox gene AFLA_069100, designated as hbx1, in two morphologically different A. flavus strains, CA14 and AF70, resulted in complete loss of production of conidia and sclerotia as well as aflatoxins B1 and B2, cyclopiazonic acid and aflatrem. Microscopic examination showed that the Δhbx1 mutants did not produce conidiophores. The inability of Δhbx1 mutants to produce conidia was related to downregulation of brlA (bristle and abaA (abacus, regulatory genes for conidiophore development. These mutants also had significant downregulation of the aflatoxin pathway biosynthetic genes aflC, aflD, aflM and the cluster-specific regulatory gene, aflR. Our results demonstrate that hbx1 not only plays a significant role in controlling A. flavus development but is also critical for the production of secondary metabolites, such as aflatoxins.

  10. NHR-23 dependent collagen and hedgehog-related genes required for molting

    International Nuclear Information System (INIS)

    Kouns, Nathaniel A.; Nakielna, Johana; Behensky, Frantisek; Krause, Michael W.; Kostrouch, Zdenek; Kostrouchova, Marta

    2011-01-01

    Highlights: → NHR-23 is a critical regulator of nematode development and molting. → The manuscript characterizes the loss-of-function phenotype of an nhr-23 mutant. → Whole genome expression analysis identifies new potential targets of NHR-23. → Hedgehog-related genes are identified as NHR-23 dependent genes. → New link between sterol mediated signaling and regulation by NHR-23 is found. -- Abstract: NHR-23, a conserved member of the nuclear receptor family of transcription factors, is required for normal development in Caenorhabditis elegans where it plays a critical role in growth and molting. In a search for NHR-23 dependent genes, we performed whole genome comparative expression microarrays on both control and nhr-23 inhibited synchronized larvae. Genes that decreased in response to nhr-23 RNAi included several collagen genes. Unexpectedly, several hedgehog-related genes were also down-regulated after nhr-23 RNAi. A homozygous nhr-23 deletion allele was used to confirm the RNAi knockdown phenotypes and the changes in gene expression. Our results indicate that NHR-23 is a critical co-regulator of functionally linked genes involved in growth and molting and reveal evolutionary parallels among the ecdysozoa.

  11. NHR-23 dependent collagen and hedgehog-related genes required for molting

    Energy Technology Data Exchange (ETDEWEB)

    Kouns, Nathaniel A.; Nakielna, Johana; Behensky, Frantisek [Laboratory of Model Systems, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague (Czech Republic); Krause, Michael W. [Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD (United States); Kostrouch, Zdenek [Laboratory of Model Systems, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague (Czech Republic); Kostrouchova, Marta, E-mail: marta.kostrouchova@lf1.cuni.cz [Laboratory of Model Systems, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague (Czech Republic)

    2011-10-07

    Highlights: {yields} NHR-23 is a critical regulator of nematode development and molting. {yields} The manuscript characterizes the loss-of-function phenotype of an nhr-23 mutant. {yields} Whole genome expression analysis identifies new potential targets of NHR-23. {yields} Hedgehog-related genes are identified as NHR-23 dependent genes. {yields} New link between sterol mediated signaling and regulation by NHR-23 is found. -- Abstract: NHR-23, a conserved member of the nuclear receptor family of transcription factors, is required for normal development in Caenorhabditis elegans where it plays a critical role in growth and molting. In a search for NHR-23 dependent genes, we performed whole genome comparative expression microarrays on both control and nhr-23 inhibited synchronized larvae. Genes that decreased in response to nhr-23 RNAi included several collagen genes. Unexpectedly, several hedgehog-related genes were also down-regulated after nhr-23 RNAi. A homozygous nhr-23 deletion allele was used to confirm the RNAi knockdown phenotypes and the changes in gene expression. Our results indicate that NHR-23 is a critical co-regulator of functionally linked genes involved in growth and molting and reveal evolutionary parallels among the ecdysozoa.

  12. Aerosol challenge of calves with Haemophilus somnus and Mycoplasma dispar

    DEFF Research Database (Denmark)

    Tegtmeier, C.; Angen, Øystein; Grell, S.N.

    2000-01-01

    The aim of the study was to examine the ability of Haemophilus somnus and Mycoplasma dispar to induce pneumonia in healthy calves under conditions closely resembling the supposed natural way of infection, viz, by inhalation of aerosol droplets containing the microorganisms. The infections were...

  13. The costs and benefits of a vaccination programme for Haemophilus ...

    African Journals Online (AJOL)

    The US Food and. Drug Administration ... reduce their risk of death, regardless of their earning ... weight within the Consumer Price Index), and then converted to rands at ..... Research Council and Lederle Laboratories. REFERENCES. ,. Wifert C. Epidemiology of Haemophilus inf/uenzae type b infections. Pediatrics. 1990 ...

  14. Haemophilus influenzae type f meningitis in a previously healthy boy

    DEFF Research Database (Denmark)

    Ronit, Andreas; Berg, Ronan M G; Bruunsgaard, Helle

    2013-01-01

    Non-serotype b strains of Haemophilus influenzae are extremely rare causes of acute bacterial meningitis in immunocompetent individuals. We report a case of acute bacterial meningitis in a 14-year-old boy, who was previously healthy and had been immunised against H influenzae serotype b (Hib...

  15. Safety and immunogenicity of two Haemophilus influenzae type b ...

    African Journals Online (AJOL)

    Objectives. Haemophilus influenzae type b (Hib) infection remains a major public health problem inthe developing world. We evaluated the safety and immunogenicity of a new PRP-CRM197 conjugate Hib vaccine (Vaxem Hib, Chiron Vacdnes), compared with theHibTITER vaccine (WyethLederle Vaccines), following the ...

  16. Chronic cutaneous ulcers secondary to Haemophilus ducreyi infection.

    Science.gov (United States)

    Peel, Trisha N; Bhatti, Deepak; De Boer, Jim C; Stratov, Ivan; Spelman, Denis W

    2010-03-15

    Haemophilus ducreyi is a well recognised causative agent of genital ulcers and chancroid. We report two unusual cases of non-sexually transmitted H. ducreyi infection leading to chronic lower limb ulcers. Both patients were Australian expatriates visiting Australia from the Pacific Islands--one from Papua New Guinea and the other from Vanuatu.

  17. Haemophilus influenzae Type a Meningitis in Immunocompetent Child, Oman, 2015.

    Science.gov (United States)

    Sawardekar, Kiran P

    2017-07-01

    Meningitis caused by Haemophilus influenzae type b (Hib) was eliminated in Oman after the introduction of Hib vaccine in 2001. However, a case of H. influenzae type a meningitis was diagnosed in a child from Oman in 2015, which highlights the need to monitor the incidence of invasive non-Hib H. influenzae disease.

  18. [Severe Haemophilus influenzae b infection in healthy male adult

    DEFF Research Database (Denmark)

    Vilmar, A.C.; Gjorup, I.; David, Kim Peter

    2008-01-01

    Haemophilus influenzae b (Hib) can be the cause of serious infections, and is mainly observed affecting children and immuno-compromised patients. We report a case of a healthy 49-year old male with a severe Hib infection complicated by septicaemia, meningitis and anuria. The risk of invasive Hib...

  19. OxyR of Haemophilus parasuis is a global transcriptional regulator important in oxidative stress resistance and growth.

    Science.gov (United States)

    Wen, Yongping; Wen, Yiping; Wen, Xintian; Cao, Sanjie; Huang, Xiaobo; Wu, Rui; Zhao, Qin; Liu, Mafeng; Huang, Yong; Yan, Qigui; Han, Xinfeng; Ma, Xiaoping; Dai, Ke; Ding, Lingqiang; Liu, Sitong; Yang, Jian

    2018-02-15

    Haemophilus parasuis is an opportunistic pathogen and the causative agent of Glässer's disease in swine. This disease has high morbidity and mortality rates in swine populations, and is responsible for major economic losses worldwide. Survival of H. parasuis within the host requires mechanisms for coping with oxidative stress conditions. In many bacteria, OxyR is known to mediate protection against oxidative stress; however, little is known about the role of OxyR in H. parasuis. In the current study, an oxyR mutant strain was constructed in H. parasuis strain SC1401 and designated H. parasuis SC1401∆oxyR. The oxyR mutant strain had a slower growth rate and impaired biofilm formation compared to the wild type strain. Complementation restored the growth-associated phenotypes to wild type levels. Oxidative stress susceptibility testing, using a range of concentrations of H 2 O 2 , indicated that H. parasuis SC1401∆oxyR was more sensitive to oxidative stress than the wild type strain. RNA sequencing transcriptome analysis comparing H. parasuis SC1401 with H. parasuis SC1401∆oxyR identified 466 differentially expressed genes. These genes were involved in a wide range of biological processes, including: oxidative stress, transcriptional regulation, and DNA replication, recombination, and repair. These findings provide a foundation for future research to examine the role of OxyR as a global transcriptional regulator and to better define its role in oxidative stress resistance in H. parasuis. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. A genetic screen identifies interferon-α effector genes required to suppress hepatitis C virus replication.

    Science.gov (United States)

    Fusco, Dahlene N; Brisac, Cynthia; John, Sinu P; Huang, Yi-Wen; Chin, Christopher R; Xie, Tiao; Zhao, Hong; Jilg, Nikolaus; Zhang, Leiliang; Chevaliez, Stephane; Wambua, Daniel; Lin, Wenyu; Peng, Lee; Chung, Raymond T; Brass, Abraham L

    2013-06-01

    Hepatitis C virus (HCV) infection is a leading cause of end-stage liver disease. Interferon-α (IFNα) is an important component of anti-HCV therapy; it up-regulates transcription of IFN-stimulated genes, many of which have been investigated for their antiviral effects. However, all of the genes required for the antiviral function of IFNα (IFN effector genes [IEGs]) are not known. IEGs include not only IFN-stimulated genes, but other nontranscriptionally induced genes that are required for the antiviral effect of IFNα. In contrast to candidate approaches based on analyses of messenger RNA (mRNA) expression, identification of IEGs requires a broad functional approach. We performed an unbiased genome-wide small interfering RNA screen to identify IEGs that inhibit HCV. Huh7.5.1 hepatoma cells were transfected with small interfering RNAs incubated with IFNα and then infected with JFH1 HCV. Cells were stained using HCV core antibody, imaged, and analyzed to determine the percent infection. Candidate IEGs detected in the screen were validated and analyzed further. The screen identified 120 previously unreported IEGs. From these, we more fully evaluated the following: asparagine-linked glycosylation 10 homolog (yeast, α-1,2-glucosyltransferase); butyrylcholinesterase; dipeptidyl-peptidase 4 (CD26, adenosine deaminase complexing protein 2); glucokinase (hexokinase 4) regulator; guanylate cyclase 1, soluble, β 3; MYST histone acetyltransferase 1; protein phosphatase 3 (formerly 2B), catalytic subunit, β isoform; peroxisomal proliferator-activated receptor-γ-DBD-interacting protein 1; and solute carrier family 27 (fatty acid transporter), member 2; and demonstrated that they enabled IFNα-mediated suppression of HCV at multiple steps of its life cycle. Expression of these genes had more potent effects against flaviviridae because a subset was required for IFNα to suppress dengue virus but not influenza A virus. In addition, many of the host genes detected in this

  1. Gene Expression Profiling of Cecropin B-Resistant Haemophilus parasuis

    NARCIS (Netherlands)

    Wang, Chunmei; Chen, Fangzhou; Hu, Han; Li, Wentao; Wang, Yang; Chen, Pin; Liu, Yingyu; Ku, Xugang; He, Qigai; Chen, Huanchun; Xue, Feiqun

    2014-01-01

    Synthetically designed antimicrobial peptides (AMPs) present the potential of replacing antibiotics in the treatment of bacterial infections. However, microbial resistance to AMPs has been reported and little is known regarding the underlying mechanism of such resistance. The naturally occurring AMP

  2. Haemophilus responses to nutritional immunity: epigenetic and morphological contribution to biofilm architecture, invasion, persistence and disease severity.

    Directory of Open Access Journals (Sweden)

    Blake R Szelestey

    Full Text Available In an effort to suppress microbial outgrowth, the host sequesters essential nutrients in a process termed nutritional immunity. However, inflammatory responses to bacterial insult can restore nutritional resources. Given that nutrient availability modulates virulence factor production and biofilm formation by other bacterial species, we hypothesized that fluctuations in heme-iron availability, particularly at privileged sites, would similarly influence Haemophilus biofilm formation and pathogenesis. Thus, we cultured Haemophilus through sequential heme-iron deplete and heme-iron replete media to determine the effect of transient depletion of internal stores of heme-iron on multiple pathogenic phenotypes. We observed that prior heme-iron restriction potentiates biofilm changes for at least 72 hours that include increased peak height and architectural complexity as compared to biofilms initiated from heme-iron replete bacteria, suggesting a mechanism for epigenetic responses that participate in the changes observed. Additionally, in a co-infection model for human otitis media, heme-iron restricted Haemophilus, although accounting for only 10% of the inoculum (90% heme-iron replete, represented up to 99% of the organisms recovered at 4 days. These data indicate that fluctuations in heme-iron availability promote a survival advantage during disease. Filamentation mediated by a SulA-related ortholog was required for optimal biofilm peak height and persistence during experimental otitis media. Moreover, severity of disease in response to heme-iron restricted Haemophilus was reduced as evidenced by lack of mucosal destruction, decreased erythema, hemorrhagic foci and vasodilatation. Transient restriction of heme-iron also promoted productive invasion events leading to the development of intracellular bacterial communities. Taken together, these data suggest that nutritional immunity, may, in fact, foster long-term phenotypic changes that better equip

  3. Inflammatory response of Haemophilus influenzae biotype aegyptius causing Brazilian Purpuric Fever

    Directory of Open Access Journals (Sweden)

    Gisele Cristiane Gentile Cury

    2014-12-01

    Full Text Available The Brazilian Purpuric Fever (BPF is a systemic disease with many clinical features of meningococcal sepsis and is usually preceded by purulent conjunctivitis. The illness is caused by Haemophilus influenza biogroup aegyptius, which was associated exclusively with conjunctivitis. In this work construction of the las gene, hypothetically responsible for this virulence, were fusioned with ermAM cassette in Neisseria meningitidis virulent strains and had its DNA transfer to non BPF H. influenzae strains. The effect of the las transfer was capable to increase the cytokines TNFα and IL10 expression in Hec-1B cells line infected with these transformed mutants (in eight log scale of folding change RNA expression. This is the first molecular study involving the las transfer to search an elucidation of the pathogenic factors by horizontal intergeneric transfer from meningococci to H. influenzae.

  4. Identification of two gene clusters and a transcriptional regulator required for Pseudomonas aeruginosa glycine betaine catabolism.

    Science.gov (United States)

    Wargo, Matthew J; Szwergold, Benjamin S; Hogan, Deborah A

    2008-04-01

    Glycine betaine (GB), which occurs freely in the environment and is an intermediate in the catabolism of choline and carnitine, can serve as a sole source of carbon or nitrogen in Pseudomonas aeruginosa. Twelve mutants defective in growth on GB as the sole carbon source were identified through a genetic screen of a nonredundant PA14 transposon mutant library. Further growth experiments showed that strains with mutations in two genes, gbcA (PA5410) and gbcB (PA5411), were capable of growth on dimethylglycine (DMG), a catabolic product of GB, but not on GB itself. Subsequent nuclear magnetic resonance (NMR) experiments with 1,2-(13)C-labeled choline indicated that these genes are necessary for conversion of GB to DMG. Similar experiments showed that strains with mutations in the dgcAB (PA5398-PA5399) genes, which exhibit homology to genes that encode other enzymes with demethylase activity, are required for the conversion of DMG to sarcosine. Mutant analyses and (13)C NMR studies also confirmed that the soxBDAG genes, predicted to encode a sarcosine oxidase, are required for sarcosine catabolism. Our screen also identified a predicted AraC family transcriptional regulator, encoded by gbdR (PA5380), that is required for growth on GB and DMG and for the induction of gbcA, gbcB, and dgcAB in response to GB or DMG. Mutants defective in the previously described gbt gene (PA3082) grew on GB with kinetics similar to those of the wild type in both the PAO1 and PA14 strain backgrounds. These studies provided important insight into both the mechanism and the regulation of the catabolism of GB in P. aeruginosa.

  5. Comprehensive identification of Vibrio vulnificus genes required for growth in human serum.

    Science.gov (United States)

    Carda-Diéguez, M; Silva-Hernández, F X; Hubbard, T P; Chao, M C; Waldor, M K; Amaro, C

    2018-12-31

    Vibrio vulnificus can be a highly invasive pathogen capable of spreading from an infection site to the bloodstream, causing sepsis and death. To survive and proliferate in blood, the pathogen requires mechanisms to overcome the innate immune defenses and metabolic limitations of this host niche. We created a high-density transposon mutant library in YJ016, a strain representative of the most virulent V. vulnificus lineage (or phylogroup) and used transposon insertion sequencing (TIS) screens to identify loci that enable the pathogen to survive and proliferate in human serum. Initially, genes underrepresented for insertions were used to estimate the V. vulnificus essential gene set; comparisons of these genes with similar TIS-based classification of underrepresented genes in other vibrios enabled the compilation of a common Vibrio essential gene set. Analysis of the relative abundance of insertion mutants in the library after exposure to serum suggested that genes involved in capsule biogenesis are critical for YJ016 complement resistance. Notably, homologues of two genes required for YJ016 serum-resistance and capsule biogenesis were not previously linked to capsule biogenesis and are largely absent from other V. vulnificus strains. The relative abundance of mutants after exposure to heat inactivated serum was compared with the findings from the serum screen. These comparisons suggest that in both conditions the pathogen relies on its Na + transporting NADH-ubiquinone reductase (NQR) complex and type II secretion system to survive/proliferate within the metabolic constraints of serum. Collectively, our findings reveal the potency of comparative TIS screens to provide knowledge of how a pathogen overcomes the diverse limitations to growth imposed by serum.

  6. tlpA gene expression is required for arginine and bicarbonate chemotaxis in Helicobacter pylori.

    Science.gov (United States)

    Cerda, Oscar A; Núñez-Villena, Felipe; Soto, Sarita E; Ugalde, José Manuel; López-Solís, Remigio; Toledo, Héctor

    2011-01-01

    About half of the human population is infected with Helicobacter pylori, a bacterium causing gastritis, peptic ulcer and progression to gastric cancer. Chemotaxis and flagellar motility are required for colonization and persistence of H. pylori in the gastric mucus layer. It is not completely clear which chemical gradients are used by H. pylori to maintain its position. TlpA, a chemotaxis receptor for arginine/ bicarbonate, has been identified. This study aimed to find out whether tlpA gene expression is required for the chemotactic response to arginine/bicarbonate. Wild-type motile H. pylori ATCC 700392 and H. pylori ATCC 43504, a strain having an interrupted tlpA gene, were used. Also, a tlpA-knockout mutant of H. pylori 700392 (H. pylori 700-tlpA::cat) was produced by homologous recombination. Expression of tlpA was assessed by a Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay. Chemotaxis was measured as a Relative Chemotaxis Response (RCR) by a modified capillary assay. H. pylori 700392 presented chemotaxis to arginine and sodium bicarbonate. H. pylori 700-tlpA::cat showed neither tlpA gene expression nor chemotaxis towards arginine and bicarbonate. Besides confirming that TlpA is a chemotactic receptor for arginine/bicarbonate in H. pylori, this study showed that tlpA gene expression is required for arginine/bicarbonate chemotaxis.

  7. tlpA gene expression is required for arginine and bicarbonate chemotaxis in Helicobacter pylori

    Directory of Open Access Journals (Sweden)

    Oscar A Cerda

    2011-01-01

    Full Text Available About half of the human population is infected with Helicobacter pylori, a bacterium causing gastritis, peptic ulcer and progression to gastric cancer. Chemotaxis and flagellar motility are required for colonization and persistence of H. pylori in the gastric mucus layer. It is not completely clear which chemical gradients are used by H. pylori to maintain its position. TlpA, a chemotaxis receptor for arginine/ bicarbonate, has been identified. This study aimed to find out whether tlpA gene expression is required for the chemotactic response to arginine/bicarbonate. Wild-type motile H. pylori ATCC 700392 and H. pylori ATCC 43504, a strain having an interrupted tlpA gene, were used. Also, a tlpA-knockout mutant of H. pylori 700392 (H. pylori 700-tlpA::cat was produced by homologous recombination. Expression of tlpA was assessed by a Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR assay. Chemotaxis was measured as a Relative Chemotaxis Response (RCR by a modified capillary assay. H. pylori 700392 presented chemotaxis to arginine and sodium bicarbonate. H. pylori 700-tlpA::cat showed neither tlpA gene expression nor chemotaxis towards arginine and bicarbonate. Besides confirming that TlpA is a chemotactic receptor for arginine/bicarbonate in H. pylori, this study showed that tlpA gene expression is required for arginine/bicarbonate chemotaxis.

  8. The Arabidopsis histone chaperone FACT is required for stress-induced expression of anthocyanin biosynthetic genes.

    Science.gov (United States)

    Pfab, Alexander; Breindl, Matthias; Grasser, Klaus D

    2018-03-01

    The histone chaperone FACT is involved in the expression of genes encoding anthocyanin biosynthetic enzymes also upon induction by moderate high-light and therefore contributes to the stress-induced plant pigmentation. The histone chaperone FACT consists of the SSRP1 and SPT16 proteins and associates with transcribing RNAPII (RNAPII) along the transcribed region of genes. FACT can promote transcriptional elongation by destabilising nucleosomes in the path of RNA polymerase II, thereby facilitating efficient transcription of chromatin templates. Transcript profiling of Arabidopsis plants depleted in SSRP1 or SPT16 demonstrates that only a small subset of genes is differentially expressed relative to wild type. The majority of these genes is either up- or down-regulated in both the ssrp1 and spt16 plants. Among the down-regulated genes, those encoding enzymes of the biosynthetic pathway of the plant secondary metabolites termed anthocyanins (but not regulators of the pathway) are overrepresented. Upon exposure to moderate high-light stress several of these genes are up-regulated to a lesser extent in ssrp1/spt16 compared to wild type plants, and accordingly the mutant plants accumulate lower amounts of anthocyanin pigments. Moreover, the expression of SSRP1 and SPT16 is induced under these conditions. Therefore, our findings indicate that FACT is a novel factor required for the accumulation of anthocyanins in response to light-induction.

  9. Complete Deletion of the Fucose Operon in Haemophilus influenzae Is Associated with a Cluster in Multilocus Sequence Analysis-Based Phylogenetic Group II Related to Haemophilus haemolyticus: Implications for Identification and Typing.

    Science.gov (United States)

    de Gier, Camilla; Kirkham, Lea-Ann S; Nørskov-Lauritsen, Niels

    2015-12-01

    Nonhemolytic variants of Haemophilus haemolyticus are difficult to differentiate from Haemophilus influenzae despite a wide difference in pathogenic potential. A previous investigation characterized a challenging set of 60 clinical strains using multiple PCRs for marker genes and described strains that could not be unequivocally identified as either species. We have analyzed the same set of strains by multilocus sequence analysis (MLSA) and near-full-length 16S rRNA gene sequencing. MLSA unambiguously allocated all study strains to either of the two species, while identification by 16S rRNA sequence was inconclusive for three strains. Notably, the two methods yielded conflicting identifications for two strains. Most of the "fuzzy species" strains were identified as H. influenzae that had undergone complete deletion of the fucose operon. Such strains, which are untypeable by the H. influenzae multilocus sequence type (MLST) scheme, have sporadically been reported and predominantly belong to a single branch of H. influenzae MLSA phylogenetic group II. We also found evidence of interspecies recombination between H. influenzae and H. haemolyticus within the 16S rRNA genes. Establishing an accurate method for rapid and inexpensive identification of H. influenzae is important for disease surveillance and treatment. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. Molecular basis of antimicrobial resistance in non-typable Haemophilus influenzae.

    Science.gov (United States)

    Sánchez, L; Leranoz, S; Puig, M; Lorén, J G; Nikaido, H; Viñas, M

    1997-09-01

    Strains of the facultative anaerobe Haemophilus influenzae, both type b and non typable strains, are frequently multiresistant. The measurement of the antibiotic permeability of Haemophilus influenzae outer membrane (OM) shows that antibiotics can cross through the OM easily. Thus, enzymatic activity or efflux pumps could be responsible for multiresistance. An efflux system closely related to AcrAB of Escherichia coli is present in Haemophilus influenzae. However, their role in multiresistance seems irrelevant. Classical mechanisms such as plasmid exchange seems to be playing a major role in the multidrug resistance in Haemophilus influenzae.

  11. Comparative transcriptional profiling of tildipirosin-resistant and sensitive Haemophilus parasuis.

    Science.gov (United States)

    Lei, Zhixin; Fu, Shulin; Yang, Bing; Liu, Qianying; Ahmed, Saeed; Xu, Lei; Xiong, Jincheng; Cao, Jiyue; Qiu, Yinsheng

    2017-08-08

    Numerous studies have been conducted to examine the molecular mechanism of Haemophilus parasuis resistance to antibiotic, but rarely to tildipirosin. In the current study, transcriptional profiling was applied to analyse the variation in gene expression of JS0135 and tildipirosin-resistant JS32. The growth curves showed that JS32 had a higher growth rate but fewer bacteria than JS0135. The cell membranes of JS32 and a resistant clinical isolate (HB32) were observed to be smoother than those of JS0135. From the comparative gene expression profile 349 up- and 113 downregulated genes were observed, covering 37 GO and 63 KEGG pathways which are involved in biological processes (11), cellular components (17), molecular function (9), cellular processes (1), environmental information processing (4), genetic information processing (9) and metabolism (49) affected in JS32. In addition, the relative overexpression of genes of the metabolism pathway (HAPS_RS09315, HAPS_RS09320), ribosomes (HAPS_RS07815) and ABC transporters (HAPS_RS10945) was detected, particularly the metabolism pathway, and verified with RT-qPCR. Collectively, the gene expression profile in connection with tildipirosin resistance factors revealed unique and highly resistant determinants of H. parasuis to macrolides that warrant further attention due to the significant threat of bacterial resistance.

  12. Epidemiology, clinical features, diagnosis and treatment of Haemophilus ducreyi - a disappearing pathogen?

    Science.gov (United States)

    Lewis, David A

    2014-06-01

    Chancroid, caused by Haemophilus ducreyi, has declined in importance as a sexually transmitted pathogen in most countries where it was previously endemic. The global prevalence of chancroid is unknown as most countries lack the required laboratory diagnostic capacity and surveillance systems to determine this. H. ducreyi has recently emerged as a cause of chronic skin ulceration in some South Pacific islands. Although no antimicrobial susceptibility data for H. ducreyi have been published for two decades, it is still assumed that the infection will respond successfully to treatment with recommended cephalosporin, macrolide or fluoroquinolone-based regimens. HIV-1-infected patients require careful follow-up due to reports of treatment failure with single dose regimens. Buboes may need additional treatment with either aspiration or excision and drainage.

  13. Identification and Characterization of msf, a Novel Virulence Factor in Haemophilus influenzae.

    Directory of Open Access Journals (Sweden)

    Jennifer M Kress-Bennett

    Full Text Available Haemophilus influenzae is an opportunistic pathogen. The emergence of virulent, non-typeable strains (NTHi emphasizes the importance of developing new interventional targets. We screened the NTHi supragenome for genes encoding surface-exposed proteins suggestive of immune evasion, identifying a large family containing Sel1-like repeats (SLRs. Clustering identified ten SLR-containing gene subfamilies, each with various numbers of SLRs per gene. Individual strains also had varying numbers of SLR-containing genes from one or more of the subfamilies. Statistical genetic analyses of gene possession among 210 NTHi strains typed as either disease or carriage found a significant association between possession of the SlrVA subfamily (which we have termed, macrophage survival factor, msf and the disease isolates. The PittII strain contains four chromosomally contiguous msf genes. Deleting all four of these genes (msfA1-4 (KO resulted in a highly significant decrease in phagocytosis and survival in macrophages; which was fully complemented by a single copy of the msfA1 gene. Using the chinchilla model of otitis media and invasive disease, the KO strain displayed a significant decrease in fitness compared to the WT in co-infections; and in single infections, the KO lost its ability to invade the brain. The singly complemented strain showed only a partial ability to compete with the WT suggesting gene dosage is important in vivo. The transcriptional profiles of the KO and WT in planktonic growth were compared using the NTHi supragenome array, which revealed highly significant changes in the expression of operons involved in virulence and anaerobiosis. These findings demonstrate that the msfA1-4 genes are virulence factors for phagocytosis, persistence, and trafficking to non-mucosal sites.

  14. Extracellular Matrix-Regulated Gene Expression RequiresCooperation of SWI/SNF and Transcription Factors

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ren; Spencer, Virginia A.; Bissell, Mina J.

    2006-05-25

    Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation (ChIP) assays and mammary-specific genes as models, we show here that extracellular matrix (ECM) molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the {beta}- and ?-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both {beta}- and ?-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. ChIP analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Coimmunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, C/EBP{beta}, and glucocorticoid receptor (GR). Thus, ECM- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors.

  15. Quorum signaling and sensing by nontypeable Haemophilus influenzae.

    Science.gov (United States)

    Swords, W Edward

    2012-01-01

    Quorum signals are diffusible factors produced by bacteria that coordinate communal responses. For nontypeable Haemophilus influenzae (NTHi), a series of recent papers indicate that production and sensing of quorum signals are determinants of biofilm formation/maturation and persistence in vivo. In this mini-review I will summarize the current knowledge about quorum signaling/sensing by this organism, and identify specific topics for additional study.

  16. Quorum signaling and sensing by nontypeable Haemophilus influenzae

    Directory of Open Access Journals (Sweden)

    W Edward Swords

    2012-07-01

    Full Text Available Quorum signals are diffusible factors produced by bacteria that coordinate communal responses. For Haemophilus influenzae, a series of recent papers indicate that production and sensing of quorum signals are determinants of biofilm formation/maturation and persistence in vivo. In this mini-review I will summarize the current knowledge about quorum signaling/sensing by H. influenzae, and identify specific topics for additional study.

  17. Meningitis y artritis por Haemophilus influenzae en un adulto

    Directory of Open Access Journals (Sweden)

    Javier Molina

    1988-02-01

    Full Text Available Tradicionalmente el Haemophilus influenzae ha sido considerado un germen causante de infecciones en niños; en adultos se lo ha relacionado con Infecciones respiratorias, pero en los últimos tiempos se han descrito en ellos infecciones severas cuando hay algunos factores predisponentes. Se describe un paciente drogadicto de 30 años con cuadro de meningitis y artritis y prueba de látex y cultivo de LCR positivos para HaemophiIus influenzae, quien recibió tratamiento con ampicilina, 2 gramos Intravenosos cada 4 horas y evolucionó a la mejoría sin secuelas. Se plantea la necesidad de tener en cuenta al Haemophilus influenzae como patógeno del adulto y más en aquellas personas con factores predisponentes.

    Haemophilus influenzae has traditionally been considered as an infectious agent that predominantly affects children; instead, in adults It has been Linked either to respiratory infections or to gevere infections occurring when predisposing factors are present. We describe a 30 year-old drug adict patient that presented with meningitis and arthritis; both latex test and cerebrospinal fluid culture were positive for Haemophilus influenzae. He was treated with ampicilin 2 gm, I. V. every four hours and improved without sequelae. This microorganism must be considered among those affecting adult patients specially when predisposing factors for infection are present.

  18. Epiglottitis with an abscess caused by Haemophilus parainfluenzae

    DEFF Research Database (Denmark)

    Juul, Marie Louise; Johansen, Helle Krogh; Homøe, Preben

    2014-01-01

    A healthy 23-year-old man was admitted under the diagnosis of acute epiglottitis. Flexible fiber laryngoscopic examination showed a swollen epiglottis with an abscess. Microbiologic swab showed Haemophilus parainfluenzae, non-haemolytic Streptococcus and non-haemolytic Streptococcus salivarius. O....... Only in 1984 a case of acute epiglottitis due to H. parainfluenzae has been described in the literature. Still, in this case we think that H. parainfluenzae was the most likely pathogen causing the abscess....

  19. To tilfaelde af invasive infektioner med Haemophilus influenzae type f

    DEFF Research Database (Denmark)

    Nielsen, Jette Dettmann; Lind, Jens Wentzel; Bruun, Britta

    2009-01-01

    Two cases of invasive Haemophilus influenzae type f infection are presented: a three-week-old boy with meningitis and a 62-year-old woman with arthritis and bacteremia. Since 1993 vaccination against H. influenzae type b (Hib) has been offered to Danish children. The result has been a remarkable...... decrease in invasive Hib disease. However, physicians need to be aware of the existence of non-type b invasive H. influenzae disease. Udgivelsesdato: 2009-Jan...

  20. To tilfaelde af invasive infektioner med Haemophilus influenzae type f

    DEFF Research Database (Denmark)

    Nielsen, Jette Dettmann; Lind, Jens; Bruun, Brita

    2009-01-01

    Two cases of invasive Haemophilus influenzae type f infection are presented: a three-week-old boy with meningitis and a 62-year-old woman with arthritis and bacteremia. Since 1993 vaccination against H. influenzae type b (Hib) has been offered to Danish children. The result has been a remarkable...... decrease in invasive Hib disease. However, physicians need to be aware of the existence of non-type b invasive H. influenzae disease. Udgivelsesdato: 2009-Jan-19...

  1. [Two cases of invasive Haemophilus influenzae type f infection

    DEFF Research Database (Denmark)

    Nielsen, J.D.; Lind, J.W.; Bruun, B.

    2009-01-01

    Two cases of invasive Haemophilus influenzae type f infection are presented: a three-week-old boy with meningitis and a 62-year-old woman with arthritis and bacteremia. Since 1993 vaccination against H. influenzae type b (Hib) has been offered to Danish children. The result has been a remarkable...... decrease in invasive Hib disease. However, physicians need to be aware of the existence of non-type b invasive H. influenzae disease Udgivelsesdato: 2009/1/19...

  2. Are common symbiosis genes required for endophytic rice-rhizobial interactions?

    Science.gov (United States)

    Chen, Caiyan; Zhu, Hongyan

    2013-09-01

    Legume plants are able to establish root nodule symbioses with nitrogen-fixing bacteria, called rhizobia. Recent studies revealed that the root nodule symbiosis has co-opted the signaling pathway that mediates the ancestral mycorrhizal symbiosis that occurs in most land plants. Despite being unable to induce nodulation, rhizobia have been shown to be able to infect and colonize the roots of non-legumes such as rice. One fascinating question is whether establishment of such associations requires the common symbiosis (Sym) genes that are essential for infection of plant cells by mycorrhizal fungi and rhizobia in legumes. Here, we demonstrated that the common Sym genes are not required for endophytic colonization of rice roots by nitrogen-fixing rhizobia.

  3. CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

    Science.gov (United States)

    Převorovský, Martin; Oravcová, Martina; Zach, Róbert; Jordáková, Anna; Bähler, Jürg; Půta, František; Folk, Petr

    2016-11-16

    For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.

  4. The characterization and geographical distribution of the genes responsible for vernalization requirement in Chinese bread wheat.

    Science.gov (United States)

    Sun, Qing-Ming; Zhou, Rong-Hua; Gao, Li-Feng; Zhao, Guang-Yao; Jia, Ji-Zeng

    2009-04-01

    The frequency and distribution of the major vernalization requirement genes and their effects on growth habits were studied. Of the 551 bread wheat genotypes tested, seven allelic combinations of the three Vrn-1 genes were found to be responsible for the spring habit, three for the facultative habit and one for the winter habit. The three Vrn-1 genes behaved additively with the dominant allele of Vrn-A1 exerting the strongest effect. The allele combinations of the facultative genotypes and the discovery of spring genotypes with "winter" allele of Vrn-1 implied the presence of as yet unidentified alleles/genes for vernalization response. The dominant alleles of the three Vrn-1 genes were found in all ten ecological regions where wheat is cultivated in China, with Vrn-D1 as the most common allele in nine and Vrn-A1 in one. The combination of vrn-A1vrn-B1Vrn-D1 was the predominant genotype in seven of the regions. Compared with landraces, improved varieties contain a higher proportion of the spring type. This was attributed by a higher frequency of the dominant Vrn-A1 and Vrn-B1 alleles in the latter. Correlations between Vrn-1 allelic constitutions and heading date, spike length, plant type as well as cold tolerance were established.

  5. Haemophilus ducreyi Seeks Alternative Carbon Sources and Adapts to Nutrient Stress and Anaerobiosis during Experimental Infection of Human Volunteers.

    Science.gov (United States)

    Gangaiah, Dharanesh; Zhang, Xinjun; Baker, Beth; Fortney, Kate R; Gao, Hongyu; Holley, Concerta L; Munson, Robert S; Liu, Yunlong; Spinola, Stanley M

    2016-05-01

    Haemophilus ducreyi causes the sexually transmitted disease chancroid in adults and cutaneous ulcers in children. In humans, H. ducreyi resides in an abscess and must adapt to a variety of stresses. Previous studies (D. Gangaiah, M. Labandeira-Rey, X. Zhang, K. R. Fortney, S. Ellinger, B. Zwickl, B. Baker, Y. Liu, D. M. Janowicz, B. P. Katz, C. A. Brautigam, R. S. Munson, Jr., E. J. Hansen, and S. M. Spinola, mBio 5:e01081-13, 2014, http://dx.doi.org/10.1128/mBio.01081-13) suggested that H. ducreyi encounters growth conditions in human lesions resembling those found in stationary phase. However, how H. ducreyi transcriptionally responds to stress during human infection is unknown. Here, we determined the H. ducreyi transcriptome in biopsy specimens of human lesions and compared it to the transcriptomes of bacteria grown to mid-log, transition, and stationary phases. Multidimensional scaling showed that the in vivo transcriptome is distinct from those of in vitro growth. Compared to the inoculum (mid-log-phase bacteria), H. ducreyi harvested from pustules differentially expressed ∼93 genes, of which 62 were upregulated. The upregulated genes encode homologs of proteins involved in nutrient transport, alternative carbon pathways (l-ascorbate utilization and metabolism), growth arrest response, heat shock response, DNA recombination, and anaerobiosis. H. ducreyi upregulated few genes (hgbA, flp-tad, and lspB-lspA2) encoding virulence determinants required for human infection. Most genes regulated by CpxRA, RpoE, Hfq, (p)ppGpp, and DksA, which control the expression of virulence determinants and adaptation to a variety of stresses, were not differentially expressed in vivo, suggesting that these systems are cycling on and off during infection. Taken together, these data suggest that the in vivo transcriptome is distinct from those of in vitro growth and that adaptation to nutrient stress and anaerobiosis is crucial for H. ducreyi survival in humans. Copyright © 2016

  6. Isolation of Haemophilus influenzae and Haemophilus parainfluenzae in urethral exudates from men with acute urethritis: a descriptive study of 52 cases.

    Science.gov (United States)

    Deza, Gustavo; Martin-Ezquerra, Gemma; Gómez, Julià; Villar-García, Judit; Supervia, August; Pujol, Ramon M

    2016-02-01

    To describe the clinical characteristics and therapeutic outcomes from male patients diagnosed of Haemophilus spp urethritis. A chart review of patients who presented to our hospital from January 2013 to December 2014 with symptoms of acute urethritis in which Haemophilus spp was isolated in their urethral samples was performed. Haemophilus spp was isolated in 52 out of 413 urethral samples (12.6%) received in our laboratory from patients with symptoms of acute urethritis during the study period. Seven cases corresponded to Haemophilus influenzae and 45 cases to Haemophilus parainfluenzae. The most common clinical presentation was mucopurulent urethral discharge (71%). Eight per cent were HIV-infected patients, and 60% were men who have sex with men. Haemophilus spp was isolated as a single pathogen in 6.8% (28 of 413) of cases. Seventeen per cent of Haemophilus spp were β-lactamase producers. All patients reported having practiced unprotected insertive oral sex the month before consultation, and five of them denied having had another sexual contact apart from this exposure. In all cases in which follow-up was available, empirical treatment achieved a complete clinical resolution. Haemophilus spp was considered a pathogen in at least 6.8% of the patients from the evaluated area. It affected men regardless their sexual orientation or HIV status. Unprotected oral sex could play a role in its transmission. The limitations of the study (small sample size and lack of a representative control group) do not allow to prove the true pathogenic role of Haemophilus spp in acute urethritis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  7. The Immediate Early Gene Egr3 Is Required for Hippocampal Induction of Bdnf by Electroconvulsive Stimulation

    Directory of Open Access Journals (Sweden)

    Kimberly T. Meyers

    2018-05-01

    Full Text Available Early growth response 3 (Egr3 is an immediate early gene (IEG that is regulated downstream of a cascade of genes associated with risk for psychiatric disorders, and dysfunction of Egr3 itself has been implicated in schizophrenia, bipolar disorder, and depression. As an activity-dependent transcription factor, EGR3 is poised to regulate the neuronal expression of target genes in response to environmental events. In the current study, we sought to identify a downstream target of EGR3 with the goal of further elucidating genes in this biological pathway relevant for psychiatric illness risk. We used electroconvulsive stimulation (ECS to induce high-level expression of IEGs in the brain, and conducted expression microarray to identify genes differentially regulated in the hippocampus of Egr3-deficient (-/- mice compared to their wildtype (WT littermates. Our results replicated previous work showing that ECS induces high-level expression of the brain-derived neurotrophic factor (Bdnf in the hippocampus of WT mice. However, we found that this induction is absent in Egr3-/- mice. Quantitative real-time PCR (qRT-PCR validated the microarray results (performed in males and replicated the findings in two separate cohorts of female mice. Follow-up studies of activity-dependent Bdnf exons demonstrated that ECS-induced expression of both exons IV and VI requires Egr3. In situ hybridization demonstrated high-level cellular expression of Bdnf in the hippocampal dentate gyrus following ECS in WT, but not Egr3-/-, mice. Bdnf promoter analysis revealed eight putative EGR3 binding sites in the Bdnf promoter, suggesting a mechanism through which EGR3 may directly regulate Bdnf gene expression. These findings do not appear to result from a defect in the development of hippocampal neurons in Egr3-/- mice, as cell counts in tissue sections stained with anti-NeuN antibodies, a neuron-specific marker, did not differ between Egr3-/- and WT mice. In addition, Sholl

  8. Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction.

    Science.gov (United States)

    Abdeldaim, Guma M K; Strålin, Kristoffer; Kirsebom, Leif A; Olcén, Per; Blomberg, Jonas; Herrmann, Björn

    2009-08-01

    A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 10(4) DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

  9. Epidemiology of Haemophilus influenzae bacteremia: A multi-national population-based assessment

    DEFF Research Database (Denmark)

    Laupland, Kevin B; Schønheyder, Henrik C; Østergaard, Christian

    2011-01-01

    OBJECTIVES: Haemophilus influenzae is an important cause of invasive infection but contemporary data in non-selected populations is limited. METHODS: Population-based surveillance for Haemophilus influenzae bacteremia was conducted in seven regions in Australia, Canada, and Denmark during 2000-20...

  10. Geminin is required for zygotic gene expression at the Xenopus mid-blastula transition.

    Directory of Open Access Journals (Sweden)

    Sarah L Kerns

    Full Text Available In many organisms early development is under control of the maternal genome and zygotic gene expression is delayed until the mid-blastula transition (MBT. As zygotic transcription initiates, cell cycle checkpoints become activated and the tempo of cell division slows. The mechanisms that activate zygotic transcription at the MBT are incompletely understood, but they are of interest because they may resemble mechanisms that cause stem cells to stop dividing and terminally differentiate. The unstable regulatory protein Geminin is thought to coordinate cell division with cell differentiation. Geminin is a bi-functional protein. It prevents a second round of DNA replication during S and G2 phase by binding and inhibiting the essential replication factor Cdt1. Geminin also binds and inhibits a number of transcription factors and chromatin remodeling proteins and is thought to keep dividing cells in an undifferentiated state. We previously found that the cells of Geminin-deficient Xenopus embryos arrest in G2 phase just after the MBT then disintegrate at the onset of gastrulation. Here we report that they also fail to express most zygotic genes. The gene expression defect is cell-autonomous and is reproduced by over-expressing Cdt1 or by incubating the embryos in hydroxyurea. Geminin deficient and hydroxyurea-treated blastomeres accumulate DNA damage in the form of double stranded breaks. Bypassing the Chk1 pathway overcomes the cell cycle arrest caused by Geminin depletion but does not restore zygotic gene expression. In fact, bypassing the Chk1 pathway by itself induces double stranded breaks and abolishes zygotic transcription. We did not find evidence that Geminin has a replication-independent effect on transcription. We conclude that Geminin is required to maintain genome integrity during the rapid cleavage divisions, and that DNA damage disrupts zygotic gene transcription at the MBT, probably through activation of DNA damage checkpoint pathways.

  11. Deletion of HAPS_2096 Increases Sensitivity to Cecropin B in Haemophilus parasuis.

    Science.gov (United States)

    Chen, Fanjie; Hu, Han; Li, Zhonghua; Huang, Jiacheng; Cai, Xuwang; Wang, Chunmei; He, Qigai; Cao, Jiyue

    2015-01-01

    Cecropin B (CB) is a very effective natural antimicrobial peptide that has shown great potential for future antimicrobial drug development. HAPS_2096 is a Haemophilus parasuis gene that encodes the periplasmic substrate-binding protein of an ATP-binding cassette-type amino acid transporter. In this research, we constructed and verified an HAPS_2096 deletion mutant and a complementary HAPS_2096 mutant of H. parasuis JS0135. A bactericidal assay revealed that the HAPS_2096 deletion mutant was significantly more sensitive than the wild-type strain to 0.25-0.5 µg/ml CB. However, the gene complementation alleviated the CB sensitivity of the mutant. Immunoelectron microscopy observation following a 30-min treatment with a sublethal concentration of CB (0.25 μg/ml) revealed more extensive morphological damage in the mutant strain than in the wild-type strain. Hence, our results suggest that the HAPS_2096 gene contributes to H. parasuis resistance to CB. © 2015 S. Karger AG, Basel.

  12. Msx genes define a population of mural cell precursors required for head blood vessel maturation.

    Science.gov (United States)

    Lopes, Miguel; Goupille, Olivier; Saint Cloment, Cécile; Lallemand, Yvan; Cumano, Ana; Robert, Benoît

    2011-07-01

    Vessels are primarily formed from an inner endothelial layer that is secondarily covered by mural cells, namely vascular smooth muscle cells (VSMCs) in arteries and veins and pericytes in capillaries and veinules. We previously showed that, in the mouse embryo, Msx1(lacZ) and Msx2(lacZ) are expressed in mural cells and in a few endothelial cells. To unravel the role of Msx genes in vascular development, we have inactivated the two Msx genes specifically in mural cells by combining the Msx1(lacZ), Msx2(lox) and Sm22α-Cre alleles. Optical projection tomography demonstrated abnormal branching of the cephalic vessels in E11.5 mutant embryos. The carotid and vertebral arteries showed an increase in caliber that was related to reduced vascular smooth muscle coverage. Taking advantage of a newly constructed Msx1(CreERT2) allele, we demonstrated by lineage tracing that the primary defect lies in a population of VSMC precursors. The abnormal phenotype that ensues is a consequence of impaired BMP signaling in the VSMC precursors that leads to downregulation of the metalloprotease 2 (Mmp2) and Mmp9 genes, which are essential for cell migration and integration into the mural layer. Improper coverage by VSMCs secondarily leads to incomplete maturation of the endothelial layer. Our results demonstrate that both Msx1 and Msx2 are required for the recruitment of a population of neural crest-derived VSMCs.

  13. Comprehensive identification of Salmonella enterica serovar typhimurium genes required for infection of BALB/c mice.

    Directory of Open Access Journals (Sweden)

    Roy R Chaudhuri

    2009-07-01

    Full Text Available Genes required for infection of mice by Salmonella Typhimurium can be identified by the interrogation of random transposon mutant libraries for mutants that cannot survive in vivo. Inactivation of such genes produces attenuated S. Typhimurium strains that have potential for use as live attenuated vaccines. A quantitative screen, Transposon Mediated Differential Hybridisation (TMDH, has been developed that identifies those members of a large library of transposon mutants that are attenuated. TMDH employs custom transposons with outward-facing T7 and SP6 promoters. Fluorescently-labelled transcripts from the promoters are hybridised to whole-genome tiling microarrays, to allow the position of the transposon insertions to be determined. Comparison of microarray data from the mutant library grown in vitro (input with equivalent data produced after passage of the library through mice (output enables an attenuation score to be determined for each transposon mutant. These scores are significantly correlated with bacterial counts obtained during infection of mice using mutants with individual defined deletions of the same genes. Defined deletion mutants of several novel targets identified in the TMDH screen are effective live vaccines.

  14. Polymicrobial infective endocarditis caused by Neisseria sicca and Haemophilus parainfluenzae

    Directory of Open Access Journals (Sweden)

    Nikoloz Koshkelashvili

    2016-01-01

    Full Text Available Infective endocarditis is a common clinical problem in industrialized countries. Risk factors include abnormal cardiac valves, a history of endocarditis, intracardiac devices, prosthetic valves and intravenous drug use. We report a case of polymicrobial infective endocarditis in a 33 year-old female with a history chronic heroin use caused by Neisseria sicca and Haemophilus parainfluenzae. We believe the patient was exposed to these microbes by cleansing her skin with saliva prior to injection. Pairing a detailed history with the consideration of atypical agents is crucial in the proper diagnosis and management of endocarditis in patients with high-risk injection behaviors.

  15. Functional characterization of human COQ4, a gene required for Coenzyme Q10 biosynthesis

    International Nuclear Information System (INIS)

    Casarin, Alberto; Jimenez-Ortega, Jose Carlos; Trevisson, Eva; Pertegato, Vanessa; Doimo, Mara; Ferrero-Gomez, Maria Lara; Abbadi, Sara; Artuch, Rafael; Quinzii, Catarina; Hirano, Michio; Basso, Giuseppe; Ocana, Carlos Santos; Navas, Placido; Salviati, Leonardo

    2008-01-01

    Defects in genes involved in coenzyme Q (CoQ) biosynthesis cause primary CoQ deficiency, a severe multisystem disorders presenting as progressive encephalomyopathy and nephropathy. The COQ4 gene encodes an essential factor for biosynthesis in Saccharomyces cerevisiae. We have identified and cloned its human ortholog, COQ4, which is located on chromosome 9q34.13, and is transcribed into a 795 base-pair open reading frame, encoding a 265 amino acid (aa) protein (Isoform 1) with a predicted N-terminal mitochondrial targeting sequence. It shares 39% identity and 55% similarity with the yeast protein. Coq4 protein has no known enzymatic function, but may be a core component of multisubunit complex required for CoQ biosynthesis. The human transcript is detected in Northern blots as a ∼1.4 kb single band and is expressed ubiquitously, but at high levels in liver, lung, and pancreas. Transcription initiates at multiple sites, located 333-23 nucleotides upstream of the ATG. A second group of transcripts originating inside intron 1 of the gene encodes a 241 aa protein, which lacks the mitochondrial targeting sequence (isoform 2). Expression of GFP-fusion proteins in HeLa cells confirmed that only isoform 1 is targeted to mitochondria. The functional significance of the second isoform is unknown. Human COQ4 isoform 1, expressed from a multicopy plasmid, efficiently restores both growth in glycerol, and CoQ content in COQ4 null yeast strains. Human COQ4 is an interesting candidate gene for patients with isolated CoQ 10 deficiency

  16. Microarray Analysis of Transposon Insertion Mutants in Bacillus Anthracis: Global Identification of Genes Required for Sporulation and Germination

    National Research Council Canada - National Science Library

    Day , Jr., William A; Rasmussen, Suzanne L; Carpenter, Beth M; Peterson, Scott N; Friedlander, Arthur M

    2007-01-01

    .... The system, used to identify genes required for generation of the infectious anthrax spore, spore germination and optimal growth on rich medium, was predictive of the contribution of two conserved...

  17. Haemophilus paragallinarum in chickens in Indonesia: III. Antimicrobial drug sensitivity test ofHaemophilus paragallinarum from chickens suffering of coryza

    Directory of Open Access Journals (Sweden)

    Sri Poernomo

    1998-12-01

    Full Text Available An agar disc diffusion method was used to examine the sensitivity of 27 Haemophilus paragallinarum (Hpg isolates consisted of 23 local isolates, 4 standard isolates (serotype A and Escherichia coli ATCC 24922 as a control to eight antimicrobial drugs (ampicillin, erythromycin, oxytetracycline, doxycycline, neomycin, streptomycin, colistine and sulphanlethoxazole-trimethoprim . Twenty one out of 23 local isolates of Hpg were sensitive to doxycycline, 19 isolates to ampsllin, 18 isolates to oxytetracycline, 17 isolates to sulphametoxazole-trimethoprim, 16 isolates to erythromycin, and 13 isolates to neomycin, while 13 isolates were resistance to colistine and 11 isolates were also resistance to streptomycin .

  18. Mms Sensitivity of All Amino Acid-Requiring Mutants in Aspergillus and Its Suppression by Mutations in a Single Gene

    OpenAIRE

    Käfer, Etta

    1987-01-01

    All available amino acid-requiring mutants of Aspergillus nidulans were found to be hypersensitive to MMS (methyl methanesulfonate) to various degrees. On MMS media, secondary mutations could be selected which suppress this MMS sensitivity but do not affect the requirement. Many such mutations were analyzed and found to be alleles of one gene, smsA (= suppressor of MMS sensitivity), which mapped distal on the right arm of chromosome V. This gene is more likely to be involved in general regula...

  19. Development and validation of an Haemophilus influenzae supragenome hybridization (SGH array for transcriptomic analyses.

    Directory of Open Access Journals (Sweden)

    Benjamin A Janto

    Full Text Available We previously carried out the design and testing of a custom-built Haemophilus influenzae supragenome hybridization (SGH array that contains probe sequences to 2,890 gene clusters identified by whole genome sequencing of 24 strains of H. influenzae. The array was originally designed as a tool to interrogate the gene content of large numbers of clinical isolates without the need for sequencing, however, the data obtained is quantitative and is thus suitable for transcriptomic analyses. In the current study RNA was extracted from H. influenzae strain CZ4126/02 (which was not included in the design of the array converted to cDNA, and labelled and hybridized to the SGH arrays to assess the quality and reproducibility of data obtained from these custom-designed chips to serve as a tool for transcriptomics. Three types of experimental replicates were analyzed with all showing very high degrees of correlation, thus validating both the array and the methods used for RNA profiling. A custom filtering pipeline for two-condition unpaired data using five metrics was developed to minimize variability within replicates and to maximize the identification of the most significant true transcriptional differences between two samples. These methods can be extended to transcriptional analysis of other bacterial species utilizing supragenome-based arrays.

  20. Haemophilus influenzae genome evolution during persistence in the human airways in chronic obstructive pulmonary disease.

    Science.gov (United States)

    Pettigrew, Melinda M; Ahearn, Christian P; Gent, Janneane F; Kong, Yong; Gallo, Mary C; Munro, James B; D'Mello, Adonis; Sethi, Sanjay; Tettelin, Hervé; Murphy, Timothy F

    2018-04-03

    Nontypeable Haemophilus influenzae (NTHi) exclusively colonize and infect humans and are critical to the pathogenesis of chronic obstructive pulmonary disease (COPD). In vitro and animal models do not accurately capture the complex environments encountered by NTHi during human infection. We conducted whole-genome sequencing of 269 longitudinally collected cleared and persistent NTHi from a 15-y prospective study of adults with COPD. Genome sequences were used to elucidate the phylogeny of NTHi isolates, identify genomic changes that occur with persistence in the human airways, and evaluate the effect of selective pressure on 12 candidate vaccine antigens. Strains persisted in individuals with COPD for as long as 1,422 d. Slipped-strand mispairing, mediated by changes in simple sequence repeats in multiple genes during persistence, regulates expression of critical virulence functions, including adherence, nutrient uptake, and modification of surface molecules, and is a major mechanism for survival in the hostile environment of the human airways. A subset of strains underwent a large 400-kb inversion during persistence. NTHi does not undergo significant gene gain or loss during persistence, in contrast to other persistent respiratory tract pathogens. Amino acid sequence changes occurred in 8 of 12 candidate vaccine antigens during persistence, an observation with important implications for vaccine development. These results indicate that NTHi alters its genome during persistence by regulation of critical virulence functions primarily by slipped-strand mispairing, advancing our understanding of how a bacterial pathogen that plays a critical role in COPD adapts to survival in the human respiratory tract.

  1. Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl

    Science.gov (United States)

    Zhu, Wei; Pao, Gerald M; Satoh, Akira; Cummings, Gillian; Monaghan, James R; Harkins, Timothy T; Bryant, Susan V; Voss, S Randal; Gardiner, David M; Hunter, Tony

    2013-01-01

    The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration – wound healing, cellular dedifferentiation and blastemal formation – will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells to a germline-like state. Two genes that are required for self-renewal of germ cells in mice and flies, Piwi-like 1 (PL1) and Piwi-like 2 (PL2), are expressed in limb blastemal cells, the basal layer keratinocytes and the thickened apical epithelial cap in the wound epidermis in the regenerating limb. Depletion of PL1 and PL2 by morpholino oligonucleotides decreased cell proliferation and increased cell death in the blastema leading to a significant retardation of regeneration. Examination of key molecules that are known to be required for limb development or regeneration further revealed that FGF8 is transcriptionally downregulated in the presence of the morpholino oligos, indicating PL1 and PL2 might participate in FGF signaling during limb regeneration. Given the requirement for FGF signaling in limb development and regeneration, the results suggest that PL1 and PL2 function to establish a unique germline-like state that is associated with successful regeneration. PMID:22841627

  2. Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways

    Science.gov (United States)

    Viadas, Cristina; Ruiz de los Mozos, Igor; Valle, Jaione; Bengoechea, José Antonio; Garmendia, Junkal

    2015-01-01

    Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence. PMID:25894755

  3. Efficient transcription of the glycolytic gene ADH1 and three translational component genes requires the GCR1 product, which can act through TUF/GRF/RAP binding sites.

    OpenAIRE

    Santangelo, G M; Tornow, J

    1990-01-01

    Glycolytic gene expression in Saccharomyces cerevisiae is thought to be activated by the GCR and TUF proteins. We tested the hypothesis that GCR function is mediated by TUF/GRF/RAP binding sites (UASRPG elements). We found that UASRPG-dependent activation of a heterologous gene and transcription of ADH1, TEF1, TEF2, and RP59 were sensitive to GCR1 disruption. GCR is not required for TUF/GRF/RAP expression or in vitro DNA-binding activity.

  4. PRMT1 mediated methylation of TAF15 is required for its positive gene regulatory function

    Energy Technology Data Exchange (ETDEWEB)

    Jobert, Laure; Argentini, Manuela [Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), CNRS UMR 7104, INSERM U 596, Universite Louis Pasteur de Strasbourg, BP 10142 - 67404 Illkirch Cedex, CU de Strasbourg (France); Tora, Laszlo, E-mail: laszlo@igbmc.u-strasbg.fr [Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), CNRS UMR 7104, INSERM U 596, Universite Louis Pasteur de Strasbourg, BP 10142 - 67404 Illkirch Cedex, CU de Strasbourg (France)

    2009-04-15

    TAF15 (formerly TAF{sub II}68) is a nuclear RNA-binding protein that is associated with a distinct population of TFIID and RNA polymerase II complexes. TAF15 harbours an N-terminal activation domain, an RNA recognition motif (RRM) and many Arg-Gly-Gly (RGG) repeats at its C-terminal end. The N-terminus of TAF15 serves as an essential transforming domain in the fusion oncoprotein created by chromosomal translocation in certain human chondrosarcomas. Post-transcriptional modifications (PTMs) of proteins are known to regulate their activity, however, nothing is known on how PTMs affect TAF15 function. Here we demonstrate that endogenous human TAF15 is methylated in vivo at its numerous RGG repeats. Furthermore, we identify protein arginine N-methyltransferase 1 (PRMT1) as a TAF15 interactor and the major PRMT responsible for its methylation. In addition, the RGG repeat-containing C-terminus of TAF15 is responsible for the shuttling between the nucleus and the cytoplasm and the methylation of RGG repeats affects the subcellular localization of TAF15. The methylation of TAF15 by PRMT1 is required for the ability of TAF15 to positively regulate the expression of the studied endogenous TAF15-target genes. Our findings demonstrate that arginine methylation of TAF15 by PRMT1 is a crucial event determining its proper localization and gene regulatory function.

  5. Transportin-SR is required for proper splicing of resistance genes and plant immunity.

    Directory of Open Access Journals (Sweden)

    Shaohua Xu

    2011-06-01

    Full Text Available Transportin-SR (TRN-SR is a member of the importin-β super-family that functions as the nuclear import receptor for serine-arginine rich (SR proteins, which play diverse roles in RNA metabolism. Here we report the identification and cloning of mos14 (modifier of snc1-1, 14, a mutation that suppresses the immune responses conditioned by the auto-activated Resistance (R protein snc1 (suppressor of npr1-1, constitutive 1. MOS14 encodes a nuclear protein with high similarity to previously characterized TRN-SR proteins in animals. Yeast two-hybrid assays showed that MOS14 interacts with AtRAN1 via its N-terminus and SR proteins via its C-terminus. In mos14-1, localization of several SR proteins to the nucleus was impaired, confirming that MOS14 functions as a TRN-SR. The mos14-1 mutation results in altered splicing patterns of SNC1 and another R gene RPS4 and compromised resistance mediated by snc1 and RPS4, suggesting that nuclear import of SR proteins by MOS14 is required for proper splicing of these two R genes and is important for their functions in plant immunity.

  6. Herbaspirillum seropedicae rfbB and rfbC genes are required for maize colonization.

    Science.gov (United States)

    Balsanelli, Eduardo; Serrato, Rodrigo V; de Baura, Valter A; Sassaki, Guilherme; Yates, Marshall G; Rigo, Liu Un; Pedrosa, Fábio O; de Souza, Emanuel M; Monteiro, Rose A

    2010-08-01

    In this study we disrupted two Herbaspirillum seropedicae genes, rfbB and rfbC, responsible for rhamnose biosynthesis and its incoporation into LPS. GC-MS analysis of the H. seropedicae wild-type strain LPS oligosaccharide chain showed that rhamnose, glucose and N-acetyl glucosamine are the predominant monosaccharides, whereas rhamnose and N-acetyl glucosamine were not found in the rfbB and rfbC strains. The electrophoretic pattern of the mutants LPS was drastically altered when compared with the wild type. Knockout of rfbB or rfbC increased the sensitivity towards SDS, polymyxin B sulfate and salicylic acid. The mutants attachment capacity to maize root surface plantlets was 100-fold lower than the wild type. Interestingly, the wild-type capacity to attach to maize roots was reduced to a level similar to that of the mutants when the assay was performed in the presence of isolated wild-type LPS, glucosamine or N-acetyl glucosamine. The mutant strains were also significantly less efficient in endophytic colonization of maize. Expression analysis indicated that the rfbB gene is upregulated by naringenin, apigenin and CaCl(2). Together, the results suggest that intact LPS is required for H. seropedicae attachment to maize root and internal colonization of plant tissues. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

  7. PRMT1 mediated methylation of TAF15 is required for its positive gene regulatory function

    International Nuclear Information System (INIS)

    Jobert, Laure; Argentini, Manuela; Tora, Laszlo

    2009-01-01

    TAF15 (formerly TAF II 68) is a nuclear RNA-binding protein that is associated with a distinct population of TFIID and RNA polymerase II complexes. TAF15 harbours an N-terminal activation domain, an RNA recognition motif (RRM) and many Arg-Gly-Gly (RGG) repeats at its C-terminal end. The N-terminus of TAF15 serves as an essential transforming domain in the fusion oncoprotein created by chromosomal translocation in certain human chondrosarcomas. Post-transcriptional modifications (PTMs) of proteins are known to regulate their activity, however, nothing is known on how PTMs affect TAF15 function. Here we demonstrate that endogenous human TAF15 is methylated in vivo at its numerous RGG repeats. Furthermore, we identify protein arginine N-methyltransferase 1 (PRMT1) as a TAF15 interactor and the major PRMT responsible for its methylation. In addition, the RGG repeat-containing C-terminus of TAF15 is responsible for the shuttling between the nucleus and the cytoplasm and the methylation of RGG repeats affects the subcellular localization of TAF15. The methylation of TAF15 by PRMT1 is required for the ability of TAF15 to positively regulate the expression of the studied endogenous TAF15-target genes. Our findings demonstrate that arginine methylation of TAF15 by PRMT1 is a crucial event determining its proper localization and gene regulatory function.

  8. Invasive Haemophilus Influenzae Disease, Europe, 1996–2006

    Centers for Disease Control (CDC) Podcasts

    This podcast describes monitoring of Haemophilus influenzae disease in Europe from 1996 through 2006. CDC epidemiologist Stacey Martin discusses what researchers learned about the effect of vaccination on disease prevalence.

  9. A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression

    Directory of Open Access Journals (Sweden)

    Amsterdam Adam

    2006-06-01

    Full Text Available Abstract Background Craniofacial birth defects result from defects in cranial neural crest (NC patterning and morphogenesis. The vertebrate craniofacial skeleton is derived from cranial NC cells and the patterning of these cells occurs within the pharyngeal arches. Substantial efforts have led to the identification of several genes required for craniofacial skeletal development such as the endothelin-1 (edn1 signaling pathway that is required for lower jaw formation. However, many essential genes required for craniofacial development remain to be identified. Results Through screening a collection of insertional zebrafish mutants containing approximately 25% of the genes essential for embryonic development, we present the identification of 15 essential genes that are required for craniofacial development. We identified 3 genes required for hyomandibular development. We also identified zebrafish models for Campomelic Dysplasia and Ehlers-Danlos syndrome. To further demonstrate the utility of this method, we include a characterization of the wdr68 gene. We show that wdr68 acts upstream of the edn1 pathway and is also required for formation of the upper jaw equivalent, the palatoquadrate. We also present evidence that the level of wdr68 activity required for edn1 pathway function differs between the 1st and 2nd arches. Wdr68 interacts with two minibrain-related kinases, Dyrk1a and Dyrk1b, required for embryonic growth and myotube differentiation, respectively. We show that a GFP-Wdr68 fusion protein localizes to the nucleus with Dyrk1a in contrast to an engineered loss of function mutation Wdr68-T284F that no longer accumulated in the cell nucleus and failed to rescue wdr68 mutant animals. Wdr68 homologs appear to exist in all eukaryotic genomes. Notably, we found that the Drosophila wdr68 homolog CG14614 could substitute for the vertebrate wdr68 gene even though insects lack the NC cell lineage. Conclusion This work represents a systematic

  10. The PCP genes Celsr1 and Vangl2 are required for normal lung branching morphogenesis

    Science.gov (United States)

    Yates, Laura L.; Schnatwinkel, Carsten; Murdoch, Jennifer N.; Bogani, Debora; Formstone, Caroline J.; Townsend, Stuart; Greenfield, Andy; Niswander, Lee A.; Dean, Charlotte H.

    2010-01-01

    The lungs are generated by branching morphogenesis as a result of reciprocal signalling interactions between the epithelium and mesenchyme during development. Mutations that disrupt formation of either the correct number or shape of epithelial branches affect lung function. This, in turn, can lead to congenital abnormalities such as cystadenomatoid malformations, pulmonary hypertension or lung hypoplasia. Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis. Identifying the signalling pathways which drive epithelial tube formation will likely shed light on both congenital and adult lung disease. Here we show that mutations in the planar cell polarity (PCP) genes Celsr1 and Vangl2 lead to disrupted lung development and defects in lung architecture. Lungs from Celsr1Crsh and Vangl2Lp mouse mutants are small and misshapen with fewer branches, and by late gestation exhibit thickened interstitial mesenchyme and defective saccular formation. We observe a recapitulation of these branching defects following inhibition of Rho kinase, an important downstream effector of the PCP signalling pathway. Moreover, epithelial integrity is disrupted, cytoskeletal remodelling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10. We further show that Celsr1 and Vangl2 proteins are present in restricted spatial domains within lung epithelium. Our data show that the PCP genes Celsr1 and Vangl2 are required for foetal lung development thereby revealing a novel signalling pathway critical for this process that will enhance our understanding of congenital and adult lung diseases and may in future lead to novel therapeutic strategies. PMID:20223754

  11. Nontypeable Haemophilus influenzae biofilms: role in chronic airway infections

    Directory of Open Access Journals (Sweden)

    W Edward Swords

    2012-07-01

    Full Text Available Like many pathogens inhabiting mucosal surfaces, nontypeable Haemophilus influenzae (NTHi forms multicellular biofilm communities both in vitro and in various infection models. In the past 15 years much has been learned about determinants of biofilm formation by this organism and potential roles in bacterial virulence, especially in the context of chronic and recurrent infections. However, this concept has not been without some degree of controversy, and in the past some have expressed doubts about the relevance of NTHi biofilms to disease. In this review, I will summarize the present information on the composition and potential role(s of NTHi biofilms in different clinical contexts, as well as highlight potential areas for future work.

  12. Nontypeable Haemophilus influenzae biofilms: role in chronic airway infections.

    Science.gov (United States)

    Swords, W Edward

    2012-01-01

    Like many pathogens inhabiting mucosal surfaces, nontypeable Haemophilus influenzae (NTHi) forms multicellular biofilm communities both in vitro and in various infection models. In the past 15 years much has been learned about determinants of biofilm formation by this organism and potential roles in bacterial virulence, especially in the context of chronic and recurrent infections. However, this concept has not been without some degree of controversy, and in the past some have expressed doubts about the relevance of NTHi biofilms to disease. In this review, I will summarize the present information on the composition and potential role(s) of NTHi biofilms in different clinical contexts, as well as highlight potential areas for future work.

  13. Mutation induction in Haemophilus influenzae by ICR-191. Pt. 1

    International Nuclear Information System (INIS)

    Perdue, S.W.; Kimball, R.F.; McGray, P.C.; Tennessee Univ., Oak Ridge

    1981-01-01

    The investigation of mutagenic mechanisms in Haemophilus influenzae has been confined until now to mutagens that normally produce mainly base pair substitutions. This paper describes the development of a system suitable for detecting frameshift mutations induced by ICR-191. The system involves reversions from thymidine dependence to thymidine independence. Evidence is presented from a comparison of the responses to ICR-191 and to N-methyl-N'-nitro-N-nitrosoguanidine that the system is specific for frameshift mutations. The genetic recombination involved in transformation leads to a marked increase in spontaneous reversion of the frameshift mutations but not of the base substitution mutations. Presumably, this is a consequence of mispairing, with consequent change in the number of bases, during the recombination. (orig.)

  14. A comparative study of preservation and storage of Haemophilus influenzae

    Directory of Open Access Journals (Sweden)

    Olga C Aulet de Saab

    2001-05-01

    Full Text Available The aim of this study was to compare the efficacy of conservation by freezing the strains of Haemophilus influenzae at -20ºC and -70ºC. Skim milk supplemented with glucose, yeast extract and glycerol allowed highest viability of H. influenzae both at -20ºC and -70ºC from the media analyzed. Trypticase soy broth and brain heart infusion broth supplemented with glycerol, allowed excellent recovery. Use of cotton swaps as supporting material, with or without addition of cryoprotective agents, did not modify H. influenzae viability after six months of storage. Concentration of the initial inoculum positively affected viability when stored at -20ºC. Initial concentration did not influence survival after storage at -70ºC. Thawing at room temperature should not exceed 3 h as to get highest survival percentage.

  15. Use of signature-tagged mutagenesis to identify virulence determinants in Haemophilus ducreyi responsible for ulcer formation.

    Science.gov (United States)

    Yeung, Angela; Cameron, D William; Desjardins, Marc; Lee, B Craig

    2011-02-01

    Elucidating the molecular mechanisms responsible for chancroid, a genital ulcer disease caused by Haemophilus ducreyi, has been hampered in part by the relative genetic intractability of the organism. A whole genome screen using signature-tagged mutagenesis in the temperature-dependent rabbit model (TDRM) of H. ducreyi infection uncovered 26 mutants with a presumptive attenuated phenotype. Insertions in two previously recognized virulence determinants, hgbA and lspA1, validated this genome scanning technique. Database interrogation allowed assignment of 24 mutants to several functional classes, including transport, metabolism, DNA repair, stress response and gene regulation. The attenuated virulence for a 3 strain with a mutation in hicB was confirmed by individual infection in the TDRM. The results from this preliminary study indicate that this high throughput strategy will further the understanding of the pathogenesis of H. ducreyi infection. Copyright © 2010 Elsevier B.V. All rights reserved.

  16. Simplified PCR for detection of Haemophilus ducreyi and diagnosis of chancroid.

    Science.gov (United States)

    West, B; Wilson, S M; Changalucha, J; Patel, S; Mayaud, P; Ballard, R C; Mabey, D

    1995-01-01

    A simplified PCR was developed for detection of Haemophilus ducreyi in samples from chancroid patients. The strategy included a straightforward chloroform extraction sample preparation method, a one-tube nested PCR to minimize contamination risks, and a colorimetric method for detection of products. Primers were designed from published nucleotide sequences of the 16S rRNA gene of H. ducreyi, with longer outer primers for annealing at a higher temperature and shorter inner primers labelled with biotin and digoxigenin for binding with avidin and colorimetric detection. The PCR technique detected all 35 strains of H. ducreyi tested, from four different geographical regions, and was negative for other, related strains of bacteria and for the common contaminating bacteria tested. Of 25 samples from H. ducreyi culture-positive chancroid patients, 24 were PCR positive and 1 produced a weak reaction. Of 83 samples from clinical cases of chancroid in the Republic of South Africa, 69 were PCR positive. The sensitivity of PCR compared with that of clinical diagnosis was 83%. All 50 negative control samples were negative. Encouraging results were also obtained with a consecutive series of 25 genital ulcer patients in Tanzania, of whom 9 were PCR positive. The adaptations of this simplified PCR strategy, at the sensitivity and specificity levels obtained, mean it will be useful for detection of H. ducreyi in areas where the organism is endemic, particularly where testing by culture is difficult or impossible. PMID:7540625

  17. Molecular phylogenetic analysis of non-sexually transmitted strains of Haemophilus ducreyi.

    Science.gov (United States)

    Gaston, Jordan R; Roberts, Sally A; Humphreys, Tricia L

    2015-01-01

    Haemophilus ducreyi, the etiologic agent of chancroid, has been previously reported to show genetic variance in several key virulence factors, placing strains of the bacterium into two genetically distinct classes. Recent studies done in yaws-endemic areas of the South Pacific have shown that H. ducreyi is also a major cause of cutaneous limb ulcers (CLU) that are not sexually transmitted. To genetically assess CLU strains relative to the previously described class I, class II phylogenetic hierarchy, we examined nucleotide sequence diversity at 11 H. ducreyi loci, including virulence and housekeeping genes, which encompass approximately 1% of the H. ducreyi genome. Sequences for all 11 loci indicated that strains collected from leg ulcers exhibit DNA sequences homologous to class I strains of H. ducreyi. However, sequences for 3 loci, including a hemoglobin receptor (hgbA), serum resistance protein (dsrA), and a collagen adhesin (ncaA) contained informative amounts of variation. Phylogenetic analyses suggest that these non-sexually transmitted strains of H. ducreyi comprise a sub-clonal population within class I strains of H. ducreyi. Molecular dating suggests that CLU strains are the most recently developed, having diverged approximately 0.355 million years ago, fourteen times more recently than the class I/class II divergence. The CLU strains' divergence falls after the divergence of humans from chimpanzees, making it the first known H. ducreyi divergence event directly influenced by the selective pressures accompanying human hosts.

  18. Molecular phylogenetic analysis of non-sexually transmitted strains of Haemophilus ducreyi.

    Directory of Open Access Journals (Sweden)

    Jordan R Gaston

    Full Text Available Haemophilus ducreyi, the etiologic agent of chancroid, has been previously reported to show genetic variance in several key virulence factors, placing strains of the bacterium into two genetically distinct classes. Recent studies done in yaws-endemic areas of the South Pacific have shown that H. ducreyi is also a major cause of cutaneous limb ulcers (CLU that are not sexually transmitted. To genetically assess CLU strains relative to the previously described class I, class II phylogenetic hierarchy, we examined nucleotide sequence diversity at 11 H. ducreyi loci, including virulence and housekeeping genes, which encompass approximately 1% of the H. ducreyi genome. Sequences for all 11 loci indicated that strains collected from leg ulcers exhibit DNA sequences homologous to class I strains of H. ducreyi. However, sequences for 3 loci, including a hemoglobin receptor (hgbA, serum resistance protein (dsrA, and a collagen adhesin (ncaA contained informative amounts of variation. Phylogenetic analyses suggest that these non-sexually transmitted strains of H. ducreyi comprise a sub-clonal population within class I strains of H. ducreyi. Molecular dating suggests that CLU strains are the most recently developed, having diverged approximately 0.355 million years ago, fourteen times more recently than the class I/class II divergence. The CLU strains' divergence falls after the divergence of humans from chimpanzees, making it the first known H. ducreyi divergence event directly influenced by the selective pressures accompanying human hosts.

  19. DNA repair in Haemophilus influenzae: isolation and characterization of an ultraviolet sensitive mutator mutant

    International Nuclear Information System (INIS)

    Walter, R.B.

    1985-01-01

    DNA repair in Haemophilus influenzae appears to be quite different from that seen in Escherichia coli in that H. influenzae shows neither SOS nor adaptation phenomena. Repair of DNA lesions in H. influenzae has been seen to occur via recombinational, excision, and mismatch repair pathways acting independently of one another. The author has isolated an ultraviolet (UV)-sensitive mutator mutant (mutB1) of H. influenzae Rd which shows deficiencies in both recombinational and mismatch repair pathways. This mutant is sensitive to a variety of DNA damaging agents as well as being hypermutable by alkylating agents and base analogues. MutB1 cells do not show post-UV DNA breakdown but do begin excision after UV irradiation. Genetic transformation with UV-irradiated DNA on mut B1 recipients shows that high (HE) and low (LE) efficiency markers are transformed at a ratio of 1.0 as in the mismatch repair deficient hex 1 mutant; however, kinetics of UV-inactivation experiments indicate that HE markers are sensitized and act as LE markers do on wild type recipients. Thus, the mutB gene product appears to play a role in both DNA repair and genetic transformation. A model is outlined which presents a role for a DNA helicase in both DNA repair and genetic transformation of H. influenzae

  20. Evolution of New cis-Regulatory Motifs Required for Cell-Specific Gene Expression in Caenorhabditis.

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    Michalis Barkoulas

    2016-09-01

    Full Text Available Patterning of C. elegans vulval cell fates relies on inductive signaling. In this induction event, a single cell, the gonadal anchor cell, secretes LIN-3/EGF and induces three out of six competent precursor cells to acquire a vulval fate. We previously showed that this developmental system is robust to a four-fold variation in lin-3/EGF genetic dose. Here using single-molecule FISH, we find that the mean level of expression of lin-3 in the anchor cell is remarkably conserved. No change in lin-3 expression level could be detected among C. elegans wild isolates and only a low level of change-less than 30%-in the Caenorhabditis genus and in Oscheius tipulae. In C. elegans, lin-3 expression in the anchor cell is known to require three transcription factor binding sites, specifically two E-boxes and a nuclear-hormone-receptor (NHR binding site. Mutation of any of these three elements in C. elegans results in a dramatic decrease in lin-3 expression. Yet only a single E-box is found in the Drosophilae supergroup of Caenorhabditis species, including C. angaria, while the NHR-binding site likely only evolved at the base of the Elegans group. We find that a transgene from C. angaria bearing a single E-box is sufficient for normal expression in C. elegans. Even a short 58 bp cis-regulatory fragment from C. angaria with this single E-box is able to replace the three transcription factor binding sites at the endogenous C. elegans lin-3 locus, resulting in the wild-type expression level. Thus, regulatory evolution occurring in cis within a 58 bp lin-3 fragment, results in a strict requirement for the NHR binding site and a second E-box in C. elegans. This single-cell, single-molecule, quantitative and functional evo-devo study demonstrates that conserved expression levels can hide extensive change in cis-regulatory site requirements and highlights the evolution of new cis-regulatory elements required for cell-specific gene expression.

  1. Murine craniofacial development requires Hdac3-mediated repression of Msx gene expression.

    Science.gov (United States)

    Singh, Nikhil; Gupta, Mudit; Trivedi, Chinmay M; Singh, Manvendra K; Li, Li; Epstein, Jonathan A

    2013-05-15

    Craniofacial development is characterized by reciprocal interactions between neural crest cells and neighboring cell populations of ectodermal, endodermal and mesodermal origin. Various genetic pathways play critical roles in coordinating the development of cranial structures by modulating the growth, survival and differentiation of neural crest cells. However, the regulation of these pathways, particularly at the epigenomic level, remains poorly understood. Using murine genetics, we show that neural crest cells exhibit a requirement for the class I histone deacetylase Hdac3 during craniofacial development. Mice in which Hdac3 has been conditionally deleted in neural crest demonstrate fully penetrant craniofacial abnormalities, including microcephaly, cleft secondary palate and dental hypoplasia. Consistent with these abnormalities, we observe dysregulation of cell cycle genes and increased apoptosis in neural crest structures in mutant embryos. Known regulators of cell cycle progression and apoptosis in neural crest, including Msx1, Msx2 and Bmp4, are upregulated in Hdac3-deficient cranial mesenchyme. These results suggest that Hdac3 serves as a critical regulator of craniofacial morphogenesis, in part by repressing core apoptotic pathways in cranial neural crest cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. MMS sensitivity of all amino acid-requiring mutants in aspergillus and its suppression by mutations in a single gene.

    Science.gov (United States)

    Käfer, E

    1987-04-01

    All available amino acid-requiring mutants of Aspergillus nidulans were found to be hypersensitive to MMS (methyl methanesulfonate) to various degrees. On MMS media, secondary mutations could be selected which suppress this MMS sensitivity but do not affect the requirement. Many such mutations were analyzed and found to be alleles of one gene, smsA (= suppressor of MMS sensitivity), which mapped distal on the right arm of chromosome V. This gene is more likely to be involved in general regulation of amino acid biosynthesis than MMS uptake, since a variety of pathway interactions were clearly modified by smsA suppressors in the absence of MMS.

  3. Efficient transcription of the glycolytic gene ADH1 and three translational component genes requires the GCR1 product, which can act through TUF/GRF/RAP binding sites.

    Science.gov (United States)

    Santangelo, G M; Tornow, J

    1990-01-01

    Glycolytic gene expression in Saccharomyces cerevisiae is thought to be activated by the GCR and TUF proteins. We tested the hypothesis that GCR function is mediated by TUF/GRF/RAP binding sites (UASRPG elements). We found that UASRPG-dependent activation of a heterologous gene and transcription of ADH1, TEF1, TEF2, and RP59 were sensitive to GCR1 disruption. GCR is not required for TUF/GRF/RAP expression or in vitro DNA-binding activity. Images PMID:2405258

  4. A CHROMATIN MODIFYING ENZYME, SDG8, IS REQUIRED FOR MORPHOLOGICAL, GENE EXPRESSION, AND EPIGENETIC RESPONSES TO MECHANICAL STIMULATION

    Directory of Open Access Journals (Sweden)

    Christopher Ian Cazzonelli

    2014-10-01

    Full Text Available Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzyme, SDG8/ASHH2, which can regulate the expression of many touch responsive genes identified in Arabidopsis. SDG8 is required for the permissive expression of touch induced genes; and the loss of function of sdg8 perturbs the maximum levels of induction on selected touch gene targets. SDG8 is required to maintain permissive H3K4 trimethylation marks surrounding the Arabidopsis touch-inducible gene TOUCH 3 (TCH3, which encodes a calmodulin-like protein (CML12. The gene neighbouring was also slightly down regulated, revealing a new target for SDG8 mediated chromatin modification. Finally, sdg8 mutants show perturbed morphological response to wind-agitated mechanical stimuli, implicating an epigenetic memory-forming process in the acclimation response of thigmomorphogenesis.

  5. Sequence analysis of putative swrW gene required for surfactant ...

    African Journals Online (AJOL)

    owner

    2012-07-17

    Jul 17, 2012 ... These nucleotide and protein sequence analysis of the putative swrW gene provides vital information on the versatility .... chain reaction (PCR) products were stored at 4°C. Presence of ... identical to the same gene with an E-value of 0.0. .... The Prokaryotes-A Handbook on the Biol. of Bacteria:Ecophysiol.

  6. The Heat Shock Protein 26 Gene is Required for Ethanol Tolerance in Drosophila

    Directory of Open Access Journals (Sweden)

    Awoyemi A. Awofala

    2011-01-01

    Full Text Available Stress plays an important role in drug- and addiction-related behaviours. However, the mechanisms underlying these behavioural responses are still poorly understood. In the light of recent reports that show consistent regulation of many genes encoding stress proteins including heat shock proteins following ethanol exposure in Drosophila , it was hypothesised that transition to alcohol dependence may involve the dysregulation of the circuits that mediate behavioural responses to stressors. Thus, behavioural genetic methodologies were used to investigate the role of the Drosophila hsp26 gene, a small heat shock protein coding gene which is induced in response to various stresses, in the development of rapid tolerance to ethanol sedation. Rapid tolerance was quantified as the percentage difference in the mean sedation times between the second and first ethanol exposure. Two independently isolated P-element mutations near the hsp26 gene eliminated the capacity for tolerance. In addition, RNAi-mediated functional knockdown of hsp26 expression in the glial cells and the whole nervous system also caused a defect in tolerance development. The rapid tolerance phenotype of the hsp26 mutants was rescued by the expression of the wild-type hsp26 gene in the nervous system. None of these manipulations of the hsp26 gene caused changes in the rate of ethanol absorption. Hsp26 genes are evolutionary conserved, thus the role of hsp26 in ethanol tolerance may present a new direction for research into alcohol dependency.

  7. Analysis of an ordered, comprehensive STM mutant library in infectious Borrelia burgdorferi: insights into the genes required for mouse infectivity.

    Directory of Open Access Journals (Sweden)

    Tao Lin

    Full Text Available The identification of genes important in the pathogenesis of Lyme disease Borrelia has been hampered by exceedingly low transformation rates in low-passage, infectious organisms. Using the infectious, moderately transformable B. burgdorferi derivative 5A18NP1 and signature-tagged versions of the Himar1 transposon vector pGKT, we have constructed a defined transposon library for the efficient genome-wide investigation of genes required for wild-type pathogenesis, in vitro growth, physiology, morphology, and plasmid replication. To facilitate analysis, the insertion sites of 4,479 transposon mutants were determined by sequencing. The transposon insertions were widely distributed across the entire B. burgdorferi genome, with an average of 2.68 unique insertion sites per kb DNA. The 10 linear plasmids and 9 circular plasmids had insertions in 33 to 100 percent of their predicted genes. In contrast, only 35% of genes in the 910 kb linear chromosome had incapacitating insertions; therefore, the remaining 601 chromosomal genes may represent essential gene candidates. In initial signature-tagged mutagenesis (STM analyses, 434 mutants were examined at multiple tissue sites for infectivity in mice using a semi-quantitative, Luminex-based DNA detection method. Examples of genes found to be important in mouse infectivity included those involved in motility, chemotaxis, the phosphoenolpyruvate phosphotransferase system, and other transporters, as well as putative plasmid maintenance genes. Availability of this ordered STM library and a high-throughput screening method is expected to lead to efficient assessment of the roles of B. burgdorferi genes in the infectious cycle and pathogenesis of Lyme disease.

  8. Crystal structure of the Haemophilus influenzae Hap adhesin reveals an intercellular oligomerization mechanism for bacterial aggregation

    Science.gov (United States)

    Meng, Guoyu; Spahich, Nicole; Kenjale, Roma; Waksman, Gabriel; St Geme, Joseph W

    2011-01-01

    Bacterial biofilms are complex microbial communities that are common in nature and are being recognized increasingly as an important determinant of bacterial virulence. However, the structural determinants of bacterial aggregation and eventual biofilm formation have been poorly defined. In Gram-negative bacteria, a major subgroup of extracellular proteins called self-associating autotransporters (SAATs) can mediate cell–cell adhesion and facilitate biofilm formation. In this study, we used the Haemophilus influenzae Hap autotransporter as a prototype SAAT to understand how bacteria associate with each other. The crystal structure of the H. influenzae HapS passenger domain (harbouring the SAAT domain) was determined to 2.2 Å by X-ray crystallography, revealing an unprecedented intercellular oligomerization mechanism for cell–cell interaction. The C-terminal SAAT domain folds into a triangular-prism-like structure that can mediate Hap–Hap dimerization and higher degrees of multimerization through its F1–F2 edge and F2 face. The intercellular multimerization can give rise to massive buried surfaces that are required for overcoming the repulsive force between cells, leading to bacterial cell–cell interaction and formation of complex microcolonies. PMID:21841773

  9. Experimental infection of human volunteers with Haemophilus ducreyi: fifteen years of clinical data and experience.

    Science.gov (United States)

    Janowicz, Diane M; Ofner, Susan; Katz, Barry P; Spinola, Stanley M

    2009-06-01

    Haemophilus ducreyi causes chancroid, which facilitates transmission of human immunodeficiency virus type 1. To better understand the biology of H. ducreyi, we developed a human inoculation model. In the present article, we describe clinical outcomes for 267 volunteers who were infected with H. ducreyi. There was a relationship between papule formation and estimated delivered dose. The outcome (either pustule formation or resolution) of infected sites for a given subject was not independent; the most important determinants of pustule formation were sex and host effects. When 41 subjects were infected a second time, their outcomes segregated toward their initial outcome, confirming the host effect. Subjects with pustules developed local symptoms that required withdrawal from the study after a mean of 8.6 days. There were 191 volunteers who had tissue biopsy performed, 173 of whom were available for follow-up analysis; 28 (16.2%) of these developed hypertrophic scars, but the model was otherwise safe. Mutant-parent trials confirmed key features in H. ducreyi pathogenesis, and the model has provided an opportunity to study differential human susceptibility to a bacterial infection.

  10. Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae.

    OpenAIRE

    Preston, R A; Manolson, M F; Becherer, K; Weidenhammer, E; Kirkpatrick, D; Wright, R; Jones, E W

    1991-01-01

    The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX...

  11. Crystal Structure of Homoserine Transacetylase from Haemophilus Influenzae Reveals a New Family of alpha/beta-Hydrolases

    Energy Technology Data Exchange (ETDEWEB)

    Mirza,I.; Nazi, I.; Korczynska, M.; Wright, G.; Berghuis, A.

    2005-01-01

    Homoserine transacetylase catalyzes one of the required steps in the biosynthesis of methionine in fungi and several bacteria. We have determined the crystal structure of homoserine transacetylase from Haemophilus influenzae to a resolution of 1.65 A. The structure identifies this enzyme to be a member of the alpha/beta-hydrolase structural superfamily. The active site of the enzyme is located near the end of a deep tunnel formed by the juxtaposition of two domains and incorporates a catalytic triad involving Ser143, His337, and Asp304. A structural basis is given for the observed double displacement kinetic mechanism of homoserine transacetylase. Furthermore, the properties of the tunnel provide a rationale for how homoserine transacetylase catalyzes a transferase reaction vs. hydrolysis, despite extensive similarity in active site architecture to hydrolytic enzymes.

  12. Identification of Two Gene Clusters and a Transcriptional Regulator Required for Pseudomonas aeruginosa Glycine Betaine Catabolism▿ †

    Science.gov (United States)

    Wargo, Matthew J.; Szwergold, Benjamin S.; Hogan, Deborah A.

    2008-01-01

    Glycine betaine (GB), which occurs freely in the environment and is an intermediate in the catabolism of choline and carnitine, can serve as a sole source of carbon or nitrogen in Pseudomonas aeruginosa. Twelve mutants defective in growth on GB as the sole carbon source were identified through a genetic screen of a nonredundant PA14 transposon mutant library. Further growth experiments showed that strains with mutations in two genes, gbcA (PA5410) and gbcB (PA5411), were capable of growth on dimethylglycine (DMG), a catabolic product of GB, but not on GB itself. Subsequent nuclear magnetic resonance (NMR) experiments with 1,2-13C-labeled choline indicated that these genes are necessary for conversion of GB to DMG. Similar experiments showed that strains with mutations in the dgcAB (PA5398-PA5399) genes, which exhibit homology to genes that encode other enzymes with demethylase activity, are required for the conversion of DMG to sarcosine. Mutant analyses and 13C NMR studies also confirmed that the soxBDAG genes, predicted to encode a sarcosine oxidase, are required for sarcosine catabolism. Our screen also identified a predicted AraC family transcriptional regulator, encoded by gbdR (PA5380), that is required for growth on GB and DMG and for the induction of gbcA, gbcB, and dgcAB in response to GB or DMG. Mutants defective in the previously described gbt gene (PA3082) grew on GB with kinetics similar to those of the wild type in both the PAO1 and PA14 strain backgrounds. These studies provided important insight into both the mechanism and the regulation of the catabolism of GB in P. aeruginosa. PMID:17951379

  13. Cloning and characterization of a gene (UVR3) required for photorepair of 6-4 photoproducts in Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Nakajima, S.; Sugiyama, M.; Iwai, S.; Hitomi, K.; Otoshi, E.; Kim SangTae; Jiang CaiZhong; Todo, T.; Britt, A.B.; Yamamoto, K.

    1998-01-01

    UV radiation induces two major classes of pyrimidine dimers: the pyrimidine [6-4] pyrimidone photoproduct (6-4 product) and the cyclobutane pyrimidine dimer (CPD). Many organisms produce enzymes, termed photolyases, that specifically bind to these damage products and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage. These photolyases are specific for either CPDs or 6-4 products. A gene that expresses a protein with 6-4 photolyase activity in vitro was recently cloned from Drosophila melanogaster and Xenopus laevis. We report here the isolation of a homolog of this gene, cloned on the basis of sequence similarity, from the higher plant Arabidopsis thaliana. This cloned gene produces a protein with 6-4 photolyase activity when expressed in Escherichia coli. We also find that a previously described mutant of Arabidopsis (uvr3) that is defective in photoreactivation of 6-4 products carries a nonsense mutation in this 6-4 photolyase homolog. We have therefore termed this gene UVR3. Although homologs of this gene have previously been shown to produce a functional 6-4 photolyase when expressed in heterologous systems, this is the first demonstration of a requirement for this gene for photoreactivation of 6-4 products in vivo

  14. Structural Determinants of Autoproteolysis of the Haemophilus influenzae Hap Autotransporter▿

    Science.gov (United States)

    Kenjale, Roma; Meng, Guoyu; Fink, Doran L.; Juehne, Twyla; Ohashi, Tomoo; Erickson, Harold P.; Waksman, Gabriel; St. Geme, Joseph W.

    2009-01-01

    Haemophilus influenzae is a gram-negative bacterium that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hap autotransporter protein mediates adherence, invasion, and microcolony formation in assays with respiratory epithelial cells and presumably facilitates colonization. The serine protease activity of Hap is associated with autoproteolytic cleavage and extracellular release of the HapS passenger domain, leaving the Hapβ C-terminal domain embedded in the outer membrane. Cleavage occurs most efficiently at the LN1036-37 peptide bond and to a lesser extent at three other sites. In this study, we utilized site-directed mutagenesis, homology modeling, and assays with a peptide library to characterize the structural determinants of Hap proteolytic activity and cleavage specificity. In addition, we used homology modeling to predict the S1, S2, and S4 subsite residues of the Hap substrate groove. Our results indicate that the P1 and P2 positions at the Hap cleavage sites are critical for cleavage, with leucine preferred over larger hydrophobic residues or other amino acids in these positions. The substrate groove is formed by L263 and N274 at the S1 subsite, R264 at the S2 subsite, and E265 at the S4 subsite. This information may facilitate design of approaches to block Hap activity and interfere with H. influenzae colonization. PMID:19687208

  15. Antimicrobial susceptibility patterns of Brazilian Haemophilus parasuis field isolates

    Directory of Open Access Journals (Sweden)

    Michela Miani

    Full Text Available ABSTRACT: Haemophilus parasuis is the etiological agent of Glässer’s disease (GD, an ubiquitous infection of swine characterized by systemic fibrinous polyserositis, polyarthritis and meningitis. Intensive use of antimicrobial agents in swine husbandries during the last years triggered the development of antibiotic resistances in bacterial pathogens. Thus, regular susceptibility testing is crucial to ensure efficacy of different antimicrobial agents to this porcine pathogen. In this study, 50 clinical isolates from South Brazilian pig herds were characterized and analyzed for their susceptibility to commonly used antibiotic. The identification and typing of clinical isolates was carried out by a modified indirect hemagglutination assay combined with a multiplex PCR. The susceptibility of each isolate was analyzed by broth microdilution method against a panel of 21 antimicrobial compounds. We found that field isolates are highly resistance to gentamycin, bacitracin, lincomycin and tiamulin, but sensitive to ampicillin, clindamycin, neomycin, penicillin, danofloxacin and enrofloxacin. Furthermore, an individual susceptibility analysis indicated that enrofloxacin is effective to treat clinical isolates with the exception of those classified as serovar 1. The results presented here firstly demonstrate the susceptibility of Brazilian clinical isolates of H. parasuis to antimicrobials widely used by swine veterinary practitioners and strengthen the need to perform susceptibility test prior to antibiotic therapy during GD outbreaks. In addition, because only six antimicrobial drugs (28.6% were found effective against field isolates, a continuous surveillance of the susceptibility profile should be of major concern to the swine industry.

  16. Nontypeable Haemophilus influenzae initiates formation of neutrophil extracellular traps.

    Science.gov (United States)

    Juneau, Richard A; Pang, Bing; Weimer, Kristin E D; Armbruster, Chelsie E; Swords, W Edward

    2011-01-01

    Nontypeable Haemophilus influenzae (NTHI) is a leading cause of otitis media infections, which are often chronic and/or recurrent in nature. NTHI and other bacterial species persist in vivo within biofilms during otitis media and other persistent infections. These biofilms have a significant host component that includes neutrophil extracellular traps (NETs). These NETs do not mediate clearance of NTHI, which survives within NET structures by means of specific subpopulations of lipooligosaccharides on the bacterial surface that are determinants of biofilm formation in vitro. In this study, the ability of NTHI and NTHI components to initiate NET formation was examined using an in vitro model system. Both viable and nonviable NTHI strains were shown to promote NET formation, as did preparations of bacterial DNA, outer membrane proteins, and lipooligosaccharide (endotoxin). However, only endotoxin from a parental strain of NTHI exhibited equivalent potency in NET formation to that of NTHI. Additional studies showed that NTHI entrapped within NET structures is resistant to both extracellular killing within NETs and phagocytic killing by incoming neutrophils, due to oligosaccharide moieties within the lipooligosaccharides. Thus, we concluded that NTHI elicits NET formation by means of multiple pathogen-associated molecular patterns (most notably endotoxin) and is highly resistant to killing within NET structures. These data support the conclusion that, for NTHI, formation of NET structures may be a persistence determinant by providing a niche within the middle-ear chamber.

  17. Altered indirect hemagglutination method for easy serotyping of Haemophilus parasuis

    Directory of Open Access Journals (Sweden)

    M.S. Lorenson

    Full Text Available ABSTRACT Glässer's disease is an emergent bacterial disease that affects swine husbandries worldwide causing important economic losses. The aetiological agent, Haemophilus parasuis, is currently divided in fifteen serovars but an increasing number of non-typeable serovars have been reported. Indirect hemagglutination (IHA is indicated as a serotyping method for H. parasuis. In the present study, we describe an additional step that aims to work around a possible obstacle in the original protocol that may compromise the outcome of this assay. We observed that the choice of anticoagulant for blood collection influences and/or impairs spontaneous adsorption of H. parasuis antigens on sheep red blood cells (SRBCs. However, regardless of the anticoagulant used, chemical treatment of SRBCs with tannic acid induces a stable antigen adsorption (sensitization step. The addition of 1% BSA to SRBCs washing buffer and to antisera dilution augments IHA specificity. Tannic acid treated SRBCs combined with thermo-resistant H. parasuis antigens increases the assay resolution. Thus, our results demonstrate an improvement in the technique of H. parasuis serotyping that will prove valuable to understand Glässer's disease epidemiology and to better characterize serovars involved in outbreaks.

  18. Transformation of natural genetic variation into Haemophilus influenzae genomes.

    Directory of Open Access Journals (Sweden)

    Joshua Chang Mell

    2011-07-01

    Full Text Available Many bacteria are able to efficiently bind and take up double-stranded DNA fragments, and the resulting natural transformation shapes bacterial genomes, transmits antibiotic resistance, and allows escape from immune surveillance. The genomes of many competent pathogens show evidence of extensive historical recombination between lineages, but the actual recombination events have not been well characterized. We used DNA from a clinical isolate of Haemophilus influenzae to transform competent cells of a laboratory strain. To identify which of the ~40,000 polymorphic differences had recombined into the genomes of four transformed clones, their genomes and their donor and recipient parents were deep sequenced to high coverage. Each clone was found to contain ~1000 donor polymorphisms in 3-6 contiguous runs (8.1±4.5 kb in length that collectively comprised ~1-3% of each transformed chromosome. Seven donor-specific insertions and deletions were also acquired as parts of larger donor segments, but the presence of other structural variation flanking 12 of 32 recombination breakpoints suggested that these often disrupt the progress of recombination events. This is the first genome-wide analysis of chromosomes directly transformed with DNA from a divergent genotype, connecting experimental studies of transformation with the high levels of natural genetic variation found in isolates of the same species.

  19. Localization of Haemophilus ducreyi in naturally acquired chancroidal ulcers.

    Science.gov (United States)

    Bauer, Margaret E; Townsend, Carisa A; Ronald, Allan R; Spinola, Stanley M

    2006-08-01

    Haemophilus ducreyi causes the sexually transmitted genital ulcer disease chancroid. In human inoculation experiments, bacteria colocalize with neutrophils and macrophages but remain extracellular. The organism also colocalizes with collagen and fibrin but not with keratinocytes, fibroblasts, laminin, or fibronectin. These relationships are established by 48 h postinoculation and persist through the pustular stage of disease. To extend these observations to the ulcerative stage of disease, and to compare results in the human model with those of natural disease, we obtained biopsies from patients with naturally acquired chancroid. All ulcers were culture positive for H. ducreyi and histologically very similar to pustules from the human model. Staining with H. ducreyi-specific monoclonal antibodies demonstrated H. ducreyi within 5 biopsies. The organism was chiefly found within the granulocytic infiltrate of the ulcer. Dual staining for H. ducreyi and eukaryotic tissue components showed that H. ducreyi colocalized with neutrophils and fibrin at the ulcerative stage of disease. No bacteria were associated with keratinocytes, fibroblasts, or collagen. Overall, these findings are consistent with results from the human model. This is the first reported study to localize bacteria specifically identified as H. ducreyi within naturally acquired chancroid.

  20. Assessing the antibiotic potential of essential oils against Haemophilus ducreyi.

    Science.gov (United States)

    Lindeman, Zachary; Waggoner, Molly; Batdorff, Audra; Humphreys, Tricia L

    2014-05-27

    Haemophilus ducreyi is the bacterium responsible for the genital ulcer disease chancroid, a cofactor for the transmission of HIV, and it is resistant to many antibiotics. With the goal of exploring possible alternative treatments, we tested essential oils (EOs) for their efficacy as antimicrobial agents against H. ducreyi. We determine the minimum inhibitory concentration (MIC) of Cinnamomum verum (cinnamon), Eugenia caryophyllus (clove) and Thymus satureioides (thyme) oil against 9 strains of H. ducreyi using the agar dilution method. We also determined the minimum lethal concentration for each oil by subculturing from the MIC plates onto fresh agar without essential oil. For both tests, we used a 2-way ANOVA to evaluate whether antibiotic-resistant strains had a different sensitivity to the oils relative to non-resistant strains. All 3 oils demonstrated excellent activity against H. ducreyi, with MICs of 0.05 to 0.52 mg/mL and MLCs of 0.1-0.5 mg/mL. Antibiotic-resistant strains of H. ducreyi were equally susceptible to these 3 essential oils relative to non-resistant strains (p=0.409). E. caryophyllus, C. verum and T. satureioides oils are promising alternatives to antibiotic treatment for chancroid.

  1. Haemophilus influenzae: an underrated cause of vulvovaginitis in young girls.

    Science.gov (United States)

    Cox, R A

    1997-01-01

    AIMS: To establish the common pathogens associated with infective vulvovaginitis in young girls in the local population and to determine current management of this condition. METHODS: A prospective laboratory based survey was carried out over 19 months. A questionnaire was then sent to local general practitioners and hospital doctors. RESULTS: One hundred and six swabs were received during the study period of which 43 (40.5%) yielded organisms recognised as causes of vulvovaginitis. The most common pathogen was group A beta haemolytic streptococcus (19), with Haemophilus influenzae the second most common (11). Candida was isolated on nine occasions. The users' questionnaire had an overall response rate of 52%. Forty one per cent of respondents nominated candida as the most common cause of this condition. Forty six per cent were aware that beta haemolytic streptococci caused juvenile vulvovaginitis, but only four (3.6%) knew that H influenzae was a possible pathogen. The most popular agent for empirical treatment of vulvovaginitis was topical clotrimazole cream, although 24 respondents (22%) prescribed antibiotics that are active against both group A beta haemolytic streptococci and H influenzae. CONCLUSIONS: Although H influenzae is the second most common infective cause of juvenile vulvovaginitis in the local population, most doctors managing these patients were unaware of its importance and may not be prescribing appropriate empirical treatment. Images PMID:9389978

  2. Effect of Multiple Mutations in the Hemoglobin- and Hemoglobin-Haptoglobin-Binding Proteins, HgpA, HgpB, and HgpC, of Haemophilus influenzae Type b

    OpenAIRE

    Morton, Daniel J.; Whitby, Paul W.; Jin, Hongfan; Ren, Zhen; Stull, Terrence L.

    1999-01-01

    Haemophilus influenzae requires heme for growth and can utilize hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified two hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA and HgpB, in H. influenzae HI689. Insertional mutation of hgpA and hgpB, either singly or together, did not abrogate the ability to utilize or bind either hemoglobin or the hemoglobin-haptoglobin complex. A hemoglobin affinity purification method was used to isolate a protein of approxi...

  3. Multiple BiP genes of Arabidopsis thaliana are required for male gametogenesis and pollen competitiveness.

    Science.gov (United States)

    Maruyama, Daisuke; Sugiyama, Tomoyuki; Endo, Toshiya; Nishikawa, Shuh-Ichi

    2014-04-01

    Immunoglobulin-binding protein (BiP) is a molecular chaperone of the heat shock protein 70 (Hsp70) family. BiP is localized in the endoplasmic reticulum (ER) and plays key roles in protein translocation, protein folding and quality control in the ER. The genomes of flowering plants contain multiple BiP genes. Arabidopsis thaliana has three BiP genes. BIP1 and BIP2 are ubiquitously expressed. BIP3 encodes a less well conserved BiP paralog, and it is expressed only under ER stress conditions in the majority of organs. Here, we report that all BiP genes are expressed and functional in pollen and pollen tubes. Although the bip1 bip2 double mutation does not affect pollen viability, the bip1 bip2 bip3 triple mutation is lethal in pollen. This result indicates that lethality of the bip1 bip2 double mutation is rescued by BiP3 expression. A decrease in the copy number of the ubiquitously expressed BiP genes correlates well with a decrease in pollen tube growth, which leads to reduced fitness of mutant pollen during fertilization. Because an increased protein secretion activity is expected to increase the protein folding demand in the ER, the multiple BiP genes probably cooperate with each other to ensure ER homeostasis in cells with active secretion such as rapidly growing pollen tubes.

  4. Mutations in sit B and sit D genes affect manganese-growth requirements in Sinorhizobium meliloti.

    Science.gov (United States)

    Platero, Raúl A; Jaureguy, Melina; Battistoni, Federico J; Fabiano, Elena R

    2003-01-21

    Two transposon-induced mutants of Sinorhizobium meliloti 242 were isolated based on their inability to grow on rich medium supplemented with the metal chelator ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) and either heme-compounds or siderophores as iron sources. Tagged loci of these mutants were identified as sit B and sit D genes. These genes encode components of an ABC (ATP-binding cassette) metal-type permease in several Gram-negative bacteria. In this work, the phenotypes of these two mutants were compared with those of two siderophore-mediated iron transport mutants. The results strongly implicate a role of the sit genes in manganese acquisition when this metal is limiting in S. meliloti.

  5. Identification of genes specifically required for the anaerobic metabolism of benzene in Geobacter metallireducens

    DEFF Research Database (Denmark)

    Zhang, Tian; Tremblay, Pier-Luc; Chaurasia, Akhilesh Kumar

    2014-01-01

    Although the biochemical pathways for the anaerobic degradation of many of the hydrocarbon constituents in petroleum reservoirs have been elucidated, the mechanisms for anaerobic activation of benzene, a very stable molecule, are not known. Previous studies have demonstrated that Geobacter...... metallireducens can anaerobically oxidize benzene to carbon dioxide with Fe(III) as the sole electron acceptor and that phenol is an intermediate in benzene oxidation. In an attempt to identify enzymes that might be involved in the conversion of benzene to phenol, whole-genome gene transcript abundance...... was compared in cells metabolizing benzene and cells metabolizing phenol. Eleven genes had significantly higher transcript abundance in benzene-metabolizing cells. Five of these genes had annotations suggesting that they did not encode proteins that could be involved in benzene metabolism and were not further...

  6. Genome-wide screen for salmonella genes required for long-term systemic infection of the mouse.

    Directory of Open Access Journals (Sweden)

    2006-02-01

    Full Text Available A microarray-based negative selection screen was performed to identify Salmonella enterica serovar Typhimurium (serovar Typhimurium genes that contribute to long-term systemic infection in 129X1/SvJ (Nramp1(r mice. A high-complexity transposon-mutagenized library was used to infect mice intraperitoneally, and the selective disappearance of mutants was monitored after 7, 14, 21, and 28 d postinfection. One hundred and eighteen genes were identified to contribute to serovar Typhimurium infection of the spleens of mice by 28 d postinfection. The negatively selected mutants represent many known aspects of Salmonella physiology and pathogenesis, although the majority of the identified genes are of putative or unknown function. Approximately 30% of the negatively selected genes correspond to horizontally acquired regions such as those within Salmonella pathogenicity islands (SPI 1-5, prophages (Gifsy-1 and -2 and remnant, and the pSLT virulence plasmid. In addition, mutations in genes responsible for outer membrane structure and remodeling, such as LPS- and PhoP-regulated and fimbrial genes, were also selected against. Competitive index experiments demonstrated that the secreted SPI2 effectors SseK2 and SseJ as well as the SPI4 locus are attenuated relative to wild-type bacteria during systemic infection. Interestingly, several SPI1-encoded type III secretion system effectors/translocases are required by serovar Typhimurium to establish and, unexpectedly, to persist systemically, challenging the present description of Salmonella pathogenesis. Moreover, we observed a progressive selection against serovar Typhimurium mutants based upon the duration of the infection, suggesting that different classes of genes may be required at distinct stages of infection. Overall, these data indicate that Salmonella long-term systemic infection in the mouse requires a diverse repertoire of virulence factors. This diversity of genes presumably reflects the fact that

  7. No control genes required: Bayesian analysis of qRT-PCR data.

    Directory of Open Access Journals (Sweden)

    Mikhail V Matz

    Full Text Available Model-based analysis of data from quantitative reverse-transcription PCR (qRT-PCR is potentially more powerful and versatile than traditional methods. Yet existing model-based approaches cannot properly deal with the higher sampling variances associated with low-abundant targets, nor do they provide a natural way to incorporate assumptions about the stability of control genes directly into the model-fitting process.In our method, raw qPCR data are represented as molecule counts, and described using generalized linear mixed models under Poisson-lognormal error. A Markov Chain Monte Carlo (MCMC algorithm is used to sample from the joint posterior distribution over all model parameters, thereby estimating the effects of all experimental factors on the expression of every gene. The Poisson-based model allows for the correct specification of the mean-variance relationship of the PCR amplification process, and can also glean information from instances of no amplification (zero counts. Our method is very flexible with respect to control genes: any prior knowledge about the expected degree of their stability can be directly incorporated into the model. Yet the method provides sensible answers without such assumptions, or even in the complete absence of control genes. We also present a natural Bayesian analogue of the "classic" analysis, which uses standard data pre-processing steps (logarithmic transformation and multi-gene normalization but estimates all gene expression changes jointly within a single model. The new methods are considerably more flexible and powerful than the standard delta-delta Ct analysis based on pairwise t-tests.Our methodology expands the applicability of the relative-quantification analysis protocol all the way to the lowest-abundance targets, and provides a novel opportunity to analyze qRT-PCR data without making any assumptions concerning target stability. These procedures have been implemented as the MCMC.qpcr package in R.

  8. No control genes required: Bayesian analysis of qRT-PCR data.

    Science.gov (United States)

    Matz, Mikhail V; Wright, Rachel M; Scott, James G

    2013-01-01

    Model-based analysis of data from quantitative reverse-transcription PCR (qRT-PCR) is potentially more powerful and versatile than traditional methods. Yet existing model-based approaches cannot properly deal with the higher sampling variances associated with low-abundant targets, nor do they provide a natural way to incorporate assumptions about the stability of control genes directly into the model-fitting process. In our method, raw qPCR data are represented as molecule counts, and described using generalized linear mixed models under Poisson-lognormal error. A Markov Chain Monte Carlo (MCMC) algorithm is used to sample from the joint posterior distribution over all model parameters, thereby estimating the effects of all experimental factors on the expression of every gene. The Poisson-based model allows for the correct specification of the mean-variance relationship of the PCR amplification process, and can also glean information from instances of no amplification (zero counts). Our method is very flexible with respect to control genes: any prior knowledge about the expected degree of their stability can be directly incorporated into the model. Yet the method provides sensible answers without such assumptions, or even in the complete absence of control genes. We also present a natural Bayesian analogue of the "classic" analysis, which uses standard data pre-processing steps (logarithmic transformation and multi-gene normalization) but estimates all gene expression changes jointly within a single model. The new methods are considerably more flexible and powerful than the standard delta-delta Ct analysis based on pairwise t-tests. Our methodology expands the applicability of the relative-quantification analysis protocol all the way to the lowest-abundance targets, and provides a novel opportunity to analyze qRT-PCR data without making any assumptions concerning target stability. These procedures have been implemented as the MCMC.qpcr package in R.

  9. Epstein-Barr Virus BKRF4 Gene Product Is Required for Efficient Progeny Production.

    Science.gov (United States)

    Masud, H M Abdullah Al; Watanabe, Takahiro; Yoshida, Masahiro; Sato, Yoshitaka; Goshima, Fumi; Kimura, Hiroshi; Murata, Takayuki

    2017-12-01

    Epstein-Barr virus (EBV), a member of human gammaherpesvirus, infects mainly B cells. EBV has two alternative life cycles, latent and lytic, and is reactivated occasionally from the latent stage to the lytic cycle. To combat EBV-associated disorders, understanding the molecular mechanisms of the EBV lytic replication cycle is also important. Here, we focused on an EBV lytic gene, BKRF4. Using our anti-BKRF4 antibody, we revealed that the BKRF4 gene product is expressed during the lytic cycle with late kinetics. To characterize the role of BKRF4, we constructed BKRF4-knockout mutants using the bacterial artificial chromosome (BAC) and CRISPR/Cas9 systems. Although disruption of the BKRF4 gene had almost no effect on viral protein expression and DNA synthesis, it significantly decreased progeny virion levels in HEK293 and Akata cells. Furthermore, we show that BKRF4 is involved not only in production of progeny virions but also in increasing the infectivity of the virus particles. Immunoprecipitation assays revealed that BKRF4 interacted with a virion protein, BGLF2. We showed that the C-terminal region of BKRF4 was critical for this interaction and for efficient progeny production. Immunofluorescence analysis revealed that BKRF4 partially colocalized with BGLF2 in the nucleus and perinuclear region. Finally, we showed that BKRF4 is a phosphorylated, possible tegument protein and that the EBV protein kinase BGLF4 may be important for this phosphorylation. Taken together, our data suggest that BKRF4 is involved in the production of infectious virions. IMPORTANCE Although the latent genes of EBV have been studied extensively, the lytic genes are less well characterized. This study focused on one such lytic gene, BKRF4, which is conserved only among gammaherpesviruses (ORF45 of Kaposi's sarcoma-associated herpesvirus or murine herpesvirus 68). After preparing the BKRF4 knockout virus using B95-8 EBV-BAC, we demonstrated that the BKRF4 gene was involved in infectious

  10. Rapid Differentiation of Haemophilus influenzae and Haemophilus haemolyticus by Use of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry with ClinProTools Mass Spectrum Analysis.

    Science.gov (United States)

    Chen, Jonathan H K; Cheng, Vincent C C; Wong, Chun-Pong; Wong, Sally C Y; Yam, Wing-Cheong; Yuen, Kwok-Yung

    2017-09-01

    Haemophilus influenzae is associated with severe invasive disease, while Haemophilus haemolyticus is considered part of the commensal flora in the human respiratory tract. Although the addition of a custom mass spectrum library into the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system could improve identification of these two species, the establishment of such a custom database is technically complicated and requires a large amount of resources, which most clinical laboratories cannot afford. In this study, we developed a mass spectrum analysis model with 7 mass peak biomarkers for the identification of H. influenzae and H. haemolyticus using the ClinProTools software. We evaluated the diagnostic performance of this model using 408 H. influenzae and H. haemolyticus isolates from clinical respiratory specimens from 363 hospitalized patients and compared the identification results with those obtained with the Bruker IVD MALDI Biotyper. The IVD MALDI Biotyper identified only 86.9% of H. influenzae (311/358) and 98.0% of H. haemolyticus (49/50) clinical isolates to the species level. In comparison, the ClinProTools mass spectrum model could identify 100% of H. influenzae (358/358) and H. haemolyticus (50/50) clinical strains to the species level and significantly improved the species identification rate (McNemar's test, P mass spectrometry to handle closely related bacterial species when the proprietary spectrum library failed. This approach should be useful for the differentiation of other closely related bacterial species. Copyright © 2017 American Society for Microbiology.

  11. The role of cytomegalovirus, Haemophilus influenzae and Epstein Barr virus in Guillain Barre syndrome.

    Directory of Open Access Journals (Sweden)

    Shahriar Nafissi

    2013-06-01

    Full Text Available Guillain Barre Syndrome (GBS is an inflammatory, usually demyelinating, polyneuropathy; clinically characterized by acute onset of symmetric progressive muscle weakness with loss of myotatic reflexes. Thirty five patients with GBS, defined clinically according to the criteria of Asbury and Cornblath, were recruited from three hospital affiliated to Tehran University of Medical Sciences.As a control group 35 age and sex matched patients with other neurological diseases admitted to the same hospital at the same time, were included in our study. Serum samples were collected before treatment from each patient (within 4 weeks after the disease onset and controls, and stored frozen at -80ºC until serologic assays were done. Serologic testing of pretreatment serum was performed in all patients. Positive titer of virus specific IgM antibody against cytomegalovirus (CMV was found in 6 cases and 2 controls. 34 patients and 31 controls had high titer of anti Haemophilus influenzae IgG and one patient had serologic evidence of a recent Epstein Barr virus (EBV infection. The mean titer of IgG antibody against Haemophilus influenzae in cases and controls was 5.21 and 2.97 respectively. Although serologic evidence of all these infections were more frequent in cases than in controls, only Haemophilus influenzae infection appeared to be significantly related to GBS (P=0.002. Eleven cases and 3 controls had high titers of IgG antibody against Haemophilus influenzae type B (titer >8. There is significant association between high titer of IgG antibody against Haemophilus influenzae and GBS (P=0.017. Our results provide further evidence that Haemophilus influenzae and probably CMV, can be associated with GBS.

  12. Sialylation of lipooligosaccharides is dispensable for the virulence of Haemophilus ducreyi in humans.

    Science.gov (United States)

    Spinola, Stanley M; Li, Wei; Fortney, Kate R; Janowicz, Diane M; Zwickl, Beth; Katz, Barry P; Munson, Robert S

    2012-02-01

    Sialylated glycoconjugates on the surfaces of mammalian cells play important roles in intercellular communication and self-recognition. The sialic acid preferentially expressed in human tissues is N-acetylneuraminic acid (Neu5Ac). In a process called molecular mimicry, many bacterial pathogens decorate their cell surface glycolipids with Neu5Ac. Incorporation of Neu5Ac into bacterial glycolipids promotes bacterial interactions with host cell receptors called Siglecs. These interactions affect bacterial adherence, resistance to serum killing and phagocytosis, and innate immune responses. Haemophilus ducreyi, the etiologic agent of chancroid, expresses lipooligosaccharides (LOS) that are highly sialylated. However, an H. ducreyi sialyltransferase (lst) mutant, whose LOS contain reduced levels of Neu5Ac, is fully virulent in human volunteers. Recently, a second sialyltransferase gene (Hd0053) was discovered in H. ducreyi, raising the possibility that Hd0053 compensated for the loss of lst during human infection. CMP-Neu5Ac is the obligate nucleotide sugar donor for all bacterial sialyltransferases; LOS derived from an H. ducreyi CMP-Neu5Ac synthetase (neuA) mutant has no detectable Neu5Ac. Here, we compared an H. ducreyi neuA mutant to its wild-type parent in several models of pathogenesis. In human inoculation experiments, the neuA mutant formed papules and pustules at rates that were no different than those of its parent. When grown in media with and without Neu5Ac supplementation, the neuA mutant and its parent had similar phenotypes in bactericidal, macrophage uptake, and dendritic cell activation assays. Although we cannot preclude a contribution of LOS sialylation to ulcerative disease, these data strongly suggest that sialylation of LOS is dispensable for H. ducreyi pathogenesis in humans.

  13. Haemophilus influenzae from Patients with Chronic Obstructive Pulmonary Disease Exacerbation Induce More Inflammation than Colonizers

    Science.gov (United States)

    Chin, Cecilia L.; Manzel, Lori J.; Lehman, Erin E.; Humlicek, Alicia L.; Shi, Lei; Starner, Timothy D.; Denning, Gerene M.; Murphy, Timothy F.; Sethi, Sanjay; Look, Dwight C.

    2005-01-01

    Rationale: Airway infection with Haemophilus influenzae causes airway inflammation, and isolation of new strains of this bacteria is associated with increased risk of exacerbations in patients with chronic obstructive pulmonary disease (COPD). Objective: To determine whether strains of H. influenzae associated with exacerbations cause more inflammation than strains that colonize the airways of patients with COPD. Methods: Exacerbation strains of H. influenzae were isolated from patients during exacerbation of clinical symptoms with subsequent development of a homologous serum antibody response and were compared with colonization strains that were not associated with symptom worsening or an antibody response. Bacterial strains were compared using an in vivo mouse model of airway infection and in vitro cell culture model of bacterial adherence and defense gene and signaling pathway activation in primary human airway epithelial cells. Results: H. influenzae associated with exacerbations caused more airway neutrophil recruitment compared with colonization strains in the mouse model of airway bacterial infection. Furthermore, exacerbation strains adhered to epithelial cells in significantly higher numbers and induced more interleukin-8 release after interaction with airway epithelial cells. This effect was likely mediated by increased activation of the nuclear factor-κB and p38 mitogen-activated protein kinase signaling pathways. Conclusions: The results indicate that H. influenzae strains isolated from patients during COPD exacerbations often induce more airway inflammation and likely have differences in virulence compared with colonizing strains. These findings support the concept that bacteria infecting the airway during COPD exacerbations mediate increased airway inflammation and contribute to decreased airway function. PMID:15805181

  14. The rgg0182 gene encodes a transcriptional regulator required for the full Streptococcus thermophilus LMG18311 thermal adaptation.

    Science.gov (United States)

    Henry, Romain; Bruneau, Emmanuelle; Gardan, Rozenn; Bertin, Stéphane; Fleuchot, Betty; Decaris, Bernard; Leblond-Bourget, Nathalie

    2011-10-07

    Streptococcus thermophilus is an important starter strain for the production of yogurt and cheeses. The analysis of sequenced genomes of four strains of S. thermophilus indicates that they contain several genes of the rgg familly potentially encoding transcriptional regulators. Some of the Rgg proteins are known to be involved in bacterial stress adaptation. In this study, we demonstrated that Streptococcus thermophilus thermal stress adaptation required the rgg0182 gene which transcription depends on the culture medium and the growth temperature. This gene encoded a protein showing similarity with members of the Rgg family transcriptional regulator. Our data confirmed that Rgg0182 is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a Δrgg0182 mutant revealed that this protein played a role in the heat shock adaptation of Streptococcus thermophilus LMG18311. These data showed the importance of the Rgg0182 transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.

  15. The rgg0182 gene encodes a transcriptional regulator required for the full Streptococcus thermophilus LMG18311 thermal adaptation

    Directory of Open Access Journals (Sweden)

    Bertin Stéphane

    2011-10-01

    Full Text Available Abstract Background Streptococcus thermophilus is an important starter strain for the production of yogurt and cheeses. The analysis of sequenced genomes of four strains of S. thermophilus indicates that they contain several genes of the rgg familly potentially encoding transcriptional regulators. Some of the Rgg proteins are known to be involved in bacterial stress adaptation. Results In this study, we demonstrated that Streptococcus thermophilus thermal stress adaptation required the rgg0182 gene which transcription depends on the culture medium and the growth temperature. This gene encoded a protein showing similarity with members of the Rgg family transcriptional regulator. Our data confirmed that Rgg0182 is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a Δrgg0182 mutant revealed that this protein played a role in the heat shock adaptation of Streptococcus thermophilus LMG18311. Conclusions These data showed the importance of the Rgg0182 transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.

  16. The noncoding RNA taurine upregulated gene 1 is required for differentiation of the murine retina.

    Science.gov (United States)

    Young, T L; Matsuda, T; Cepko, C L

    2005-03-29

    With the advent of genome-wide analyses, it is becoming evident that a large number of noncoding RNAs (ncRNAs) are expressed in vertebrates. However, of the thousands of ncRNAs identified, the functions of relatively few have been established. In a screen for genes upregulated by taurine in developing retinal cells, we identified a gene that appears to be a ncRNA. Taurine Upregulated Gene 1 (TUG1) is a spliced, polyadenylated RNA that does not encode any open reading frame greater than 82 amino acids in its full-length, 6.7 kilobase (kb) RNA sequence. Analyses of Northern blots and in situ hybridization revealed that TUG1 is expressed in the developing retina and brain, as well as in adult tissues. In the newborn retina, knockdown of TUG1 with RNA interference (RNAi) resulted in malformed or nonexistent outer segments of transfected photoreceptors. Immunofluorescent staining and microarray analyses suggested that this loss of proper photoreceptor differentiation is a result of the disregulation of photoreceptor gene expression. A function for a newly identified ncRNA, TUG1, has been established. TUG1 is necessary for the proper formation of photoreceptors in the developing rodent retina.

  17. A Genome-Wide Screen for Dendritically Localized RNAs Identifies Genes Required for Dendrite Morphogenesis

    Directory of Open Access Journals (Sweden)

    Mala Misra

    2016-08-01

    Full Text Available Localizing messenger RNAs at specific subcellular sites is a conserved mechanism for targeting the synthesis of cytoplasmic proteins to distinct subcellular domains, thereby generating the asymmetric protein distributions necessary for cellular and developmental polarity. However, the full range of transcripts that are asymmetrically distributed in specialized cell types, and the significance of their localization, especially in the nervous system, are not known. We used the EP-MS2 method, which combines EP transposon insertion with the MS2/MCP in vivo fluorescent labeling system, to screen for novel localized transcripts in polarized cells, focusing on the highly branched Drosophila class IV dendritic arborization neurons. Of a total of 541 lines screened, we identified 55 EP-MS2 insertions producing transcripts that were enriched in neuronal processes, particularly in dendrites. The 47 genes identified by these insertions encode molecularly diverse proteins, and are enriched for genes that function in neuronal development and physiology. RNAi-mediated knockdown confirmed roles for many of the candidate genes in dendrite morphogenesis. We propose that the transport of mRNAs encoded by these genes into the dendrites allows their expression to be regulated on a local scale during the dynamic developmental processes of dendrite outgrowth, branching, and/or remodeling.

  18. In Lactobacillus pentosus, the olive brine adaptation genes are required for biofilm formation.

    Science.gov (United States)

    Perpetuini, G; Pham-Hoang, B N; Scornec, H; Tofalo, R; Schirone, M; Suzzi, G; Cavin, J F; Waché, Y; Corsetti, A; Licandro-Seraut, H

    2016-01-04

    Lactobacillus pentosus is one of the few lactic acid bacteria (LAB) species capable of surviving in olive brine, and thus desirable during table olive fermentation. We have recently generated mutants of the efficient strain L. pentosus C11 by transposon mutagenesis and identified five mutants unable to survive and adapt to olive brine conditions. Since biofilm formation represents one of the main bacterial strategy to survive in stressful environments, in this study, the capacity of adhesion and formation of biofilm on olive skin was investigated for this strain and five derivative mutants which are interrupted in metabolic genes (enoA1 and gpi), and in genes of unknown function ("oba" genes). Confocal microscopy together with bacteria count revealed that the sessile state represented the prevailing L. pentosus C11 life-style during table olive fermentation. The characterization of cell surface properties showed that mutants present less hydrophobic and basic properties than the wild type (WT). In fact, their ability to adhere to both abiotic (polystyrene plates) and biotic (olive skin) surfaces was lower than that of the WT. Confocal microscopy revealed that mutants adhered sparsely to the olive skin instead of building a thin, multilayer biofilm. Moreover, RT-qPCR showed that the three genes enoA1, gpi and obaC were upregulated in the olive biofilm compared to the planktonic state. Thus enoA1, gpi and "oba" genes are necessary in L. pentosus to form an organized biofilm on the olive skin. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Genome-wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid

    Directory of Open Access Journals (Sweden)

    Sá-Correia Isabel

    2010-10-01

    Full Text Available Abstract Background Acetic acid is a byproduct of Saccharomyces cerevisiae alcoholic fermentation. Together with high concentrations of ethanol and other toxic metabolites, acetic acid may contribute to fermentation arrest and reduced ethanol productivity. This weak acid is also a present in lignocellulosic hydrolysates, a highly interesting non-feedstock substrate in industrial biotechnology. Therefore, the better understanding of the molecular mechanisms underlying S. cerevisiae tolerance to acetic acid is essential for the rational selection of optimal fermentation conditions and the engineering of more robust industrial strains to be used in processes in which yeast is explored as cell factory. Results The yeast genes conferring protection against acetic acid were identified in this study at a genome-wide scale, based on the screening of the EUROSCARF haploid mutant collection for susceptibility phenotypes to this weak acid (concentrations in the range 70-110 mM, at pH 4.5. Approximately 650 determinants of tolerance to acetic acid were identified. Clustering of these acetic acid-resistance genes based on their biological function indicated an enrichment of genes involved in transcription, internal pH homeostasis, carbohydrate metabolism, cell wall assembly, biogenesis of mitochondria, ribosome and vacuole, and in the sensing, signalling and uptake of various nutrients in particular iron, potassium, glucose and amino acids. A correlation between increased resistance to acetic acid and the level of potassium in the growth medium was found. The activation of the Snf1p signalling pathway, involved in yeast response to glucose starvation, is demonstrated to occur in response to acetic acid stress but no evidence was obtained supporting the acetic acid-induced inhibition of glucose uptake. Conclusions Approximately 490 of the 650 determinants of tolerance to acetic acid identified in this work are implicated, for the first time, in tolerance to

  20. Ethylene-induced senescence-related gene expression requires protein synthesis

    International Nuclear Information System (INIS)

    Lawton, K.A.; Raghothama, K.G.; Woodson, W.R.

    1990-01-01

    We have investigated the effects of inhibiting protein synthesis on the ethylene-induced expression of 3 carnation senescence-related genes, pSR5, pSR8, and pSR12. Treatment of preclimacteric carnation petal discs with 1μg/ml of cycloheximide, a cytoplasmic protein synthesis inhibitor, for 3h inhibited protein synthesis by >80% as quantitated by the incorporation of [35S]methionine into protein. Pre-treatment of petal discs with cycloheximide prevented ethylene-induced SR transcript accumulation. Cycloheximide treatment of petal discs held in air did not result in increased levels of SR mRNA. These results indicate that ethylene does not interact with pre-formed factors but rather that the activation of SR gene expression by ethylene is mediated by labile protein factor(s) synthesized on cytoplasmic ribosomes. Experiments are currently underway to determine if cycloheximide exerts its effect at the transcriptional or post-transcriptional level

  1. Resistance to organic hydroperoxides requires ohr and ohrR genes in Sinorhizobium meliloti

    Directory of Open Access Journals (Sweden)

    Dufour Virginie

    2011-05-01

    Full Text Available Abstract Background Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Medicago sativa. During nodule formation bacteria have to withstand oxygen radicals produced by the plant. Resistance to H2O2 and superoxides has been extensively studied in S. meliloti. In contrast resistance to organic peroxides has not been investigated while S. meliloti genome encodes putative organic peroxidases. Organic peroxides are produced by plants and are highly toxic. The resistance to these oxygen radicals has been studied in various bacteria but never in plant nodulating bacteria. Results In this study we report the characterisation of organic hydroperoxide resistance gene ohr and its regulator ohrR in S. meliloti. The inactivation of ohr affects resistance to cumene and ter-butyl hydroperoxides but not to hydrogen peroxide or menadione in vitro. The expression of ohr and ohrR genes is specifically induced by organic peroxides. OhrR binds to the intergenic region between the divergent genes ohr and ohrR. Two binding sites were characterised. Binding to the operator is prevented by OhrR oxidation that promotes OhrR dimerisation. The inactivation of ohr did not affect symbiosis and nitrogen fixation, suggesting that redundant enzymatic activity exists in this strain. Both ohr and ohrR are expressed in nodules suggesting that they play a role during nitrogen fixation. Conclusions This report demonstrates the significant role Ohr and OhrR proteins play in bacterial stress resistance against organic peroxides in S. meliloti. The ohr and ohrR genes are expressed in nodule-inhabiting bacteroids suggesting a role during nodulation.

  2. Cloning of genes required for hypersensitivity and pathogenicity in Pseudomonas syringae pv. aptata.

    Science.gov (United States)

    Minardi, P

    1995-01-01

    A genomic library of Pseudomonas syringae pv. aptata strain NCPPB 2664, which causes bacterial blight of sugar beet, lettuce and other plants, was constructed in the cosmid vector pCPP31. The 13.4 kb EcoRI fragment of the cosmid pHIR11, containing the hrp (hypersensitive response and pathogenicity) gene cluster of the closely related bacterium Pseudomonas syringae pv. syringae strain 61, was used as a probe to identify a homologous hrp gene cluster in P. syringae pv. aptata. Thirty of 2500 cosmid clones, screened by colony hybridization, gave a strong hybridization signal with the probe, but none of these conferred to the non-pathogenic bacterium, Pseudomonas fluorescens, the ability to elicit the hypersensitive response (HR) in tobacco. Southern blot analysis of EcoRI-digested genomic DNA of P. syringae pv. aptata showed hybridizing bands of 12 kb and 4.4 kb. Only a 12 kb fragment hybridized in digests of the cosmids. Cosmid clone pCPP1069 was mutagenized with Tn10-minitet and marker-exchanged into the genome of P. syringae pv. aptata. Three resulting prototrophic mutant strains failed to elicit the HR in tobacco and to cause disease in lettuce. The DNA flanking the Tn10-minitet insertions from mutated derivatives of pCPP1069 hybridized with the 10.6 kb Bg/II fragment of pHIR11. These results indicate that P. syringae pv. aptata harbours hrp genes that are similar to, but arranged differently from, homologous hrp genes of P. syringae pv. syringae.

  3. PEP activity and expression of photosynthesis genes required for embryo and seed development in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Dmitry eKremnev

    2014-08-01

    Full Text Available Chloroplast biogenesis and function is essential for proper plant embryo and seed development but the molecular mechanisms underlying the role of plastids during embryogenesis are poorly understood. Expression of plastid encoded genes is dependent on two different transcription machineries; a plastid-encoded bacterial-type RNA polymerase (PEP and a nuclear-encoded phage-type RNA polymerase (NEP, which recognize distinct types of promoters. However, the division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear. We show here that PRIN2 and CSP41b, two proteins identified in plastid nucleoid preparations, are essential for proper plant embryo development. Using Co-IP assays and native PAGE we have shown a direct physical interaction between PRIN2 and CSP41b. Moreover, PRIN2 and CSP41b form a distinct protein complex in vitro that binds DNA. The prin2.2 and csp41b-2 single mutants displayed pale phenotypes, abnormal chloroplasts with reduced transcript levels of photosynthesis genes and defects in embryo development. The respective csp41b-2prin2.2 homo/heterozygote double mutants produced abnormal white colored ovules and shrunken seeds. Thus, the csp41b-2prin2.2 double mutant is embryo lethal. In silico analysis of available array data showed that a large number of genes traditionally classified as PEP dependent genes are transcribed during early embryo development from the pre-globular stage to the mature-green-stage. Taken together, our results suggest that PEP activity and consequently the switch from NEP to PEP activity, is essential during embryo development and that the PRIN2-CSP41b DNA binding protein complex possibly is important for full PEP activity during this process.

  4. Genome-Wide Search for Genes Required for Bifidobacterial Growth under Iron-Limitation

    Science.gov (United States)

    Lanigan, Noreen; Bottacini, Francesca; Casey, Pat G.; O'Connell Motherway, Mary; van Sinderen, Douwe

    2017-01-01

    Bacteria evolved over millennia in the presence of the vital micronutrient iron. Iron is involved in numerous processes within the cell and is essential for nearly all living organisms. The importance of iron to the survival of bacteria is obvious from the large variety of mechanisms by which iron may be acquired from the environment. Random mutagenesis and global gene expression profiling led to the identification of a number of genes, which are essential for Bifidobacterium breve UCC2003 survival under iron-restrictive conditions. These genes encode, among others, Fe-S cluster-associated proteins, a possible ferric iron reductase, a number of cell wall-associated proteins, and various DNA replication and repair proteins. In addition, our study identified several presumed iron uptake systems which were shown to be essential for B. breve UCC2003 growth under conditions of either ferric and/or ferrous iron chelation. Of these, two gene clusters encoding putative iron-uptake systems, bfeUO and sifABCDE, were further characterised, indicating that sifABCDE is involved in ferrous iron transport, while the bfeUO-encoded transport system imports both ferrous and ferric iron. Transcription studies showed that bfeUO and sifABCDE constitute two separate transcriptional units that are induced upon dipyridyl-mediated iron limitation. In the anaerobic gastrointestinal environment ferrous iron is presumed to be of most relevance, though a mutation in the sifABCDE cluster does not affect B. breve UCC2003's ability to colonise the gut of a murine model. PMID:28620359

  5. Genome-Wide Search for Genes Required for Bifidobacterial Growth under Iron-Limitation

    Directory of Open Access Journals (Sweden)

    Noreen Lanigan

    2017-05-01

    Full Text Available Bacteria evolved over millennia in the presence of the vital micronutrient iron. Iron is involved in numerous processes within the cell and is essential for nearly all living organisms. The importance of iron to the survival of bacteria is obvious from the large variety of mechanisms by which iron may be acquired from the environment. Random mutagenesis and global gene expression profiling led to the identification of a number of genes, which are essential for Bifidobacterium breve UCC2003 survival under iron-restrictive conditions. These genes encode, among others, Fe-S cluster-associated proteins, a possible ferric iron reductase, a number of cell wall-associated proteins, and various DNA replication and repair proteins. In addition, our study identified several presumed iron uptake systems which were shown to be essential for B. breve UCC2003 growth under conditions of either ferric and/or ferrous iron chelation. Of these, two gene clusters encoding putative iron-uptake systems, bfeUO and sifABCDE, were further characterised, indicating that sifABCDE is involved in ferrous iron transport, while the bfeUO-encoded transport system imports both ferrous and ferric iron. Transcription studies showed that bfeUO and sifABCDE constitute two separate transcriptional units that are induced upon dipyridyl-mediated iron limitation. In the anaerobic gastrointestinal environment ferrous iron is presumed to be of most relevance, though a mutation in the sifABCDE cluster does not affect B. breve UCC2003's ability to colonise the gut of a murine model.

  6. An ancient duplication of exon 5 in the Snap25 gene is required for complex neuronal development/function.

    Directory of Open Access Journals (Sweden)

    Jenny U Johansson

    2008-11-01

    Full Text Available Alternative splicing is an evolutionary innovation to create functionally diverse proteins from a limited number of genes. SNAP-25 plays a central role in neuroexocytosis by bridging synaptic vesicles to the plasma membrane during regulated exocytosis. The SNAP-25 polypeptide is encoded by a single copy gene, but in higher vertebrates a duplication of exon 5 has resulted in two mutually exclusive splice variants, SNAP-25a and SNAP-25b. To address a potential physiological difference between the two SNAP-25 proteins, we generated gene targeted SNAP-25b deficient mouse mutants by replacing the SNAP-25b specific exon with a second SNAP-25a equivalent. Elimination of SNAP-25b expression resulted in developmental defects, spontaneous seizures, and impaired short-term synaptic plasticity. In adult mutants, morphological changes in hippocampus and drastically altered neuropeptide expression were accompanied by severe impairment of spatial learning. We conclude that the ancient exon duplication in the Snap25 gene provides additional SNAP-25-function required for complex neuronal processes in higher eukaryotes.

  7. ato-Gal4 fly lines for gene function analysis: Eya is required in late progenitors for eye morphogenesis.

    Science.gov (United States)

    Yu, Linlin; Zhou, Qingxiang; Pignoni, Francesca

    2015-06-01

    The Gal4/UAS system is one of the most powerful tools for the study of cellular and developmental processes in Drosophila. Gal4 drivers can be used to induce targeted expression of dominant-negative and dominant-active proteins, histological markers, activity sensors, gene-specific dsRNAs, modulators of cell survival or proliferation, and other reagents. Here, we describe novel atonal-Gal4 lines that contain regions of the regulatory DNA of atonal, the proneural gene for photoreceptors, stretch receptors, auditory organ, and some olfactory sensilla. During neurogenesis, the atonal gene is expressed at a critical juncture, a time of transition from progenitor cell to developing neuron. Thus, these lines are particularly well suited for the study of the transcription factors and signaling molecules orchestrating this critical transition. To demonstrate their usefulness, we focus on two visual organs, the eye and the Bolwig. We demonstrate the induction of predicted eye phenotypes when expressing the dominant-negative EGF receptor or a dsRNA against Notch in the developing eye disc. In another example, we show the deletion of the Bolwig's organ using the proapoptotic factor Hid. Finally, we investigate the function of the eye specification factor Eyes absent or Eya in late retinal progenitors, shortly before they begin morphogenesis. We show that Eya is still required in these late progenitors to promote eye formation, and show failure to induce the target gene atonal and consequent lack of neuron formation. © 2015 Wiley Periodicals, Inc.

  8. Genes Required for Growth at High Hydrostatic Pressure in Escherichia coli K-12 Identified by Genome-Wide Screening

    Science.gov (United States)

    Black, S. Lucas; Dawson, Angela; Ward, F. Bruce; Allen, Rosalind J.

    2013-01-01

    Despite the fact that much of the global microbial biosphere is believed to exist in high pressure environments, the effects of hydrostatic pressure on microbial physiology remain poorly understood. We use a genome-wide screening approach, combined with a novel high-throughput high-pressure cell culture method, to investigate the effects of hydrostatic pressure on microbial physiology in vivo. The Keio collection of single-gene deletion mutants in Escherichia coli K-12 was screened for growth at a range of pressures from 0.1 MPa to 60 MPa. This led to the identification of 6 genes, rodZ, holC, priA, dnaT, dedD and tatC, whose products were required for growth at 30 MPa and a further 3 genes, tolB, rffT and iscS, whose products were required for growth at 40 MPa. Our results support the view that the effects of pressure on cell physiology are pleiotropic, with DNA replication, cell division, the cytoskeleton and cell envelope physiology all being potential failure points for cell physiology during growth at elevated pressure. PMID:24040140

  9. First isolation of Haemophilus parasuis and other NAD-dependent Pasteurellaceae of swine from European wild boars.

    Science.gov (United States)

    Olvera, A; Cerdà-Cuéllar, M; Mentaberre, G; Casas-Diaz, E; Lavin, S; Marco, I; Aragon, V

    2007-11-15

    Haemophilus parasuis is a colonizer of the upper respiratory tract of pigs and the etiological agent of Glässer's disease, which is characterized by a fibrinous polyserositis, meningitis and arthritis. Glässer's disease has never been reported in wild boar (Sus scrofa), although antibodies against H. parasuis have been detected. The goal of this study was to confirm the presence of this bacterium in wild boar by bacterial isolation and to compare the strains to H. parasuis from domesticated pigs. Therefore, nasal swabs from 42 hunted wild boars were processed for bacterial isolation and subsequent H. parasuis identification by specific PCR, biochemical tests and 16S rRNA gene sequencing. Two different strains of H. parasuis from two wild boars were isolated. These strains belonged to serotype 2 and were included by 16S rRNA gene sequencing and MLST analysis in a cluster with other H. parasuis strains of nasal origin from domestic pigs. During this study, Actinobacillus minor and Actinobacillus indolicus, which are NAD-dependent Pasteurellaceae closely related to H. parasuis, were also isolated. Our results indicate similarities in the respiratory microbiota of wild boars and domestic pigs, and although H. parasuis was isolated from wild boars, more studies are needed to determine if this could be a source of H. parasuis infection for domestic pigs.

  10. Genes Required for Free Phage Production are Essential for Pseudomonas aeruginosa Chronic Lung Infections.

    Science.gov (United States)

    Lemieux, Andrée-Ann; Jeukens, Julie; Kukavica-Ibrulj, Irena; Fothergill, Joanne L; Boyle, Brian; Laroche, Jérôme; Tucker, Nicholas P; Winstanley, Craig; Levesque, Roger C

    2016-02-01

    The opportunistic pathogen Pseudomonas aeruginosa causes chronic lung infection in patients with cystic fibrosis. The Liverpool Epidemic Strain LESB58 is highly resistant to antibiotics, transmissible, and associated with increased morbidity and mortality. Its genome contains 6 prophages and 5 genomic islands. We constructed a polymerase chain reaction (PCR)-based signature-tagged mutagenesis library of 9216 LESB58 mutants and screened the mutants in a rat model of chronic lung infection. A total of 162 mutants were identified as defective for in vivo maintenance, with 11 signature-tagged mutagenesis mutants having insertions in prophage and genomic island genes. Many of these mutants showed both diminished virulence and reduced phage production. Transcription profiling by quantitative PCR and RNA-Seq suggested that disruption of these prophages had a widespread trans-acting effect on the transcriptome. This study demonstrates that temperate phages play a pivotal role in the establishment of infection through modulation of bacterial host gene expression. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  11. Identification of new genes required for meiotic recombination in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Ajimura, M.; Lee, S.H.; Ogawa, H.

    1993-01-01

    Mutants defective in meiotic recombination were isolated from a disomic haploid strain of Saccharomyces cerevisiae by examining recombination within the leu2 and his4 heteroalleles located on chromosome III. The mutants were classified into two new complementation groups (MRE2 and MRE11) and eight previously identified groups, which include SPO11, HOP1, REC114, MRE4/MEK1 and genes in the RAD52 epistasis group. All of the mutants, in which the mutations in the new complementation groups are homozygous and diploid, can undergo premeiotic DNA synthesis and produce spores. The spores are, however, not viable. The mre2 and mre11 mutants produce viable spores in a spo13 background, in which meiosis I is bypassed, suggesting that these mutants are blocked at an early step in meiotic recombination. The mre2 mutant does not exhibit any unusual phenotype during mitosis and it is, thus, considered to have a mutation in a meiosis-specific gene. By contrast, the mre11 mutant is sensitive to damage to DNA by methyl methanesulfonate and exhibits a hyperrecombination phenotype in mitosis. Among six alleles of HOP1 that were isolated, an unusual pattern of intragenic complementation was observed

  12. glpx Gene in Mycobacterium tuberculosis Is Required for In Vitro Gluconeogenic Growth and In Vivo Survival.

    Directory of Open Access Journals (Sweden)

    Hiten J Gutka

    Full Text Available Several enzymes involved in central carbon metabolism and gluconeogenesis play a critical role in survival and pathogenesis of Mycobacterium tuberculosis (Mtb. The only known functional fructose 1,6-bisphosphatase (FBPase in Mtb is encoded by the glpX gene and belongs to the Class II sub-family of FBPase. We describe herein the generation of a ΔglpX strain using homologous recombination. Although the growth profile of ΔglpX is comparable to that of wild type Mtb when grown on the standard enrichment media, its growth is dysgonic with individual gluconeogenic substrates such as oleic acid, glycerol and acetate. In mice lung CFU titers of ΔglpX were 2-3 log10 lower than the wild-type Mtb strain. The results indicate that glpX gene encodes a functional FBPase and is essential for both in vitro and in vivo growth and survival of Mtb. Loss of glpX results in significant reduction of FBPase activity but not complete abolition. These findings verify that the glpX encoded FBPase II in Mtb can be a potential target for drug discovery.

  13. Changes in outer membrane proteins of nontypable Haemophilus influenzae in patients with chronic obstructive pulmonary disease

    NARCIS (Netherlands)

    Groeneveld, K.; van Alphen, L.; Eijk, P. P.; Jansen, H. M.; Zanen, H. C.

    1988-01-01

    Five individual colonies of Haemophilus influenzae were isolated from each of one to three cultures of sputum collected from 18 patients with chronic obstructive pulmonary disease (COPD). The isolates were studied to investigate whether the major outer membrane proteins (MOMPs) changed during

  14. Immunological characterization of conjugated Haemophilus influenzae type b vaccine failure in infants

    NARCIS (Netherlands)

    Breukels, M. A.; Spanjaard, L.; Sanders, L. A.; Rijkers, G. T.

    2001-01-01

    Infant vaccination with conjugated Haemophilus influenzae type b (Hib) vaccine is highly effective in protecting against invasive Hib infections, but vaccine failures do occur. Twenty-one vaccine failures are reported since the introduction of the Hib conjugate vaccine in The Netherlands. Of the 14

  15. Vaccine-Induced Waning of Haemophilus influenzae Empyema and Meningitis, Angola

    Science.gov (United States)

    Peltola, Heikki; Bernardino, Luis; Monteiro, Lurdes; Silvestre, Silvia da Conceição; Anjos, Elizabete; Cruzeiro, Manuel Leite; Pitkäranta, Anne; Roine, Irmeli

    2014-01-01

    In Angola during 2003–2012, we detected Haemophilus influenzae in 18% of 2,634 and 26% of 2,996 bacteriologically positive pleural or cerebrospinal fluid samples, respectively, from children. After vaccination launch in 2006, H. influenzae empyema declined by 83% and meningitis by 86%. Severe H. influenzae pneumonia and meningitis are preventable by vaccination. PMID:25340259

  16. In vitro capability of faropenem to select for resistant mutants of Streptococcus pneumoniae and Haemophilus influenzae.

    Science.gov (United States)

    Kosowska-Shick, Klaudia; Clark, Catherine; Credito, Kim; Dewasse, Bonifacio; Beachel, Linda; Ednie, Lois; Appelbaum, Peter C

    2008-02-01

    When tested against nine strains of pneumococci and six of Haemophilus influenzae of various resistotypes, faropenem failed to select for resistant mutants after 50 days of consecutive subculture in subinhibitory concentrations. Faropenem also yielded low rates of spontaneous mutations against all organisms of both species. By comparison, resistant clones were obtained with macrolides, ketolides, and quinolones.

  17. Paracytosis of Haemophilus influenzae through cell layers of NCI-H292 lung epithelial cells

    NARCIS (Netherlands)

    van Schilfgaarde, M.; van Alphen, L.; Eijk, P.; Everts, V.; Dankert, J.

    1995-01-01

    Haemophilus influenzae penetrates the respiratory epithelium during carriage and invasive disease, including respiratory tract infections. We developed an in vitro model system consisting of lung epithelial NCI-H292 cells on permeable supports to study the passage of H. influenzae through lung

  18. The quest for cross protective factors of Haemophilus parasuis using 2-D gel electrophoresis

    Science.gov (United States)

    In swine, Haemophilus parasuis (H. parasuis) infection causes polyserositis, arthritis, and meningitis. A range of virulent to nonvirulent strains exists between and within the 15 serovars. Because of this, the pathogenicity and subsequent protection from H. parasuis disease has yet to be elucidated...

  19. Antibody response to Haemophilus influenzae type b capsular polysaccharide conjugated to tetanus toxoid in preterm infants

    DEFF Research Database (Denmark)

    Kristensen, Kim; Gyhrs, A; Lausen, B

    1996-01-01

    OBJECTIVE: To evaluate the antibody response to a Haemophilus influenzae type b capsular polysaccharide (HibCP) tetanus toxoid (TT) conjugate vaccine (HibCP-TT) in preterm infants. SUBJECTS: Thirty-five healthy preterm infants with gestational ages (GA) from 27 to 36 weeks and birth weights from...

  20. Bioenergetics-based modeling of Plasmodium falciparum metabolism reveals its essential genes, nutritional requirements, and thermodynamic bottlenecks

    Science.gov (United States)

    Chiappino-Pepe, Anush; Ataman, Meriç

    2017-01-01

    Novel antimalarial therapies are urgently needed for the fight against drug-resistant parasites. The metabolism of malaria parasites in infected cells is an attractive source of drug targets but is rather complex. Computational methods can handle this complexity and allow integrative analyses of cell metabolism. In this study, we present a genome-scale metabolic model (iPfa) of the deadliest malaria parasite, Plasmodium falciparum, and its thermodynamics-based flux analysis (TFA). Using previous absolute concentration data of the intraerythrocytic parasite, we applied TFA to iPfa and predicted up to 63 essential genes and 26 essential pairs of genes. Of the 63 genes, 35 have been experimentally validated and reported in the literature, and 28 have not been experimentally tested and include previously hypothesized or novel predictions of essential metabolic capabilities. Without metabolomics data, four of the genes would have been incorrectly predicted to be non-essential. TFA also indicated that substrate channeling should exist in two metabolic pathways to ensure the thermodynamic feasibility of the flux. Finally, analysis of the metabolic capabilities of P. falciparum led to the identification of both the minimal nutritional requirements and the genes that can become indispensable upon substrate inaccessibility. This model provides novel insight into the metabolic needs and capabilities of the malaria parasite and highlights metabolites and pathways that should be measured and characterized to identify potential thermodynamic bottlenecks and substrate channeling. The hypotheses presented seek to guide experimental studies to facilitate a better understanding of the parasite metabolism and the identification of targets for more efficient intervention. PMID:28333921

  1. Developmentally-Regulated Excision of the SPβ Prophage Reconstitutes a Gene Required for Spore Envelope Maturation in Bacillus subtilis

    Science.gov (United States)

    Abe, Kimihiro; Kawano, Yuta; Iwamoto, Keito; Arai, Kenji; Maruyama, Yuki; Eichenberger, Patrick; Sato, Tsutomu

    2014-01-01

    Temperate phages infect bacteria by injecting their DNA into bacterial cells, where it becomes incorporated into the host genome as a prophage. In the genome of Bacillus subtilis 168, an active prophage, SPβ, is inserted into a polysaccharide synthesis gene, spsM. Here, we show that a rearrangement occurs during sporulation to reconstitute a functional composite spsM gene by precise excision of SPβ from the chromosome. SPβ excision requires a putative site-specific recombinase, SprA, and an accessory protein, SprB. A minimized SPβ, where all the SPβ genes were deleted, except sprA and sprB, retained the SPβ excision activity during sporulation, demonstrating that sprA and sprB are necessary and sufficient for the excision. While expression of sprA was observed during vegetative growth, sprB was induced during sporulation and upon mitomycin C treatment, which triggers the phage lytic cycle. We also demonstrated that overexpression of sprB (but not of sprA) resulted in SPβ prophage excision without triggering the lytic cycle. These results suggest that sprB is the factor that controls the timing of phage excision. Furthermore, we provide evidence that spsM is essential for the addition of polysaccharides to the spore envelope. The presence of polysaccharides on the spore surface renders the spore hydrophilic in water. This property may be beneficial in allowing spores to disperse in natural environments via water flow. A similar rearrangement occurs in Bacillus amyloliquefaciens FZB42, where a SPβ-like element is excised during sporulation to reconstitute a polysaccharide synthesis gene, suggesting that this type of gene rearrangement is common in spore-forming bacteria because it can be spread by phage infection. PMID:25299644

  2. In vivo functional requirement of the mouse Ifitm1 gene for germ cell development, interferon mediated immune response and somitogenesis.

    Directory of Open Access Journals (Sweden)

    Ingeborg Klymiuk

    Full Text Available The mammalian Interferon induced transmembrane protein 1 (Ifitm1 gene was originally identified as a member of a gene family highly inducible by type I and type II interferons. Based on expression analyses, it was suggested to be required for normal primordial germ cell migration. The knockdown of Ifitm1 in mouse embryos provided evidence for a role in somitogenesis. We generated the first targeted knockin allele of the Ifitm1 gene to systematically reassess all inferred functions. Sperm motility and the fertility of male and female mutant mice are as in wild type littermates. Embryonic somites and the adult vertebral column appear normal in homozygous Ifitm1 knockout mice, demonstrating that Ifitm1 is not essential for normal segmentation of the paraxial mesoderm. Proportions of leucocyte subsets, including granulocytes, monocytes, B-cells, T-cells, NK-cells, and NKT-cells, are unchanged in mutant mice. Based on a normal immune response to Listeria monocytogenes infection, there is no evidence for a dysfunction in downstream IFNγ signaling in Ifitm1 mutant mice. Expression from the Ifitm1 locus from E8.5 to E14.5 is highly dynamic. In contrast, in adult mice, Ifitm1 expression is highly restricted and strong in the bronchial epithelium. Intriguingly, IFITM1 is highly overexpressed in tumor epithelia cells of human squamous cell carcinomas and in adenocarcinomas of NSCLC patients. These analyses underline the general importance of targeted in vivo studies for the functional annotation of the mammalian genome. The first comprehensive description of the Ifitm1 expression pattern provides a rational basis for the further examination of Ifitm1 gene functions. Based on our data, the fact that IFITM1 can function as a negative regulator of cell proliferation, and because the gene maps to chromosome band 11p15.5, previously associated with NSCLC, it is likely that IFITM1 in man has a key role in tumor formation.

  3. Bioenergetics-based modeling of Plasmodium falciparum metabolism reveals its essential genes, nutritional requirements, and thermodynamic bottlenecks.

    Directory of Open Access Journals (Sweden)

    Anush Chiappino-Pepe

    2017-03-01

    Full Text Available Novel antimalarial therapies are urgently needed for the fight against drug-resistant parasites. The metabolism of malaria parasites in infected cells is an attractive source of drug targets but is rather complex. Computational methods can handle this complexity and allow integrative analyses of cell metabolism. In this study, we present a genome-scale metabolic model (iPfa of the deadliest malaria parasite, Plasmodium falciparum, and its thermodynamics-based flux analysis (TFA. Using previous absolute concentration data of the intraerythrocytic parasite, we applied TFA to iPfa and predicted up to 63 essential genes and 26 essential pairs of genes. Of the 63 genes, 35 have been experimentally validated and reported in the literature, and 28 have not been experimentally tested and include previously hypothesized or novel predictions of essential metabolic capabilities. Without metabolomics data, four of the genes would have been incorrectly predicted to be non-essential. TFA also indicated that substrate channeling should exist in two metabolic pathways to ensure the thermodynamic feasibility of the flux. Finally, analysis of the metabolic capabilities of P. falciparum led to the identification of both the minimal nutritional requirements and the genes that can become indispensable upon substrate inaccessibility. This model provides novel insight into the metabolic needs and capabilities of the malaria parasite and highlights metabolites and pathways that should be measured and characterized to identify potential thermodynamic bottlenecks and substrate channeling. The hypotheses presented seek to guide experimental studies to facilitate a better understanding of the parasite metabolism and the identification of targets for more efficient intervention.

  4. Sequence analysis and molecular characterization of genes required for the biosynthesis of type 1 capsular polysaccharide in Staphylococcus aureus.

    Science.gov (United States)

    Lin, W S; Cunneen, T; Lee, C Y

    1994-11-01

    We previously cloned a 19.4-kb DNA region containing a cluster of genes affecting type 1 capsule production from Staphylococcus aureus M. Subcloning experiments showed that these capsule (cap) genes are localized in a 14.6-kb region. Sequencing analysis of the 14.6-kb fragment revealed 13 open reading frames (ORFs). Using complementation tests, we have mapped a collection of Cap- mutations in 10 of the 13 ORFs, indicating that these 10 genes are involved in capsule biosynthesis. The requirement for the remaining three ORFs in the synthesis of the capsule was demonstrated by constructing site-specific mutations corresponding to each of the three ORFs. Using an Escherichia coli S30 in vitro transcription-translation system, we clearly identified 7 of the 13 proteins predicted from the ORFs. Homology search between the predicted proteins and those in the data bank showed very high homology (52.3% identity) between capL and vipA, moderate homology (29% identity) between capI and vipB, and limited homology (21.8% identity) between capM and vipC. The vipA, vipB, and vipC genes have been shown to be involved in the biosynthesis of Salmonella typhi Vi antigen, a homopolymer polysaccharide consisting of N-acetylgalactosamino uronic acid, which is also one of the components of the staphylococcal type 1 capsule. The homology between these sets of genes therefore suggests that capL, capI, and capM may be involved in the biosynthesis of amino sugar, N-acetylgalactosamino uronic acid. In addition, the search showed that CapG aligned well with the consensus sequence of a family of acetyltransferases from various prokaryotic organisms, suggesting that CapG may be an acetyltransferase. Using the isogenic Cap- and Cap+ strains constructed in this study, we have confirmed that type 1 capsule is an important virulence factor in a mouse lethality test.

  5. Drosophila convoluted/dALS is an essential gene required for tracheal tube morphogenesis and apical matrix organization.

    Science.gov (United States)

    Swanson, Lianna E; Yu, Marcus; Nelson, Kevin S; Laprise, Patrick; Tepass, Ulrich; Beitel, Greg J

    2009-04-01

    Insulin-like growth factors (IGFs) control cell and organism growth through evolutionarily conserved signaling pathways. The mammalian acid-labile subunit (ALS) is a secreted protein that complexes with IGFs to modulate their activity. Recent work has shown that a Drosophila homolog of ALS, dALS, can also complex with and modulate the activity of a Drosophila IGF. Here we report the first mutations in the gene encoding dALS. Unexpectedly, we find that these mutations are allelic to a previously described mutation in convoluted (conv), a gene required for epithelial morphogenesis. In conv mutants, the tubes of the Drosophila tracheal system become abnormally elongated without altering tracheal cell number. conv null mutations cause larval lethality, but do not disrupt several processes required for tracheal tube size control, including septate junction formation, deposition of a lumenal/apical extracellular matrix, and lumenal secretion of Vermiform and Serpentine, two putative matrix-modifying proteins. Clearance of lumenal matrix and subcellular localization of clathrin also appear normal in conv mutants. However, we show that Conv/dALS is required for the dynamic organization of the transient lumenal matrix and normal structure of the cuticle that lines the tracheal lumen. These and other data suggest that the Conv/dALS-dependent tube size control mechanism is distinct from other known processes involved in tracheal tube size regulation. Moreover, we present evidence indicating that Conv/dALS has a novel, IGF-signaling independent function in tracheal morphogenesis.

  6. Neuron-specific feeding RNAi in C. elegans and its use in a screen for essential genes required for GABA neuron function.

    Science.gov (United States)

    Firnhaber, Christopher; Hammarlund, Marc

    2013-11-01

    Forward genetic screens are important tools for exploring the genetic requirements for neuronal function. However, conventional forward screens often have difficulty identifying genes whose relevant functions are masked by pleiotropy. In particular, if loss of gene function results in sterility, lethality, or other severe pleiotropy, neuronal-specific functions cannot be readily analyzed. Here we describe a method in C. elegans for generating cell-specific knockdown in neurons using feeding RNAi and its application in a screen for the role of essential genes in GABAergic neurons. We combine manipulations that increase the sensitivity of select neurons to RNAi with manipulations that block RNAi in other cells. We produce animal strains in which feeding RNAi results in restricted gene knockdown in either GABA-, acetylcholine-, dopamine-, or glutamate-releasing neurons. In these strains, we observe neuron cell-type specific behavioral changes when we knock down genes required for these neurons to function, including genes encoding the basal neurotransmission machinery. These reagents enable high-throughput, cell-specific knockdown in the nervous system, facilitating rapid dissection of the site of gene action and screening for neuronal functions of essential genes. Using the GABA-specific RNAi strain, we screened 1,320 RNAi clones targeting essential genes on chromosomes I, II, and III for their effect on GABA neuron function. We identified 48 genes whose GABA cell-specific knockdown resulted in reduced GABA motor output. This screen extends our understanding of the genetic requirements for continued neuronal function in a mature organism.

  7. Genomic characterization of Haemophilus parasuis SH0165, a highly virulent strain of serovar 5 prevalent in China.

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    Zhuofei Xu

    Full Text Available Haemophilus parasuis can be either a commensal bacterium of the porcine respiratory tract or an opportunistic pathogen causing Glässer's disease, a severe systemic disease that has led to significant economical losses in the pig industry worldwide. We determined the complete genomic sequence of H. parasuis SH0165, a highly virulent strain of serovar 5, which was isolated from a hog pen in North China. The single circular chromosome was 2,269,156 base pairs in length and contained 2,031 protein-coding genes. Together with the full spectrum of genes detected by the analysis of metabolic pathways, we confirmed that H. parasuis generates ATP via both fermentation and respiration, and possesses an intact TCA cycle for anabolism. In addition to possessing the complete pathway essential for the biosynthesis of heme, this pathogen was also found to be well-equipped with different iron acquisition systems, such as the TonB system and ABC-type transport complexes, to overcome iron limitation during infection and persistence. We identified a number of genes encoding potential virulence factors, such as type IV fimbriae and surface polysaccharides. Analysis of the genome confirmed that H. parasuis is naturally competent, as genes related to DNA uptake are present. A nine-mer DNA uptake signal sequence (ACAAGCGGT, identical to that found in Actinobacillus pleuropneumoniae and Mannheimia haemolytica, followed by similar downstream motifs, was identified in the SH0165 genome. Genomic and phylogenetic comparisons with other Pasteurellaceae species further indicated that H. parasuis was closely related to another swine pathogenic bacteria A. pleuropneumoniae. The comprehensive genetic analysis presented here provides a foundation for future research on the metabolism, natural competence and virulence of H. parasuis.

  8. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  9. VID22 is required for transcriptional activation of the PSD2 gene in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Miyata, Non; Miyoshi, Takuya; Yamaguchi, Takanori; Nakazono, Toshimitsu; Tani, Motohiro; Kuge, Osamu

    2015-12-15

    Phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae is synthesized through decarboxylation of phosphatidylserine (PS), catalysed by PS decarboxylase 1 (Psd1p) and 2 (Psd2p) and the cytidine 5'-diphosphate (CDP)-ethanolamine (CDP-Etn) pathway. PSD1 null (psd1Δ) and PSD2 null (psd2Δ) mutants are viable in a synthetic minimal medium, but a psd1Δ psd2Δ double mutant exhibits Etn auxotrophy, which is incorporated into PE through the CDP-Etn pathway. We have previously shown that psd1Δ is synthetic lethal with deletion of VID22 (vid22Δ) [Kuroda et al. (2011) Mol. Microbiol. 80: , 248-265]. In the present study, we found that vid22Δ mutant exhibits Etn auxotrophy under PSD1-depressed conditions. Deletion of VID22 in wild-type and PSD1-depressed cells caused partial defects in PE formation through decarboxylation of PS. The enzyme activity of PS decarboxylase in an extract of vid22Δ cells was ∼70% of that in wild-type cells and similar to that in psd2Δ cells and the PS decarboxylase activity remaining in the PSD1-depressed cells became almost negligible with deletion of VID22. Thus, the vid22Δ mutation was suggested to cause a defect in the Psd2p activity. Furthermore, vid22Δ cells were shown to be defective in expression of the PSD2 gene tagged with 6×HA, the defect being ameliorated by replacement of the native promoter of the PSD2 gene with a CYC1 promoter. In addition, an α-galactosidase reporter assay revealed that the activity of the promoter of the PSD2 gene in vid22Δ cells was ∼5% of that in wild-type cells. These results showed that VID22 is required for transcriptional activation of the PSD2 gene. © 2015 Authors; published by Portland Press Limited.

  10. The milkweed pod1 gene encodes a KANADI protein that is required for abaxial/adaxial patterning in maize leaves.

    Science.gov (United States)

    Candela, Héctor; Johnston, Robyn; Gerhold, Abigail; Foster, Toshi; Hake, Sarah

    2008-08-01

    Leaf primordia initiate from the shoot apical meristem with inherent polarity; the adaxial side faces the meristem, while the abaxial side faces away from the meristem. Adaxial/abaxial polarity is thought to be necessary for laminar growth of leaves, as mutants lacking either adaxial or abaxial cell types often develop radially symmetric lateral organs. The milkweed pod1 (mwp1) mutant of maize (Zea mays) has adaxialized sectors in the sheath, the proximal part of the leaf. Ectopic leaf flaps develop where adaxial and abaxial cell types juxtapose. Ectopic expression of the HD-ZIPIII gene rolled leaf1 (rld1) correlates with the adaxialized regions. Cloning of mwp1 showed that it encodes a KANADI transcription factor. Double mutants of mwp1-R with a microRNA-resistant allele of rld1, Rld1-N1990, show a synergistic phenotype with polarity defects in sheath and blade and a failure to differentiate vascular and photosynthetic cell types in the adaxialized sectors. The sectored phenotype and timing of the defect suggest that mwp1 is required late in leaf development to maintain abaxial cell fate. The phenotype of mwp1; Rld1 double mutants shows that both genes are also required early in leaf development to delineate leaf margins as well as to initiate vascular and photosynthetic tissues.

  11. Asymmetric division and differential gene expression during a bacterial developmental program requires DivIVA.

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    Prahathees Eswaramoorthy

    2014-08-01

    Full Text Available Sporulation in the bacterium Bacillus subtilis is a developmental program in which a progenitor cell differentiates into two different cell types, the smaller of which eventually becomes a dormant cell called a spore. The process begins with an asymmetric cell division event, followed by the activation of a transcription factor, σF, specifically in the smaller cell. Here, we show that the structural protein DivIVA localizes to the polar septum during sporulation and is required for asymmetric division and the compartment-specific activation of σF. Both events are known to require a protein called SpoIIE, which also localizes to the polar septum. We show that DivIVA copurifies with SpoIIE and that DivIVA may anchor SpoIIE briefly to the assembling polar septum before SpoIIE is subsequently released into the forespore membrane and recaptured at the polar septum. Finally, using super-resolution microscopy, we demonstrate that DivIVA and SpoIIE ultimately display a biased localization on the side of the polar septum that faces the smaller compartment in which σF is activated.

  12. Nonencapsulated or nontypeable Haemophilus influenzae are more likely than their encapsulated or serotypeable counterparts to have mutations in their fucose operon.

    Science.gov (United States)

    Shuel, Michelle L; Karlowsky, Kathleen E; Law, Dennis K S; Tsang, Raymond S W

    2011-12-01

    Population biology of Haemophilus influenzae can be studied by multilocus sequence typing (MLST), and isolates are assigned sequence types (STs) based on nucleotide sequence variations in seven housekeeping genes, including fucK. However, the ST cannot be assigned if one of the housekeeping genes is absent or cannot be detected by the current protocol. Occasionally, strains of H. influenzae have been reported to lack the fucK gene. In this study, we examined the prevalence of this mutation among our collection of H. influenzae isolates. Of the 704 isolates studied, including 282 encapsulated and 422 nonencapsulated isolates, nine were not typeable by MLST owing to failure to detect the fucK gene. All nine fucK-negative isolates were nonencapsulated and belonged to various biotypes. DNA sequencing of the fucose operon region confirmed complete deletion of genes in the operon in seven of the nine isolates, while in the remaining two isolates, some of the genes were found intact or in parts. The significance of these findings is discussed.

  13. Identification of genes required for growth of Escherichia coli MG1655 at moderately low pH

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    Bram Vivijs

    2016-10-01

    Full Text Available The survival of some pathotypes of E. coli in very low pH environments like highly acidic foods and the stomach has been well documented and contributes to their success as foodborne pathogens. In contrast, the ability of E. coli to grow at moderately low pH has received less attention, although this property can be anticipated to be also very important for the safety of mildly acidic foods. Therefore, the objective of this study was to identify cellular functions required for growth of the non-pathogenic strain E. coli MG1655 at low pH. First, the role of the four E. coli amino acid decarboxylase systems, which are the major cellular mechanisms allowing extreme acid survival, was investigated using mutants defective in each of the systems. Only the lysine decarboxylase (CadA was required for low pH growth. Secondly, a screening of 8544 random transposon insertion mutants resulted in the identification of six genes affecting growth in LB broth acidified to pH 4.50 with HCl. Two of the genes, encoding the transcriptional regulator LeuO and the elongation factor P-β-lysine ligase EpmA, can be linked to CadA production. Two other genes, encoding the diadenosine tetraphosphatase ApaH and the tRNA modification GTPase MnmE, have been previously implicated in the bacterial response to stresses other than low pH. A fifth gene encodes the LPS heptosyltransferase WaaC, and its mutant has a deep rough colony phenotype, which has been linked to reduced acid tolerance in earlier work. Finally, tatC encodes a secA-independent protein translocase that exports a few dozen proteins and thus is likely to have a pleiotropic phenotype. For mnmE, apaH, epmA,and waaC, de novo in frame deletion and genetic complementation confirmed their role in low pH growth, and these deletion mutants were also affected in growth in apple juice and tomato juice. However, the mutants were not affected in survival in gastric simulation medium at pH 2.5, indicating that growth at

  14. Comparative supragenomic analyses among the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae Using a modification of the finite supragenome model

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    Yu Susan

    2011-04-01

    Full Text Available Abstract Background Staphylococcus aureus is associated with a spectrum of symbiotic relationships with its human host from carriage to sepsis and is frequently associated with nosocomial and community-acquired infections, thus the differential gene content among strains is of interest. Results We sequenced three clinical strains and combined these data with 13 publically available human isolates and one bovine strain for comparative genomic analyses. All genomes were annotated using RAST, and then their gene similarities and differences were delineated. Gene clustering yielded 3,155 orthologous gene clusters, of which 2,266 were core, 755 were distributed, and 134 were unique. Individual genomes contained between 2,524 and 2,648 genes. Gene-content comparisons among all possible S. aureus strain pairs (n = 136 revealed a mean difference of 296 genes and a maximum difference of 476 genes. We developed a revised version of our finite supragenome model to estimate the size of the S. aureus supragenome (3,221 genes, with 2,245 core genes, and compared it with those of Haemophilus influenzae and Streptococcus pneumoniae. There was excellent agreement between RAST's annotations and our CDS clustering procedure providing for high fidelity metabolomic subsystem analyses to extend our comparative genomic characterization of these strains. Conclusions Using a multi-species comparative supragenomic analysis enabled by an improved version of our finite supragenome model we provide data and an interpretation explaining the relatively larger core genome of S. aureus compared to other opportunistic nasopharyngeal pathogens. In addition, we provide independent validation for the efficiency and effectiveness of our orthologous gene clustering algorithm.

  15. Beta- Lactam Antibiotics Stimulate Biofilm Formation in Non-Typeable Haemophilus influenzae by Up-Regulating Carbohydrate Metabolism

    Science.gov (United States)

    Wu, Siva; Li, Xiaojin; Gunawardana, Manjula; Maguire, Kathleen; Guerrero-Given, Debbie; Schaudinn, Christoph; Wang, Charles; Baum, Marc M.; Webster, Paul

    2014-01-01

    Non-typeable Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth) stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10 µg/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended. PMID:25007395

  16. Beta- lactam antibiotics stimulate biofilm formation in non-typeable haemophilus influenzae by up-regulating carbohydrate metabolism.

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    Siva Wu

    Full Text Available Non-typeable Haemophilus influenzae (NTHi is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10 µg/mL of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended.

  17. Comparison of genes required for H2O2 resistance in Streptococcus gordonii and Streptococcus sanguinis

    Science.gov (United States)

    Xu, Yifan; Itzek, Andreas

    2014-01-01

    Hydrogen peroxide (H2O2) is produced by several members of the genus Streptococcus mainly through the pyruvate oxidase SpxB under aerobic growth conditions. The acute toxic nature of H2O2 raises the interesting question of how streptococci cope with intrinsically produced H2O2, which subsequently accumulates in the microenvironment and threatens the closely surrounding population. Here, we investigate the H2O2 susceptibility of oral Streptococcus gordonii and Streptococcus sanguinis and elucidate potential mechanisms of how they protect themselves from the deleterious effect of H2O2. Both organisms are considered primary colonizers and occupy the same intraoral niche making them potential targets for H2O2 produced by other species. We demonstrate that S. gordonii produces relatively more H2O2 and has a greater ability for resistance to H2O2 stress. Functional studies show that, unlike in Streptococcus pneumoniae, H2O2 resistance is not dependent on a functional SpxB and confirms the important role of the ferritin-like DNA-binding protein Dps. However, the observed increased H2O2 resistance of S. gordonii over S. sanguinis is likely to be caused by an oxidative stress protection machinery present even under anaerobic conditions, while S. sanguinis requires a longer period of time for adaptation. The ability to produce more H2O2 and be more resistant to H2O2 might aid S. gordonii in the competitive oral biofilm environment, since it is lower in abundance yet manages to survive quite efficiently in the oral biofilm. PMID:25280752

  18. Inhibitory effect of 1,2,4-triazole-ciprofloxacin hybrids on Haemophilus parainfluenzae and Haemophilus influenzae biofilm formation in vitro under stationary conditions.

    Science.gov (United States)

    Kosikowska, Urszula; Andrzejczuk, Sylwia; Plech, Tomasz; Malm, Anna

    2016-10-01

    Haemophilus parainfluenzae and Haemophilus influenzae, upper respiratory tract microbiota representatives, are able to colonize natural and artificial surfaces as biofilm. The aim of the present study was to assay the effect of ten 1,2,4-triazole-ciprofloxacin hybrids on planktonic or biofilm-forming haemophili cells in vitro under stationary conditions on the basis of MICs (minimal inhibitory concentrations) and MBICs (minimal biofilm inhibitory concentrations). In addition, anti-adhesive properties of these compounds were examined. The reference strains of H. parainfluenzae and H. influenzae were included. The broth microdilution microtiter plate (MTP) method with twofold dilution of the compounds, or ciprofloxacin (reference agent) in 96-well polystyrene microplates, was used. The optical density (OD) reading was made spectrophotometrically at a wavelength of 570 nm (OD570) both to measure bacterial growth and to detect biofilm-forming cells under the same conditions with 0.1% crystal violet. The following values of parameters were estimated for 1,2,4-triazole-ciprofloxacin hybrids - MIC = 0.03-15.63 mg/L, MBIC = 0.03-15.63 mg/L, MBIC/MIC = 0.125-8, depending on the compound, and for ciprofloxacin - MIC = 0.03-0.06 mg/L, MBIC = 0.03-0.12 mg/L, MBIC/MIC = 1-2. The observed strong anti-adhesive properties (95-100% inhibition) of the tested compounds were reversible during long-term incubation at subinhibitory concentrations. Thus, 1,2,4-triazole-ciprofloxacin hybrids may be considered as starting compounds for designing improved agents not only against planktonic but also against biofilm-forming Haemophilus spp. cells. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  19. Down-regulation of adipose tissue lipoprotein lipase during fasting requires that a gene, separate from the lipase gene, is switched on.

    Science.gov (United States)

    Bergö, Martin; Wu, Gengshu; Ruge, Toralph; Olivecrona, Thomas

    2002-04-05

    During short term fasting, lipoprotein lipase (LPL) activity in rat adipose tissue is rapidly down-regulated. This down-regulation occurs on a posttranslational level; it is not accompanied by changes in LPL mRNA or protein levels. The LPL activity can be restored within 4 h by refeeding. Previously, we showed that during fasting there is a shift in the distribution of lipase protein toward an inactive form with low heparin affinity. To study the nature of the regulatory mechanism, we determined the in vivo turnover of LPL activity, protein mass, and mRNA in rat adipose tissue. When protein synthesis was inhibited with cycloheximide, LPL activity and protein mass decreased rapidly and in parallel with half-lives of around 2 h, and the effect of refeeding was blocked. This indicates that maintaining high levels of LPL activity requires continuous synthesis of new enzyme protein. When transcription was inhibited by actinomycin, LPL mRNA decreased with half-lives of 13.3 and 16.8 h in the fed and fasted states, respectively, demonstrating slow turnover of the LPL transcript. Surprisingly, when actinomycin was given to fed rats, LPL activity was not down-regulated during fasting, indicating that actinomycin interferes with the transcription of a gene that blocks the activation of newly synthesized LPL protein. When actinomycin was given to fasted rats, LPL activity increased 4-fold within 6 h, even in the absence of refeeding. The same effect was seen with alpha-amanitin, another inhibitor of transcription. The response to actinomycin was much less pronounced in aging rats, which are obese and insulin-resistant. These data suggest a default state where LPL protein is synthesized on a relatively stable mRNA and is processed into its active form. During fasting, a gene is switched on whose product prevents the enzyme from becoming active even though synthesis of LPL protein continues unabated.

  20. The Role of α-Defensins 1–3 in Antimicrobial Protection Forming in Children with Recurrent Bronchitis Caused by Bacteria of the Genus Haemophilus

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    G.O. Lezhenko

    2013-03-01

    Full Text Available The level of α-defensins 1–3 (HNP 1–3 has been analyzed in the blood plasma of children with recurrent bronchitis caused by bacteria of the genus Haemophilus. It is shown that the level of HNP 1–3 in the blood plasma depends on the form of Haemophilus. Trigger of HNP 1–3 outflow for neutrophils was the presence of bacterial capsule while presence of L-forms of Haemophilus influenzae wasn’t associated with increase in synthesis of antimicrobial peptides that could be one of the factors of forming of Haemophilus antibiotic resistance.

  1. The GCR2 gene family is not required for ABA control of seed germination and early seedling development in Arabidopsis.

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    Jianjun Guo

    Full Text Available BACKGROUND: The plant hormone abscisic acid (ABA regulates diverse processes of plant growth and development. It has recently been proposed that GCR2 functions as a G-protein-coupled receptor (GPCR for ABA. However, the structural relationships and functionality of GCR2 have been challenged by several independent studies. A central question in this controversy is whether gcr2 mutants are insensitive to ABA, because gcr2 mutants were shown to display reduced sensitivity to ABA under one experimental condition (e.g. 22 degrees C, continuous white light with 150 micromol m(-2 s(-1 but were shown to display wild-type sensitivity under another slightly different condition (e.g. 23 degrees C, 14/10 hr photoperiod with 120 micromol m(-2 s(-1. It has been hypothesized that gcr2 appears only weakly insensitive to ABA because two other GCR2-like genes in Arabidopsis, GCL1 and GCL2, compensate for the loss of function of GCR2. PRINCIPAL FINDINGS: In order to test this hypothesis, we isolated a putative loss-of-function allele of GCL2, and then generated all possible combinations of mutations in each member of the GCR2 gene family. We found that all double mutants, including gcr2 gcl1, gcr2 gcl2, gcl1 gcl2, as well as the gcr2 gcl1 gcl2 triple mutant displayed wild-type sensitivity to ABA in seed germination and early seedling development assays, demonstrating that the GCR2 gene family is not required for ABA responses in these processes. CONCLUSION: These results provide compelling genetic evidence that GCR2 is unlikely to act as a receptor for ABA in the context of either seed germination or early seedling development.

  2. Immunization with the Haemophilus ducreyi hemoglobin receptor HgbA protects against infection in the swine model of chancroid.

    Science.gov (United States)

    Afonina, Galyna; Leduc, Isabelle; Nepluev, Igor; Jeter, Chrystina; Routh, Patty; Almond, Glen; Orndorff, Paul E; Hobbs, Marcia; Elkins, Christopher

    2006-04-01

    The etiologic agent of chancroid is Haemophilus ducreyi. To fulfill its obligate requirement for heme, H. ducreyi uses two TonB-dependent receptors: the hemoglobin receptor (HgbA) and a receptor for free heme (TdhA). Expression of HgbA is necessary for H. ducreyi to survive and initiate disease in a human model of chancroid. In this study, we used a swine model of H. ducreyi infection to demonstrate that an experimental HgbA vaccine efficiently prevents chancroid, as determined by several parameters. Histological sections of immunized animals lacked typical microscopic features of chancroid. All inoculated sites from mock-immunized pigs yielded viable H. ducreyi cells, whereas no viable H. ducreyi cells were recovered from inoculated sites of HgbA-immunized pigs. Antibodies from sera of HgbA-immunized animals bound to and initiated antibody-dependent bactericidal activity against homologous H. ducreyi strain 35000HP and heterologous strain CIP542 ATCC; however, an isogenic hgbA mutant of 35000HP was not killed, proving specificity. Anti-HgbA immunoglobulin G blocked hemoglobin binding to the HgbA receptor, suggesting a novel mechanism of protection through the limitation of heme/iron acquisition by H. ducreyi. Such a vaccine strategy might be applied to other bacterial pathogens with strict heme/iron requirements. Taken together, these data suggest continuing the development of an HgbA subunit vaccine to prevent chancroid.

  3. Dynein Heavy Chain, Encoded by Two Genes in Agaricomycetes, Is Required for Nuclear Migration in Schizophyllum commune.

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    Melanie Brunsch

    Full Text Available The white-rot fungus Schizophyllum commune (Agaricomycetes was used to study the cell biology of microtubular trafficking during mating interactions, when the two partners exchange nuclei, which are transported along microtubule tracks. For this transport activity, the motor protein dynein is required. In S. commune, the dynein heavy chain is encoded in two parts by two separate genes, dhc1 and dhc2. The N-terminal protein Dhc1 supplies the dimerization domain, while Dhc2 encodes the motor machinery and the microtubule binding domain. This split motor protein is unique to Basidiomycota, where three different sequence patterns suggest independent split events during evolution. To investigate the function of the dynein heavy chain, the gene dhc1 and the motor domain in dhc2 were deleted. Both resulting mutants were viable, but revealed phenotypes in hyphal growth morphology and mating behavior as well as in sexual development. Viability of strain Δdhc2 is due to the higher expression of kinesin-2 and kinesin-14, which was proven via RNA sequencing.

  4. Clinical and microbiological features of Haemophilus influenzae vulvovaginitis in young girls

    Science.gov (United States)

    Cox, R A; Slack, M P E

    2002-01-01

    Aims: To define the clinical and microbiological features of vulvovaginitis in prepubertal girls whose genital swabs yielded Haemophilus influenzae. Methods: Laboratory based study and retrospective collection of clinical data from the requesting doctors. Results: Thirty eight isolates of non-capsulate Haemophilus influenzae and one of H parainfluenzae were isolated from 32 girls aged 18 months to 11 years. No other pathogens, such as β haemolytic streptococci or yeasts, were present with H influenzae. The most common biotype was biotype II, comprising 57% of the 26 isolates biotyped. Six children had more than one episode of vulvovaginitis caused by H influenzae and a total of 14 children had recurrent vaginal symptoms. Conclusion: Children who have H influenzae vulvovaginitis are at risk of recurrent symptoms. Biotype II is the one most commonly associated with this condition. PMID:12461068

  5. Association of the multidrug resistance-1 gene single-nucleotide polymorphisms with the tacrolimus dose requirements in renal transplant recipients.

    Science.gov (United States)

    Anglicheau, Dany; Verstuyft, Céline; Laurent-Puig, Pierre; Becquemont, Laurent; Schlageter, Marie-Hélène; Cassinat, Bruno; Beaune, Philippe; Legendre, Christophe; Thervet, Eric

    2003-07-01

    The immunosuppressive drug tacrolimus, whose pharmacokinetic characteristics display large interindividual variations, is a substrate for P-glycoprotein (P-gp), the product of the multidrug resistance-1 (MDR1) gene. Some of the single nucleotide polymorphisms (SNP) of MDR1 reported correlated with the in vivo activity of P-gp. Because P-gp is known to control tacrolimus intestinal absorption, it was postulated that these polymorphisms are associated with tacrolimus pharmacokinetic variations in renal transplant recipients. The objective of this study was to evaluate in a retrospective study of 81 renal transplant recipients the effect on tacrolimus dosages and concentration/dose ratio of four frequent MDR1 SNP possibly associated with P-gp function (T-129C in exon 1b, 1236C>T in exon 12, 2677G>T,A in exon 21, and 3435C>T in exon 26). As in the general population, the SNP in exons 12, 21, and 26 were frequent (16, 17.3, and 22.2% for the variant homozygous genotype, respectively) and exhibited incomplete linkage disequilibrium. One month after tacrolimus introduction, exon 21 SNP correlated significantly with the daily tacrolimus dose (P < or = 0.05) and the concentration/dose ratio (P < or = 0.02). Tacrolimus dose requirements were 40% higher in homozygous than wild-type patients for this SNP. The concentration/dose ratio was 36% lower in the wild-type patients, suggesting that, for a given dose, their tacrolimus blood concentration is lower. Haplotype analysis substantiated these results and suggested that exons 26 and 21 SNP may be associated with tacrolimus dose requirements. Genotype monitoring of the MDR1 gene reliably predicts the optimal dose of tacrolimus in renal transplant recipients and may predict the initial daily dose needed by individual patients to obtain adequate immunosuppression.

  6. Elimination of hydrogen peroxide by Haemophilus somnus, a catalase-negative pathogen of cattle.

    OpenAIRE

    Sample, A K; Czuprynski, C J

    1991-01-01

    Haemophilus somnus is a catalase-negative, gram-negative pathogen of cattle which is refractory to killing by bovine neutrophils. In this report, we showed that H. somnus rapidly inhibited Luminol-dependent chemiluminescence of bovine neutrophils costimulated with opsonized zymosan or phorbol myristate acetate. We have postulated that this inhibition resulted in part from H. somnus preventing the accumulation of hydrogen peroxide (H2O2) during the oxidative burst. In support of this hypothesi...

  7. Invasive Haemophilus Influenzae Disease, Europe, 1996–2006

    Centers for Disease Control (CDC) Podcasts

    2010-03-15

    This podcast describes monitoring of Haemophilus influenzae disease in Europe from 1996 through 2006. CDC epidemiologist Stacey Martin discusses what researchers learned about the effect of vaccination on disease prevalence.  Created: 3/15/2010 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID); National Center for Immunization and Respiratory Diseases (NCIRD).   Date Released: 4/5/2010.

  8. Lower airway colonization and inflammatory response in COPD: a focus on Haemophilus influenzae

    Directory of Open Access Journals (Sweden)

    Finney LJ

    2014-10-01

    Full Text Available Lydia J Finney,1 Andrew Ritchie,1 Elizabeth Pollard,2 Sebastian L Johnston,1 Patrick Mallia1 1Airway Disease Infection Section, National Heart and Lung Institute, Imperial College, London, United Kingdom; 2King's College London, London, United Kingdom Abstract: Bacterial infection of the lower respiratory tract in chronic obstructive pulmonary disease (COPD patients is common both in stable patients and during acute exacerbations. The most frequent bacteria detected in COPD patients is Haemophilus influenzae, and it appears this organism is uniquely adapted to exploit immune deficiencies associated with COPD and to establish persistent infection in the lower respiratory tract. The presence of bacteria in the lower respiratory tract in stable COPD is termed colonization; however, there is increasing evidence that this is not an innocuous phenomenon but is associated with airway inflammation, increased symptoms, and increased risk for exacerbations. In this review, we discuss host immunity that offers protection against H. influenzae and how disturbance of these mechanisms, combined with pathogen mechanisms of immune evasion, promote persistence of H. influenzae in the lower airways in COPD. In addition, we examine the role of H. influenzae in COPD exacerbations, as well as interactions between H. influenzae and respiratory virus infections, and review the role of treatments and their effect on COPD outcomes. This review focuses predominantly on data derived from human studies but will refer to animal studies where they contribute to understanding the disease in humans. Keywords: chronic obstructive pulmonary disease, Haemophilus influenzae, nontypeable Haemophilus influenzae, respiratory viruses, vaccination

  9. Fallos vacunales a vacunas conjugadas de Streptococcus pneumoniae y Haemophilus influenzae tipo b

    Directory of Open Access Journals (Sweden)

    Gonzalo Angulo

    2016-11-01

    Full Text Available Haemophilus influenzae type b and Streptococcus pneumoniae are the main cause agents of otitis, pneumonia, sepsis and meningitis, affecting mainly children under 5 years. Conjugate vaccines for encapsulated germs have dramatically decreased, the various diseases caused by these germs. Despite the decrease in morbidity and mortality, vaccine failures were observed. Children who experienced vaccine failures to Haemophilus influenzae type b had associated comorbidities more frequently than the general population (prematurity, HIV, Down syndrome, tumors, etc.. Nevertheless, most of these children have no medical history or immunological disorders. There is no consensus on whether all patients with vaccine failures should be assessed immunologically and how. There are recommendations to indicate a booster dose to patients with certain comorbidities and patients experiencing vaccine failure even in the absence of theses. Of the vaccine preparations available for Haemophilus influenzae type b association with acellular Bordetella pertussis proved to be less immunogenic and is currently being discouraged. Streptococcus pneumoniae serotypes 6B and 19F are less immunogenics and explain most of the vaccine failures in some series.

  10. The Comparison of Haemophilus Influenza in the Throat of Healthy Infants with Different Feeding Methods

    Directory of Open Access Journals (Sweden)

    A Kazemi

    2004-06-01

    Full Text Available Background: Haemophilus influenza (HI is the most commonly found pathogenic bacteria in pediatric otitis media and lower respiratory tract infections. Bacterial attachment to pharyngeal cells and proliferation may be necessary for infection. In the presence of human milk, attachment of HI to pharyngeal cells and colonization may be inhibited. To evaluate the protecting role of breast milk, we investigated the incidence of HI isolated from the throat of healthy infants with different feeding methods. Methods: Between August 2002 and March 2003, 210 healthy infants (70 purely breast-fed, 70 purely formula-fed, 70 mixed-fed, aged 1-6 months were enrolled into the study and a throat culture was taken in all of them. The incidence of HI was evaluated using Haemophilus Test Agar Bose (HTAB plates. Results: The incidence of HI in purely breast-fed, mixed-fed and purely formula-fed infants was 2.9%, 42.9% and 75.7% respectively (P = 0.000. The mean age and weight of cases in the three groups were not statistically different. Conclusion: These data suggest that human milk protects the throat of healthy infants from HI colonization especially in purely breast-fed cases. Keywords: Breast milk, Haemophilus influenza, Throat culture

  11. Haemophilus parainfluenzae Mural Endocarditis: Case Report and Review of the Literature

    Directory of Open Access Journals (Sweden)

    Luca T. Giurgea

    2016-01-01

    Full Text Available Haemophilus parainfluenzae, which uncommonly causes endocarditis, has never been documented to cause mural involvement. A 62-year-old immunocompetent female without predisposing risk factors for endocarditis except for poor dentition presented with fever, emesis, and dysmetria. Echocardiography found a mass attached to the left ventricular wall with finger-like projections. Computed tomography showed evidence of embolic phenomena to the brain, kidneys, spleen, and colon. Cardiac MRI revealed involvement of the chordae tendineae of the anterior papillary muscles. Blood cultures grew Haemophilus parainfluenzae. The patient was treated successfully with ceftriaxone with resolution of symptoms, including neurologic deficits. After eleven days of antibiotics a worsening holosystolic murmur was discovered. Worsening mitral regurgitation on echocardiography was only found three weeks later. Nine weeks after presentation, intraoperative evaluation revealed chord rupture but no residual vegetation and mitral repair was performed. Four weeks after surgery, the patient was back to her baseline. This case illustrates the ability of Haemophilus parainfluenzae to form large mural vegetations with high propensity of embolization in otherwise normal cardiac tissue among patients with dental risk factors. It also underscores the importance of physical examination in establishing a diagnosis of endocarditis and monitoring for progression of disease.

  12. Structural requirements of the major protective antibody to Haemophilus influenzae type b

    DEFF Research Database (Denmark)

    Hougs, L; Juul, L; Svejgaard, A

    1999-01-01

    expressed as antigen-binding fragments (Fabs) in Escherichia coli, define amino acids involved in antigen binding and idiotype expression, and propose a three-dimensional structure for the variable domains. We found that canonical Fabs, unlike a noncanonical Fab, bound effectively to HibCP in the absence...

  13. The pbrB gene encodes a laccase required for DHN-melanin synthesis in conidia of Talaromyces (Penicillium) marneffei.

    Science.gov (United States)

    Sapmak, Ariya; Boyce, Kylie J; Andrianopoulos, Alex; Vanittanakom, Nongnuch

    2015-01-01

    Talaromyces marneffei (Basionym: Penicillium marneffei) is a significant opportunistic fungal pathogen in patients infected with human immunodeficiency virus in Southeast Asia. T. marneffei cells have been shown to become melanized in vivo. Melanins are pigment biopolymers which act as a non-specific protectant against various stressors and which play an important role during virulence in fungi. The synthesis of the two most commonly found melanins in fungi, the eumelanin DOPA-melanin and the allomelanin DHN-melanin, requires the action of laccase enzymes. The T. marneffei genome encodes a number of laccases and this study describes the characterization of one of these, pbrB, during growth and development. A strain carrying a PbrB-GFP fusion shows that pbrB is expressed at high levels during asexual development (conidiation) but not in cells growing vegetatively. The pbrB gene is required for the synthesis of DHN-melanin in conidia and when deleted results in brown pigmented conidia, in contrast to the green conidia of the wild type.

  14. Expression of urease by Haemophilus influenzae during human respiratory tract infection and role in survival in an acid environment

    Science.gov (United States)

    2011-01-01

    Background Nontypeable Haemophilus influenzae is a common cause of otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). Prior studies have shown that H. influenzae expresses abundant urease during growth in the middle ear of the chinchilla and in pooled human sputum, suggesting that expression of urease is important for colonization and infection in the hostile environments of the middle ear and in the airways in adults. Virtually nothing else is known about the urease of H. influenzae, which was characterized in the present study. Results Analysis by reverse transcriptase PCR revealed that the ure gene cluster is expressed as a single transcript. Knockout mutants of a urease structural gene (ureC) and of the entire ure operon demonstrated no detectable urease activity indicating that this operon is the only one encoding an active urease. The ure operon is present in all strains tested, including clinical isolates from otitis media and COPD. Urease activity decreased as nitrogen availability increased. To test the hypothesis that urease is expressed during human infection, purified recombinant urease C was used in ELISA with pre acquisition and post infection serum from adults with COPD who experienced infections caused by H. influenzae. A total of 28% of patients developed new antibodies following infection indicating that H. influenzae expresses urease during airway infection. Bacterial viability assays performed at varying pH indicate that urease mediates survival of H. influenzae in an acid environment. Conclusions The H. influenzae genome contains a single urease operon that mediates urease expression and that is present in all clinical isolates tested. Nitrogen availability is a determinant of urease expression. H. influenzae expresses urease during human respiratory tract infection and urease is a target of the human antibody response. Expression of urease enhances viability in an acid

  15. Erythroid Kruppel-like factor (EKLF) is recruited to the γ-globin gene promoter as a co-activator and is required for γ-globin gene induction by short-chain fatty acid derivatives

    Science.gov (United States)

    Perrine, Susan P.; Mankidy, Rishikesh; Boosalis, Michael S.; Bieker, James J.; Faller, Douglas V.

    2011-01-01

    Objectives The erythroid Kruppel-like factor (EKLF) is an essential transcription factor for β-type globin gene switching, and specifically activates transcription of the adult β-globin gene promoter. We sought to determine if EKLF is also required for activation of the γ-globin gene by short-chain fatty acid (SCFA) derivatives, which are now entering clinical trials. Methods The functional and physical interaction of EKLF and co-regulatory molecules with the endogenous human globin gene promoters was studied in primary human erythroid progenitors and cell lines, using chromatin immunoprecipitation (ChIP) assays and genetic manipulation of the levels of EKLF and co-regulators. Results and conclusions Knockdown of EKLF prevents SCFA-induced expression of the γ-globin promoter in a stably expressed μLCRβprRlucAγprFluc cassette, and prevents induction of the endogenous γ-globin gene in primary human erythroid progenitors. EKLF is actively recruited to endogenous γ-globin gene promoters after exposure of primary human erythroid progenitors, and murine hematopoietic cell lines, to SCFA derivatives. The core ATPase BRG1 subunit of the human SWI/WNF complex, a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure, is also required for γ-globin gene induction by SCFA derivatives. BRG1 is actively recruited to the endogenous γ-globin promoter of primary human erythroid progenitors by exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes. PMID:19220418

  16. The familial dysautonomia disease gene IKBKAP is required in the developing and adult mouse central nervous system

    Directory of Open Access Journals (Sweden)

    Marta Chaverra

    2017-05-01

    Full Text Available Hereditary sensory and autonomic neuropathies (HSANs are a genetically and clinically diverse group of disorders defined by peripheral nervous system (PNS dysfunction. HSAN type III, known as familial dysautonomia (FD, results from a single base mutation in the gene IKBKAP that encodes a scaffolding unit (ELP1 for a multi-subunit complex known as Elongator. Since mutations in other Elongator subunits (ELP2 to ELP4 are associated with central nervous system (CNS disorders, the goal of this study was to investigate a potential requirement for Ikbkap in the CNS of mice. The sensory and autonomic pathophysiology of FD is fatal, with the majority of patients dying by age 40. While signs and pathology of FD have been noted in the CNS, the clinical and research focus has been on the sensory and autonomic dysfunction, and no genetic model studies have investigated the requirement for Ikbkap in the CNS. Here, we report, using a novel mouse line in which Ikbkap is deleted solely in the nervous system, that not only is Ikbkap widely expressed in the embryonic and adult CNS, but its deletion perturbs both the development of cortical neurons and their survival in adulthood. Primary cilia in embryonic cortical apical progenitors and motile cilia in adult ependymal cells are reduced in number and disorganized. Furthermore, we report that, in the adult CNS, both autonomic and non-autonomic neuronal populations require Ikbkap for survival, including spinal motor and cortical neurons. In addition, the mice developed kyphoscoliosis, an FD hallmark, indicating its neuropathic etiology. Ultimately, these perturbations manifest in a developmental and progressive neurodegenerative condition that includes impairments in learning and memory. Collectively, these data reveal an essential function for Ikbkap that extends beyond the peripheral nervous system to CNS development and function. With the identification of discrete CNS cell types and structures that depend on

  17. Immunogenicity and effectiveness of Haemophilus influenzae type b conjugate vaccine in HIV infected and uninfected African children.

    Science.gov (United States)

    Madhi, Shabir A; Kuwanda, Locadiah; Saarinen, Leena; Cutland, Clare; Mothupi, Rosalia; Käyhty, Helena; Klugman, Keith P

    2005-12-01

    The quantitative (anti-Hib capsular polysaccharide antibody concentrations; anti-HibPS) and qualitative (bactericidal activity and avidity) aspects in immune responses to Haemophilus influenzae type b polyribosyl ribitol phospshate-CRM(197) conjugate vaccine (HibCV; HibTiter) were evaluated in 66 HIV infected children not receiving anti-retroviral therapy and 127 HIV uninfected children. Surveillance was conducted for invasive Hib disease in a cohort of 39,865 (approximately 6.4% of whom were HIV infected) children from March 1998 to June 2004. HIV infected children had lower anti-HibPS geometric mean antibody concentrations 1 month post-immunisation than HIV uninfected children (Por=1.0 microg/ml (RR 0.54; 95% CI 0.43-0.69). A lower proportion of HIV infected children than HIV uninfected children (RR 0.78; 95% CI 0.66-0.93) had measurable anti-Hib serum bactericidal activity (SBA) and the HibPS antibody concentration required for 50% killing of Hib bacteria was greater among HIV infected than HIV uninfected children (P=0.001). The estimated risk of HibCV failure was 35.1-fold greater (95% CI 14.6-84.6) amongst HIV infected than HIV uninfected children.

  18. RbsB (NTHI_0632) mediates quorum signal uptake in nontypeable Haemophilus influenzae strain 86-028NP.

    Science.gov (United States)

    Armbruster, Chelsie E; Pang, Bing; Murrah, Kyle; Juneau, Richard A; Perez, Antonia C; Weimer, Kristin E D; Swords, W Edward

    2011-11-01

    Nontypeable Haemophilus influenzae (NTHI) is a respiratory commensal and opportunistic pathogen, which persists within biofilms on airway mucosal surfaces. For many species, biofilm formation is impacted by quorum signalling. Our prior work shows that production of autoinducer-2 (AI-2) promotes biofilm development and persistence for NTHI 86-028NP. NTHI 86-028NP encodes an ABC transporter annotated as a ribose transport system that includes a protein (RbsB) with similarity to the Escherichia coli LsrB and Aggregatibacter actinomycetemcomitans RbsB proteins that bind AI-2. In this study, inactivation of rbsB significantly reduced uptake of AI-2 and the AI-2 precursor dihydroxypentanedione (DPD) by NTHI 86-028NP. Moreover, DPD uptake was not competitively inhibited by ribose or other pentose sugars. Transcript levels of rbsB increased in response to DPD and as bacteria approached stationary-phase growth. The NTHI 86-028NP rbsB mutant also formed biofilms with significantly reduced thickness and total biomass and reduced surface phosphorylcholine, similar to a luxS mutant. Infection studies revealed that loss of rbsB impaired bacterial persistence in the chinchilla middle ear, similar to our previous results with luxS mutants. Based on these data, we conclude that in NTHI 86-028NP, RbsB is a LuxS/AI-2 regulated protein that is required for uptake of and response to AI-2. © 2011 Blackwell Publishing Ltd.

  19. Capsule Typing of Haemophilus influenzae by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

    Science.gov (United States)

    Månsson, Viktor; Gilsdorf, Janet R; Kahlmeter, Gunnar; Kilian, Mogens; Kroll, J Simon; Riesbeck, Kristian; Resman, Fredrik

    2018-03-01

    Encapsulated Haemophilus influenzae strains belong to type-specific genetic lineages. Reliable capsule typing requires PCR, but a more efficient method would be useful. We evaluated capsule typing by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Isolates of all capsule types (a-f and nontypeable; n = 258) and isogenic capsule transformants (types a-d) were investigated. Principal component and biomarker analyses of mass spectra showed clustering, and mass peaks correlated with capsule type-specific genetic lineages. We used 31 selected isolates to construct a capsule typing database. Validation with the remaining isolates (n = 227) showed 100% sensitivity and 92.2% specificity for encapsulated strains (a-f; n = 61). Blinded validation of a supplemented database (n = 50) using clinical isolates (n = 126) showed 100% sensitivity and 100% specificity for encapsulated strains (b, e, and f; n = 28). MALDI-TOF mass spectrometry is an accurate method for capsule typing of H. influenzae.

  20. The Haemophilus ducreyi trimeric autotransporter adhesin DsrA protects against an experimental infection in the swine model of chancroid.

    Science.gov (United States)

    Fusco, William G; Choudhary, Neelima R; Routh, Patty A; Ventevogel, Melissa S; Smith, Valerie A; Koch, Gary G; Almond, Glen W; Orndorff, Paul E; Sempowski, Gregory D; Leduc, Isabelle

    2014-06-24

    Adherence of pathogens to cellular targets is required to initiate most infections. Defining strategies that interfere with adhesion is therefore important for the development of preventative measures against infectious diseases. As an adhesin to host extracellular matrix proteins and human keratinocytes, the trimeric autotransporter adhesin DsrA, a proven virulence factor of the Gram-negative bacterium Haemophilus ducreyi, is a potential target for vaccine development. A recombinant form of the N-terminal passenger domain of DsrA from H. ducreyi class I strain 35000HP, termed rNT-DsrAI, was tested as a vaccine immunogen in the experimental swine model of H. ducreyi infection. Viable homologous H. ducreyi was not recovered from any animal receiving four doses of rNT-DsrAI administered with Freund's adjuvant at two-week intervals. Control pigs receiving adjuvant only were all infected. All animals receiving the rNT-DsrAI vaccine developed antibody endpoint titers between 3.5 and 5 logs. All rNT-DsrAI antisera bound the surface of the two H. ducreyi strains used to challenge immunized pigs. Purified anti-rNT-DsrAI IgG partially blocked binding of fibrinogen at the surface of viable H. ducreyi. Overall, immunization with the passenger domain of the trimeric autotransporter adhesin DsrA accelerated clearance of H. ducreyi in experimental lesions, possibly by interfering with fibrinogen binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Frequency of Streptococcus pneumonia and Haemophilus influenza in acute exacerbation of chronic obstructive airway disease and their sensitivity to levofloxacin

    International Nuclear Information System (INIS)

    Furqan, S.; Paracha, S.A.U.

    2014-01-01

    Objective: To determine the frequency of Streptococcus pneumoniae and Haemophilus influenzae in acute exacerbation of chronic obstructive pulmonary disease and their sensitivity to levofloxacin. Methods: The cross-sectional study was conducted at the Department of Medicine, AbbasiShaheed Hospital, Karachi, between July 2009 and January 2010. Patients already diagnosed with chronic obstructive pulmonary disease and admitted with symptoms of acute exacerbation were included in the study and their sputum samples were sent for microbiological evaluation. SPSS 16 was used for statistical analysis. Results: Of the total 105 patients in the study, 90 (85.17%) were males. Overall mean age at presentation was 62+-10.2 years. S. pneumoniae was isolated from sputum culture of 33 (31.4%) patients, while 13 (12.4%) patients showed growth of H. influenzae. Out of the 33 sputum specimens of S. pneumoniae, 32 (97.0%) were sensitive to levofloxacin, while 1 (3.0%) was resistant. All the 13 isolates of H. influenzae were sensitive to levofloxacin. Conclusion: S. pneumoniae and H. influenzae are still the most prevalent organisms isolated in acute exacerbation of chronic obstructive pulmonary disease in our population. Levofloxacin is still considered a highly sensitive antibiotic against these common micro-organisms in our population, but S. pneumoniae has started developing resistance against levofloxacin. Therefore, intermittent surveillance regarding development of resistance pattern of common micro-organisms against commonly prescribed antibiotics is required. (author)

  2. The CUP-SHAPED COTYLEDON3 gene is required for boundary and shoot meristem formation in Arabidopsis

    DEFF Research Database (Denmark)

    Vroemen, Casper W; Mordhorst, Andreas P; Albrecht, Cathy

    2003-01-01

    From an enhancer trap screen for genes expressed in Arabidopsis embryos, we identified a gene expressed from the octant stage onward in the boundary between the two presumptive cotyledons and in a variety of postembryonic organ and meristem boundaries. This gene, CUP-SHAPED COTYLEDON3 (CUC3...

  3. Relation between enzyme-linked immunosorbent assay and radioimmunoassay for detection of antibodies to the capsular polysaccharide of Haemophilus influenzae type b

    International Nuclear Information System (INIS)

    Kristensen, K.; Weis Bentzon, M.

    1992-01-01

    The measurement of antibodies to the capsular polysaccharide (PRP) of Haemophilus influenzae type b (Hib) is important because vaccines inducing such antibodies are now available. We developed and evaluated an enzyme-linked immunosorbent assay (ELISA) for detection of these antibodies based on direct coating of the plates with tyraminated PRP. The assay fulfilled the requirements for parallel line assays; it was sensitive, specific, and reproducible with a coefficient of variation between days of 19%. Results from the ELISA were compared with results from radioimmunoassay and a correlation coefficient of 0.93 was found. Results obtained by the two methods were proportional and the relation was indepenedent of the antibody level. The relation between them was also unaffected by the contribution of different antibody isotypes, indicating that these were measured to the same extent by both methods. ELISA employing direct coating of the plates with tyraminated PRP represents a useful alternative for detection of antibodies when studying immunogenicity of Hib vaccines. (au)

  4. Role of RecA protein in untargeted UV mutagenesis of bacteriophage lambda: evidence for the requirement for the dinB gene

    International Nuclear Information System (INIS)

    Brotcorne-Lannoye, A.; Maenhaut-Michel, G.

    1986-01-01

    Untargeted UV mutagenesis of bacteriophage lambda--i.e., the increased recovery of lambda mutants when unirradiated lambda infects UV-irradiated Escherichia coli--is thought to be mediated by a transient decrease in DNA replication fidelity, generating mutations in the newly synthesized strands. Using the bacteriophage lambda cI857----lambda c mutation system, we provide evidence that the RecA protein, shown previously to be required for this mutagenic pathway, is no longer needed when the LexA protein is inactivated by mutation. We suggest that the error-prone DNA replication responsible for UV-induced untargeted mutagenesis is turned on by the presence of replication-blocking lesions in the host cell DNA and that the RecA protein is required only to derepress the relevant din gene(s). This is in contrast to mutagenesis of irradiated bacteria or irradiated phage lambda, in which activated RecA protein has a second role in mutagenesis in addition to the cleavage of the LexA protein. Among the tested din genes, the dinB gene product (in addition to the uvrA and uvrB gene products) was found to be required for untargeted mutagenesis of bacteriophage lambda. To our knowledge, a phenotype associated with the dinB gene has not been reported previously

  5. Turning off flagellum rotation requires the pleiotropic gene pleD: pleA, pleC, and pleD define two morphogenic pathways in Caulobacter crescentus.

    OpenAIRE

    Sommer, J M; Newton, A

    1989-01-01

    We have identified mutations in three pleiotropic genes, pleA, pleC, and pleD, that are required for differentiation in Caulobacter crescentus. pleA and pleC mutants were isolated in an extensive screen for strains defective in both motility and adsorption of polar bacteriophage phi CbK; using temperature-sensitive alleles, we determined the time at which the two genes act. pleA was required for a short period at 0.7 of the swarmer cell cycle for flagellum biosynthesis, whereas pleC was requi...

  6. Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation.

    Science.gov (United States)

    Yamanaka, Yuki; Winardhi, Ricksen S; Yamauchi, Erika; Nishiyama, So-Ichiro; Sowa, Yoshiyuki; Yan, Jie; Kawagishi, Ikuro; Ishihama, Akira; Yamamoto, Kaneyoshi

    2018-06-15

    The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Meningite por Haemophilus influenzae tipo b em cidades do estado do Paraná, Brasil Haemophilus influenzae type b meningitis in the state of Paraná, Brazil

    Directory of Open Access Journals (Sweden)

    Nádia S. Takemura

    2001-10-01

    Full Text Available OBJETIVO: no segundo semestre de 1996, os municípios de Londrina e Curitiba (Paraná iniciaram a vacinação contra Haemophilus influenzae b (Hib, aproximadamente trinta meses antes de sua introdução no Programa Nacional de Imunização. O presente trabalho objetivou avaliar a incidência da meningite por Hib, entre crianças, em Londrina, Curitiba e nos demais municípios do estado do Paraná, antes e após a introdução da vacina nessas duas cidades. MÉTODOS: foi realizado um estudo observacional retrospectivo de todos os casos de meningite por Hib, entre menores de 5 anos, diagnosticados pelo sistema de vigilância epidemiológica de Londrina e pela Secretaria de Estado da Saúde do Paraná, de 1992 a 1999. Taxas de incidência da meningite por Hib foram calculadas por 100.000 menores de cinco anos. RESULTADOS: comparando com o período anterior à vacinação, houve redução importante do coeficiente de incidência da meningite por Hib em Londrina, passando de 23,91, em 1996, para 2,79 por 100.000 menores de cinco anos, em 1999. Redução semelhante foi observada em Curitiba, enquanto nos demais municípios do Paraná, que não dispunham da vacina até meados de 1999, o coeficiente se manteve praticamente inalterado. CONCLUSÃO: a vacinação contra Hib foi efetiva na redução da incidência da meningite entre menores de cinco anos em Londrina e Curitiba. Para a manutenção dessa baixa incidência devem ser garantidas adequada cobertura vacinal e boa qualidade do serviço de vigilância epidemiológica.OBJECTIVE: during the second half of 1996, the municipalities of Londrina and Curitiba (State of Paraná, Brazil included Haemophilus influenzae type b (Hib vaccine into their routine vaccination regimen, approximately 30 months before its introduction into the National Immunization Program. The present study aimed at verifying the incidence of meningitis caused by Hib among children in Londrina, Curitiba, and in the remaining

  8. Vacinação contra Haemophilus influenzae tipo b: proteção a longo prazo Haemophilus influenzae type b vaccination: long term protection

    Directory of Open Access Journals (Sweden)

    Cristiana M. Nascimento-Carvalho

    2006-07-01

    Full Text Available OBJETIVO: Identificar as evidências sobre o impacto da vacina conjugada para Haemophilus influenzae tipo b (Hib na epidemiologia da doença invasiva por Hib. FONTE DOS DADOS: Pesquisa nas bases de dados do MEDLINE, LILACS, publicações técnicas de organizações internacionais, diretrizes nacionais e internacionais, nos últimos 15 anos (1991-2005, utilizando os seguintes unitermos: Haemophilus influenzae type b, immunization, impact, effectiveness. Foram incluídas as publicações que apresentaram informação para atender o objetivo deste artigo. Artigos publicados em período anterior ao da pesquisa e citados em referências dos artigos incluídos foram analisados quanto à apresentação de informação de interesse. SÍNTESE DOS DADOS: A introdução da vacina conjugada para Hib produziu grande declínio na incidência de casos de doença invasiva por Hib nos diversos países em que seu uso foi incorporado à rotina de vacinação das crianças. No entanto, o ressurgimento de casos com doença invasiva por Hib tem mobilizado vários investigadores na busca das possíveis explicações para esses eventos, bem como a identificação das medidas a serem implementadas para evitar o reaparecimento da doença. CONCLUSÕES: O uso da vacina conjugada para Hib em escala populacional tem sido extremamente efetivo. No entanto, mudanças no esquema vacinal poderão ser necessárias para a manutenção do controle da doença invasiva por Hib, frente ao atual cenário epidemiológico das infecções pelo Hib.OBJECTIVE: To identify evidence of the impact of Haemophilus influenzae type b (Hib conjugate vaccine on the epidemiology of invasive Hib disease. SOURCES OF DATA: This review was based on a search of MEDLINE, LILACS, technical reports, national and international guidelines (publications from 1991 to 2005. The keywords Haemophilus influenzae type b, immunization, impact and effectiveness, alone or in combination, were used to retrieve the

  9. Nucleolin is required for DNA methylation state and the expression of rRNA gene variants in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Frédéric Pontvianne

    2010-11-01

    Full Text Available In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1. Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre-rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

  10. Nitrogenase activity of Herbaspirillum seropedicae grown under low iron levels requires the products of nifXorf1 genes.

    Science.gov (United States)

    Klassen, Giseli; de Oliveira Pedrosa, Fábio; de Souza, Emanuel M; Yates, M Geoffrey; Rigo, Liu Un

    2003-07-29

    Herbaspirillum seropedicae strains mutated in the nifX or orf1 genes showed 90% or 50% reduction in nitrogenase activity under low levels of iron or molybdenum respectively. Mutations in nifX or orf1 genes did not affect nif gene expression since a nifH::lacZ fusion was fully active in both mutants. nifX and the contiguous gene orf1 are essential for maximum nitrogen fixation under iron limitation and are probably involved in synthesis of nitrogenase iron or iron-molybdenum clusters.

  11. RNA interference and retinoblastoma-related genes are required for repression of endogenous siRNA targets in Caenorhabditis elegans.

    Science.gov (United States)

    Grishok, Alla; Hoersch, Sebastian; Sharp, Phillip A

    2008-12-23

    In Caenorhabditis elegans, a vast number of endogenous short RNAs corresponding to thousands of genes have been discovered recently. This finding suggests that these short interfering RNAs (siRNAs) may contribute to regulation of many developmental and other signaling pathways in addition to silencing viruses and transposons. Here, we present a microarray analysis of gene expression in RNA interference (RNAi)-related mutants rde-4, zfp-1, and alg-1 and the retinoblastoma (Rb) mutant lin-35. We found that a component of Dicer complex RDE-4 and a chromatin-related zinc finger protein ZFP-1, not implicated in endogenous RNAi, regulate overlapping sets of genes. Notably, genes a) up-regulated in the rde-4 and zfp-1 mutants and b) up-regulated in the lin-35(Rb) mutant, but not the down-regulated genes are highly represented in the set of genes with corresponding endogenous siRNAs (endo-siRNAs). Our study suggests that endogenous siRNAs cooperate with chromatin factors, either C. elegans ortholog of acute lymphoblastic leukemia-1 (ALL-1)-fused gene from chromosome 10 (AF10), ZFP-1, or tumor suppressor Rb, to regulate overlapping sets of genes and predicts a large role for RNAi-based chromatin silencing in control of gene expression in C. elegans.

  12. Pollen Killer Gene S35 Function Requires Interaction with an Activator That Maps Close to S24, Another Pollen Killer Gene in Rice

    Directory of Open Access Journals (Sweden)

    Takahiko Kubo

    2016-05-01

    Full Text Available Pollen killer genes disable noncarrier pollens, and are responsible for male sterility and segregation distortion in hybrid populations of distantly related plant species. The genetic networks and the molecular mechanisms underlying the pollen killer system remain largely unknown. Two pollen killer genes, S24 and S35, have been found in an intersubspecific cross of Oryza sativa ssp. indica and japonica. The effect of S24 is counteracted by an unlinked locus EFS. Additionally, S35 has been proposed to interact with S24 to induce pollen sterility. These genetic interactions are suggestive of a single S24-centric genetic pathway (EFS–S24–S35 for the pollen killer system. To examine this hypothetical genetic pathway, the S35 and the S24 regions were further characterized and genetically dissected in this study. Our results indicated that S35 causes pollen sterility independently of both the EFS and S24 genes, but is dependent on a novel gene close to the S24 locus, named incentive for killing pollen (INK. We confirmed the phenotypic effect of the INK gene separately from the S24 gene, and identified the INK locus within an interval of less than 0.6 Mb on rice chromosome 5. This study characterized the genetic effect of the two independent genetic pathways of INK–S35 and EFS–S24 in indica–japonica hybrid progeny. Our results provide clear evidence that hybrid male sterility in rice is caused by several pollen killer networks with multiple factors positively and negatively regulating pollen killer genes.

  13. Pollen Killer Gene S35 Function Requires Interaction with an Activator That Maps Close to S24, Another Pollen Killer Gene in Rice.

    Science.gov (United States)

    Kubo, Takahiko; Yoshimura, Atsushi; Kurata, Nori

    2016-05-03

    Pollen killer genes disable noncarrier pollens, and are responsible for male sterility and segregation distortion in hybrid populations of distantly related plant species. The genetic networks and the molecular mechanisms underlying the pollen killer system remain largely unknown. Two pollen killer genes, S24 and S35, have been found in an intersubspecific cross of Oryza sativa ssp. indica and japonica The effect of S24 is counteracted by an unlinked locus EFS Additionally, S35 has been proposed to interact with S24 to induce pollen sterility. These genetic interactions are suggestive of a single S24-centric genetic pathway (EFS-S24-S35) for the pollen killer system. To examine this hypothetical genetic pathway, the S35 and the S24 regions were further characterized and genetically dissected in this study. Our results indicated that S35 causes pollen sterility independently of both the EFS and S24 genes, but is dependent on a novel gene close to the S24 locus, named incentive for killing pollen (INK). We confirmed the phenotypic effect of the INK gene separately from the S24 gene, and identified the INK locus within an interval of less than 0.6 Mb on rice chromosome 5. This study characterized the genetic effect of the two independent genetic pathways of INK-S35 and EFS-S24 in indica-japonica hybrid progeny. Our results provide clear evidence that hybrid male sterility in rice is caused by several pollen killer networks with multiple factors positively and negatively regulating pollen killer genes. Copyright © 2016 Kubo et al.

  14. Aislamiento de distintos serotipos de Haemophilus influenzae en muestras profundas de pacientes pediátricos Isolation of Haemophilus influenzae serotypes from sterile sites in sick children

    Directory of Open Access Journals (Sweden)

    B.M. Gatti

    2004-03-01

    Full Text Available Haemophilus influenzae (Hi es responsable de diversas enfermedades humanas como sepsis, meningitis, celulitis y osteoartritis. En este trabajo se investigó la recuperación de distintos serotipos de Hi en muestras profundas de pacientes pediátricos. Se estudiaron 179 aislamientos de 146 niños durante el periodo 1996-2002 en el Laboratorio de Microbiología del Hospital de Niños Superiora Sor María Ludovica, Argentina. La distribución de los serotipos fue la siguiente: 1 a, 112 b, 1 c,1 d, 4 e, 3 f y 24 no tipificables. A partir del establecimiento de la estrategia de vacunación universal anti Hi b en 1998 se observa una disminución notable del serotipo b y un aumento relativo de otros y no tipificables.Haemophilus influenzae (Hi is the causative agent of several human diseases such as sepsis, meningitis, celulitis, and osteoarthritis. We investigated the isolation of Hi serotypes from sterile sites in sick children. One hundred and seventy nine strains from 146 patients were studied, period 1996-2002, at the Microbiology Laboratory, Hospital de Niños Superiora Sor María Ludovica, Argentina. The serotype distribution was:1 a, 112 b,1 c,1 d, 4 e, 3 f y 24 no typable. Since the beginning of universal Hi b vaccination in 1998, we have observed the fast decrease of serotype b and a relative increase of other serotypes.

  15. Quantitation of antibody-secreting cells in the blood after vaccination with Haemophilus influenzae type b conjugate vaccine

    DEFF Research Database (Denmark)

    Barington, T; Heilmann, C; Andersen, V

    1990-01-01

    The human B-lymphocyte response to protein-conjugated polysaccharide antigens has not previously been studied at the cellular level. In order to do so, we developed and evaluated haemolytic plaque-forming cell assays detecting Haemophilus influenzae type b (Hib) capsular polysaccharide-specific a......The human B-lymphocyte response to protein-conjugated polysaccharide antigens has not previously been studied at the cellular level. In order to do so, we developed and evaluated haemolytic plaque-forming cell assays detecting Haemophilus influenzae type b (Hib) capsular polysaccharide...

  16. 'Haemophilus quentini' in the urethra of men complaining of urethritis symptoms.

    Science.gov (United States)

    Horie, Kengo; Ito, Shin; Hatazaki, Kyoko; Yasuda, Mitsuru; Nakano, Masahiro; Kawakami, Kyojiro; Fujita, Yasunori; Ito, Masafumi; Ezaki, Takayuki; Deguchi, Takashi

    2018-01-01

    We isolated a cryptic genospecies of Haemophilus influenzae referred to as 'Haemophilus quentini' in the urethra of 3 men complaining of urethritis symptoms. H. influenzae strains, which had been isolated from the urethra in 77 of 1518 men complaining of urethritis symptoms, identified by the conventional test, and stored, were re-cultured for this study. Sixty-seven strains surviving storage were screened by a PCR-based assay specific for the cryptic genital Haemophilus genospecies. Three strains (HI09003, HI11006, and HI14016) were screened by PCR and identified as 'H. quentini' by 16S rRNA sequencing. The men positive for HI09003 and HI11006 were diagnosed as having non-chlamydial non-gonococcal urethritis (NGU), and their demographic and clinical features were similar to those of NGU caused by other pathogens. The man positive for HI14016 was ultimately diagnosed as having condyloma acuminatum on the glans. The 3 strains of 'H. quentini' produced no β-lactamase and were susceptible to ampicillin and other antimicrobial agents, including cephalosporins, fluoroquinolones, tetracyclines, and macrolides, recommended for treatment for urethritis. 'H. quentini' would be an uncommon pathogen in men with urogenital infections. Based on the clinical features of the two patients with 'H. quentini'-positive NGU, it would be difficult to predict the presence of 'H. quentini' in the urethra. The 3 strains of 'H. quentini' were susceptible to a variety of antimicrobial agents. Further accumulation of data regarding 'H. quentini' infections is needed to characterize the pathogenic roles of this genospecies in urogenital infections and to establish appropriate management of 'H. quentini' infections. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  17. The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation.

    Directory of Open Access Journals (Sweden)

    Anders Friberg

    2015-05-01

    Full Text Available Epstein-Barr virus (EBV is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2 is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.

  18. Identification of genes required for secretion of the Francisella oxidative burst-inhibiting acid phosphatase AcpA

    Directory of Open Access Journals (Sweden)

    John S Gunn

    2016-04-01

    Full Text Available Francisella tularensis is a Tier 1 bioterror threat and the intracellular pathogen responsible for tularemia in humans and animals. Upon entry into the host, Francisella uses multiple mechanisms to evade killing. Our previous studies have shown that after entering its primary cellular host, the macrophage, Francisella immediately suppresses the oxidative burst by secreting a series of acid phosphatases including AcpA-B-C and HapA, thereby evading the innate immune response of the macrophage and enhancing survival and further infection. However, the mechanism of acid phosphatase secretion by Francisella is still unknown. In this study, we screened for genes required for AcpA secretion in Francisella. We initially demonstrated that the known secretion systems, the putative Francisella-pathogenicity island (FPI-encoded Type VI secretion system and the Type IV pili, do not secrete AcpA. Using random transposon mutagenesis in conjunction with ELISA, Western blotting and acid phosphatase enzymatic assays, a transposon library of 5450 mutants was screened for strains with a minimum 1.5-fold decrease in secreted (culture supernatant AcpA, but no defect in cytosolic AcpA. Three mutants with decreased supernatant AcpA were identified. The transposon insertion sites of these mutants were revealed by direct genomic sequencing or inverse-PCR and sequencing. One of these mutants has a severe defect in AcpA secretion (at least 85% decrease and is a predicted hypothetical inner membrane protein. Interestingly, this mutant also affected the secretion of the FPI-encoded protein, VgrG. Thus, this screen identified novel protein secretion factors involved in the subversion of host defenses.

  19. Requirement of a novel splicing variant of human histone deacetylase 6 for TGF-{beta}1-mediated gene activation

    Energy Technology Data Exchange (ETDEWEB)

    Zhuang, Yan [Department of Medicine, Tulane School of Medicine, New Orleans, LA 70112 (United States); Nguyen, Hong T. [Graduate Program in Biomedical Sciences, Tulane School of Medicine, New Orleans, LA 70112 (United States); Lasky, Joseph A. [Department of Medicine, Tulane School of Medicine, New Orleans, LA 70112 (United States); Cao, Subing [Graduate Program in Biomedical Sciences, Tulane School of Medicine, New Orleans, LA 70112 (United States); Li, Cui [Department of Medicine, Tulane School of Medicine, New Orleans, LA 70112 (United States); Xiangya Hospital, Central South University, Hunan 41008 (China); Hu, Jiyao; Guo, Xinyue; Burow, Matthew E. [Department of Medicine, Tulane School of Medicine, New Orleans, LA 70112 (United States); Shan, Bin, E-mail: bshan@tulane.edu [Department of Medicine, Tulane School of Medicine, New Orleans, LA 70112 (United States)

    2010-02-19

    Histone deacetylase 6 (HDAC6) belongs to the family of class IIb HDACs and predominantly deacetylates non-histone proteins in the cytoplasm via the C-terminal deacetylase domain of its two tandem deacetylase domains. HDAC6 modulates fundamental cellular processes via deacetylation of {alpha}-tubulin, cortactin, molecular chaperones, and other peptides. Our previous study indicates that HDAC6 mediates TGF-{beta}1-induced epithelial-mesenchymal transition (EMT) in A549 cells. In the current study, we identify a novel splicing variant of human HDAC6, hHDAC6p114. The hHDAC6p114 mRNA arises from incomplete splicing and encodes a truncated isoform of the hHDAC6p114 protein of 114 kDa when compared to the major isoform hHDAC6p131. The hHDAC6p114 protein lacks the first 152 amino acids from N-terminus in the hHDAC6p131 protein, which harbors a nuclear export signal peptide and 76 amino acids of the N-terminal deacetylase domain. hHDAC6p114 is intact in its deacetylase activity against {alpha}-tubulin. The expression hHDAC6p114 is elevated in a MCF-7 derivative that exhibits an EMT-like phenotype. Moreover, hHDAC6p114 is required for TGF-{beta}1-activated gene expression associated with EMT in A549 cells. Taken together, our results implicate that expression and function of hHDAC6p114 is differentially regulated when compared to hHDAC6p131.

  20. The fruitless gene is required for the proper formation of axonal tracts in the embryonic central nervous system of Drosophila

    NARCIS (Netherlands)

    Song, Ho-Juhn; Billeter, Jean-Christophe; Reynaud, Enrique; Carlo, Troy; Spana, Eric P; Perrimon, Norbert; Goodwin, Stephen F; Baker, Bruce S; Taylor, Barbara J

    2002-01-01

    The fruitless (fru) gene in Drosophila melanogaster is a multifunctional gene that has sex-specific functions in the regulation of male sexual behavior and sex-nonspecific functions affecting adult viability and external morphology. While much attention has focused on fru's sex-specific roles, less

  1. Antimicrobial susceptibility of Haemophilus parasuis and Histophilus somni from pigs and cattle in Denmark

    DEFF Research Database (Denmark)

    Aarestrup, Frank Møller; Seyfarth, Anne Mette; Angen, Øystein

    2004-01-01

    A total of 52 Haemophilus parasuis and 80 Histophilus somni isolates were tested for antimicrobial susceptibility by MIC-determinations. None of the isolates were resistant to ampicillin, ceftiofur, ciprofloxacin, erythromycin, florphenicol, penicillin, spectinomycin, tetracycline, tiamulin......, or tilmicosin. Two H. parasuis isolates were resistant to trimethoprim + sulfamethoxazole. Six H. parasuis isolates had reduced susceptibility (0.06-0.5 mug/ml) to ciprofloxacin and 10 reduced susceptibility to TMP + sulfamethoxazole (1-2 mug/ml). This study showed that Danish isolates of H. parasuis and H...

  2. Antimicrobial susceptibility and serotypes of Actinobacillus (Haemophilus) pleuropneumoniae recovered from Missouri swine.

    Science.gov (United States)

    Fales, W H; Morehouse, L G; Mittal, K R; Bean-Knudsen, C; Nelson, S L; Kintner, L D; Turk, J R; Turk, M A; Brown, T P; Shaw, D P

    1989-01-01

    The antimicrobial susceptibility of 73 Actinobacillus (Haemophilus) pleuropneumoniae isolates from swine in Missouri was determined with a microdilution minimal inhibitory concentration test system. Serotyping was accomplished by means of co-agglutination. Serotype 1 (39/73) and serotype 5 (30/73) were commonly found, whereas serotype 7 (4/73) was infrequently encountered. Most isolates (MIC90) were found susceptible to ampicillin (amoxicillin), cephalothin, penicillin, erythromycin, gentamicin, and kanamycin. Marked resistance was found with oxytetracycline, tylosin, and sulfadimethoxine. The data indicate that use of ampicillin (amoxicillin) or penicillin may correlate well with the favorable outcome of treatment.

  3. [Isolation of Haemophilus influenzae serotypes from deep sites in sick children].

    Science.gov (United States)

    Gatti, B M; Ramirez Gronda, G A; Etchevarría, M; Vescina, C M; Varea, A M; González Ayala, S E

    2004-01-01

    Haemophilus influenzae (Hi) is the causative agent of several human diseases such as sepsis, meningitis, celulitis, and osteoarthritis. We investigated the isolation of Hi serotypes from sterile sites in sick children. One hundred and seventy nine strains from 146 patients were studied, period 1996-2002, at the Microbiology Laboratory, Hospital de Niños Superiora Sor María Ludovica, Argentina. The serotype distribution was:1 a, 112 b,1 c,1 d, 4 e, 3 f y 24 no typable. Since the beginning of universal Hi b vaccination in 1998, we have observed the fast decrease of serotype b and a relative increase of other serotypes.

  4. Haemophilus parainfluenzae Endocarditis Associated With Maxillary Sinusitis and Complicated by Cerebral Emboli in a Young Man

    Directory of Open Access Journals (Sweden)

    Anthony E. Duzenli MD

    2017-04-01

    Full Text Available HACEK endocarditis is often difficult to diagnose given the slow-growing characteristics of the organisms involved. Haemophilus parainfluenzae, one of the HACEK organisms, is an uncommon cause of endocarditis. We describe a case of a previously healthy young man with H parainfluenzae endocarditis that was associated with maxillary sinusitis and severe systemic complications, including septic cerebral emboli and mitral valve perforation. Previously reported cases have also described a predilection for younger people, cardiac valve pathology, and a high prevalence of stroke.

  5. Clonal relationship of recent invasive Haemophilus influenzae serotype f isolates from Denmark and the United States

    DEFF Research Database (Denmark)

    Bruun, B; Gahrn-Hansen, B; Westh, H

    2004-01-01

    Surveillance performed after the introduction of general Haemophilus influenzae serotype b (Hib) vaccination in Denmark identified 13 cases of invasive bacteraemic H. influenzae serotype f (Hif) disease in adults over a period of 7 years. Bacteraemic respiratory tract infections accounted for 61...... sequences. Multilocus enzyme electrophoresis typing revealed that recent Danish and American isolates belonged to a single Hif clone, which may be undergoing expansion. The need for accurate serotyping of H. influenzae to enable reliable monitoring for Hib replacement by other capsular types is emphasized....

  6. The Schizosaccharomyces pombe map1 gene encodes an SRF/MCM1-related protein required for P-cell specific gene expression

    DEFF Research Database (Denmark)

    Nielsen, O; Friis, T; Kjaerulff, S

    1996-01-01

    Cells of Schizosaccharomyces pombe undergo mating and meiosis when starved for a nitrogen source. In this process a P and and M cell first mate to generate a diploid zygote, which subsequently enters meiosis and sporulates. The P mating type is controlled by the mat1-Pc gene at the mating type lo...... cerevisiae MCM1. The Mat1-Pc protein contains a motif characteristic for proteins that interact with MADS-box factors, suggesting that Mat-Pc and Map1 may form a heterodimer that activates the P-specific map3 gene....

  7. The "Novelty" Requirements for Gene and Genetically-Modified Organisms Inventions and the Potential Benefits of a Peer-to-Patent System

    NARCIS (Netherlands)

    Margoni, T.

    2012-01-01

    The paper focuses on the patentability requirements applicable to the case of biotechnological inventions (gene patents and other genetically modified organisms). The paper takes a comparative standpoint and analyzes North-American, European, and Japanese landscapes. Attention will be also paid to

  8. Growth hormone receptor C-terminal domains required for growth hormone-induced intracellular free Ca2+ oscillations and gene transcription

    DEFF Research Database (Denmark)

    Billestrup, N; Bouchelouche, P; Allevato, G

    1995-01-01

    of varying frequency and amplitude. GH-induced transcription of the serine protease inhibitor 2.1 gene required the same C-terminal 52-amino acid domain of the receptor as for Ca2+ signaling. Mutation of the four proline residues in the conserved box 1 region of the GHR, which is responsible for binding...

  9. Regulated expression of the human cytomegalovirus pp65 gene: Octamer sequence in the promoter is required for activation by viral gene products

    International Nuclear Information System (INIS)

    Depto, A.S.; Stenberg, R.M.

    1989-01-01

    To better understand the regulation of late gene expression in human cytomegalovirus (CMV)-infected cells, the authors examined expression of the gene that codes for the 65-kilodalton lower-matrix phosphoprotein (pp65). Analysis of RNA isolated at 72 h from cells infected with CMV Towne or ts66, a DNA-negative temperature-sensitive mutant, supported the fact that pp65 is expressed at low levels prior to viral DNA replication but maximally expressed after the initiation of viral DNA replication. To investigate promoter activation in a transient expression assay, the pp65 promoter was cloned into the indicator plasmid containing the gene for chloramphenicol acetyltransferase (CAT). Transfection of the promoter-CAT construct and subsequent superinfection with CMV resulted in activation of the promoter at early times after infection. Cotransfection with plasmids capable of expressing immediate-early (IE) proteins demonstrated that the promoter was activated by IE proteins and that both IE regions 1 and 2 were necessary. These studies suggest that interactions between IE proteins and this octamer sequence may be important for the regulation and expression of this CMV gene

  10. Grr1p is required for transcriptional induction of amino acid permease genes and proper transcriptional regulation of genes in carbon metabolism of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine; Regenberg, Birgitte; Nielsen, Jens

    2005-01-01

    and a grr1 Delta strain and adding citrulline in the exponential phase. Whole-genome transcription analyses were performed on samples from each cultivation, both immediately before and 30 min after citrulline addition. Transcriptional induction of the AAP genes AGP1, BAP2, BAP3, DIP5, GNP1 and TAT1 is fully...

  11. Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene vp3.

    Science.gov (United States)

    Iverson, Eric A; Goodman, David A; Gorchels, Madeline E; Stedman, Kenneth M

    2017-05-15

    Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae , where >90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1), which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all open reading frames. We demonstrate that almost half of the SSV1 genes are not essential for infectivity, and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology. IMPORTANCE Most of the putative genes in the spindle-shaped archaeal hyperthermophile fuselloviruses have no sequences that are clearly similar to characterized genes. In order to determine which of these SSV genes are important for function, we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known fuselloviruses, vp3 , allows the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of

  12. Rice Snl6, a cinnamoyl-CoA reductase-like gene family member, is required for NH1-mediated immunity to Xanthomonas oryzae pv. oryzae.

    Directory of Open Access Journals (Sweden)

    Rebecca S Bart

    2010-09-01

    Full Text Available Rice NH1 (NPR1 homolog 1 is a key mediator of innate immunity. In both plants and animals, the innate immune response is often accompanied by rapid cell death at the site of pathogen infection. Over-expression of NH1 in rice results in resistance to the bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo, constitutive expression of defense related genes and enhanced benzothiadiazole (BTH- mediated cell death. Here we describe a forward genetic screen that identified a suppressor of NH1-mediated lesion formation and resistance, snl6. Comparative genome hybridization and fine mapping rapidly identified the genomic location of the Snl6 gene. Snl6 is a member of the cinnamoyl-CoA reductase (CCR-like gene family. We show that Snl6 is required for NH1-mediated resistance to Xoo. Further, we show that Snl6 is required for pathogenesis-related gene expression. In contrast to previously described CCR family members, disruption of Snl6 does not result in an obvious morphologic phenotype. Snl6 mutants have reduced lignin content and increased sugar extractability, an important trait for the production of cellulosic biofuels. These results suggest the existence of a conserved group of CCR-like genes involved in the defense response, and with the potential to alter lignin content without affecting development.

  13. Integrated analysis of genetic variation and gene expression reveals novel variant for increased warfarin dose requirement in African Americans

    NARCIS (Netherlands)

    Hernandez, W.; Gamazon, E. R.; Aquino-Michaels, K.; Smithberger, E.; O'Brien, T. J.; Harralson, A. F.; Tuck, M.; Barbour, A.; Cavallari, L. H.; Perera, M. A.

    2017-01-01

    Essentials Genetic variants controlling gene regulation have not been explored in pharmacogenomics. We tested liver expression quantitative trait loci for association with warfarin dose response. A novel predictor for increased warfarin dose response in African Americans was identified. Precision

  14. Induction and requirement of gene expression in the anterior cingulate cortex and medial prefrontal cortex for the consolidation of inhibitory avoidance memory

    Directory of Open Access Journals (Sweden)

    Zhang Yue

    2011-01-01

    Full Text Available Abstract Background Memory consolidation is a process to stabilize short-term memory, generating long-term memory. A critical biochemical feature of memory consolidation is a requirement for gene expression. Previous studies have shown that fear memories are consolidated through the activation of gene expression in the amygdala and hippocampus, indicating essential roles of these brain regions in memory formation. However, it is still poorly understood whether gene expression in brain regions other than the amygdala/hippocampus is required for the consolidation of fear memory; however, several brain regions are known to play modulatory roles in fear memory formation. Results To further understand the mechanisms underlying the formation of fear memory, we first identified brain regions where gene expression is activated after learning inhibitory avoidance (IA by analyzing the expression of the immediately early genes c-fos and Arc as markers. Similarly with previous findings, the induction of c-fos and Arc expression was observed in the amygdala and hippocampus. Interestingly, we also observed the induction of c-fos and Arc expression in the medial prefrontal cortex (mPFC: prelimbic (PL and infralimbic (IL regions and Arc expression in the anterior cingulate cortex (ACC. We next examined the roles of these brain regions in the consolidation of IA memory. Consistent with previous findings, inhibiting protein synthesis in the hippocampus blocked the consolidation of IA memory. More importantly, inhibition in the mPFC or ACC also blocked the formation of IA memory. Conclusion Our observations indicated that the formation of IA memory requires gene expression in the ACC and mPFC as well as in the amygdala and hippocampus, suggesting essential roles of the ACC and mPFC in IA memory formation.

  15. Haemophilus parainfluenzae Strain ATCC 33392 Forms Biofilms In Vitro and during Experimental Otitis Media Infections.

    Science.gov (United States)

    Pang, Bing; Swords, W Edward

    2017-09-01

    Haemophilus parainfluenzae is a nutritionally fastidious, Gram-negative bacterium with an oropharyngeal/nasopharyngeal carriage niche that is associated with a range of opportunistic infections, including infectious endocarditis and otitis media (OM). These infections are often chronic/recurrent in nature and typically involve bacterial persistence within biofilm communities that are highly resistant to host clearance. This study addresses the primary hypothesis that H. parainfluenzae forms biofilm communities that are important determinants of persistence in vivo The results from in vitro biofilm studies confirmed that H. parainfluenzae formed biofilm communities within which the polymeric matrix was mainly composed of extracellular DNA and proteins. Using a chinchilla OM infection model, we demonstrated that H. parainfluenzae formed surface-associated biofilm communities containing bacterial and host components that included neutrophil extracellular trap (NET) structures and that the bacteria mainly persisted in these biofilm communities. We also used this model to examine the possible interaction between H. parainfluenzae and its close relative Haemophilus influenzae , which is also commonly carried within the same host environments and can cause OM. The results showed that coinfection with H. influenzae promoted clearance of H. parainfluenzae from biofilm communities during OM infection. The underlying mechanisms for bacterial persistence and biofilm formation by H. parainfluenzae and knowledge about the survival defects of H. parainfluenzae during coinfection with H. influenzae are topics for future work. Copyright © 2017 American Society for Microbiology.

  16. On the evolution of the sexually transmitted bacteria Haemophilus ducreyi and Klebsiella granulomatis.

    Science.gov (United States)

    Lagergård, Teresa; Bölin, Ingrid; Lindholm, Leif

    2011-08-01

    Haemophilus ducreyi and Klebsiella (Calymmatobacterium) granulomatis are sexually transmitted bacteria that cause characteristic, persisting ulceration on external genitals called chancroid and granuloma inguinale, respectively. Those ulcers are endemic in developing countries or exist, as does granuloma inguinale, only in some geographic "hot spots."H. ducreyi is placed in the genus Haemophilus (family Pasteurellacae); however, this phylogenetic position is not obvious. The multiple ways in which the bacterium may be adapted to its econiche through specialized nutrient acquisitions; defenses against the immune system; and virulence factors that increase attachment, fitness, and persistence within genital tissue are discussed below. The analysis of K. granulomatis phylogeny demonstrated a high degree of identity with other Klebsiella species, and the name K. granulomatis comb. nov. was proposed. Because of the difficulty in growing this bacterium on artificial media, its characteristics have not been sufficiently defined. More studies are needed to understand bacterial genetics related to the pathogenesis and evolution of K. granulomatis. © 2011 New York Academy of Sciences.

  17. The cytolethal distending toxin of Haemophilus ducreyi aggravates dermal lesions in a rabbit model of chancroid.

    Science.gov (United States)

    Wising, Catharina; Mölne, Lena; Jonsson, Ing-Marie; Ahlman, Karin; Lagergård, Teresa

    2005-05-01

    Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, produces a cytolethal distending toxin (HdCDT) that inhibits cultured cell proliferation, leading to cell death. A rabbit model of dermal infection was used to investigate the roles of H. ducreyi bacteria and HdCDT in the development, clinical appearance, and persistence of infection. A non-toxin producing H. ducreyi strain, and for comparison purposes a non-capsulated Haemophilus influenzae strain, were inoculated intradermally, with and without co-administration of purified HdCDT. Co-administration of HdCDT resulted in significant aggravation of H. ducreyi-induced inflammatory lesions, and development of ulcers in rabbit skin. Less pronounced inflammatory lesions and lack of epithelial eruption were observed after inoculation with H. influenzae. Histopathological sections of the H. ducreyi-induced lesions, in both the presence and absence of HdCDT, showed dense infiltrates of the same type inflammatory cells, with the exception of a prominent endothelial cell proliferation noted in sections from lesions caused by H. ducreyi and toxin. Signs of chronic inflammation with involvement of T cells, macrophages, eosinophils, and granuloma formation were observed after H. ducreyi inoculation both with and without toxin. In conclusion, H. ducreyi causes a pronounced, chronic inflammation with involvement of T cells and macrophages, and in combination with HdCDT production of ulcers in the rabbit model. These pathogenic mechanisms may promote the development and persistence of chancroid ulcers.

  18. The Lung Immune Response to Nontypeable Haemophilus influenzae (Lung Immunity to NTHi

    Directory of Open Access Journals (Sweden)

    Paul T. King

    2015-01-01

    Full Text Available Haemophilus influenzae is divided into typeable or nontypeable strains based on the presence or absence of a polysaccharide capsule. The typeable strains (such as type b are an important cause of systemic infection, whilst the nontypeable strains (designated as NTHi are predominantly respiratory mucosal pathogens. NTHi is present as part of the normal microbiome in the nasopharynx, from where it may spread down to the lower respiratory tract. In this context it is no longer a commensal and becomes an important respiratory pathogen associated with a range of common conditions including bronchitis, bronchiectasis, pneumonia, and particularly chronic obstructive pulmonary disease. NTHi induces a strong inflammatory response in the respiratory tract with activation of immune responses, which often fail to clear the bacteria from the lung. This results in recurrent/persistent infection and chronic inflammation with consequent lung pathology. This review will summarise the current literature about the lung immune response to nontypeable Haemophilus influenzae, a topic that has important implications for patient management.

  19. Structure of the N-terminal region of Haemophilus Influenzae HI0017: Implications for function

    Energy Technology Data Exchange (ETDEWEB)

    Yu Liping; Mack, Jamey; Hajduk, Phil; Fesik, Stephen W. [Abbott Laboratories, Pharmaceutical Discovery Division, D46Y, AP10/LL (United States)

    2001-06-15

    Haemophilus influenzae is a gram-negative pathogen that causes infections ranging from asymptomatic colonization of the human upper respiratory tract to serious invasive diseases such as meningitis. Although the genome of Haemophilus influenzae has been completely sequenced, the structure and function of many of these proteins are unknown. HI0017 is one of these uncharacterized proteins. Here we describe the three-dimensional solution structure of the N-terminal portion of HI0017 as determined by NMR spectroscopy. The structure consists of a five-stranded antiparallel {beta}-sheet and two short {alpha}-helices. It is similar to the C-terminal domain of Diphtheria toxin repressor (DtxR). The C-terminal portion of HI0017 has an amino acid sequence that closely resembles pyruvate formate-lyase - an enzyme that converts pyruvate and CoA into acetyl-CoA and formate by a radical mechanism. Based on structural and sequence comparisons, we propose that the C-terminus of HI0017 functions as an enzyme with a glycyl radical mechanism, while the N-terminus participates in protein/protein interactions involving an activase (iron-sulfur protein) and/or the substrate.

  20. Structure of the N-terminal region of Haemophilus Influenzae HI0017: Implications for function

    International Nuclear Information System (INIS)

    Yu Liping; Mack, Jamey; Hajduk, Phil; Fesik, Stephen W.

    2001-01-01

    Haemophilus influenzae is a gram-negative pathogen that causes infections ranging from asymptomatic colonization of the human upper respiratory tract to serious invasive diseases such as meningitis. Although the genome of Haemophilus influenzae has been completely sequenced, the structure and function of many of these proteins are unknown. HI0017 is one of these uncharacterized proteins. Here we describe the three-dimensional solution structure of the N-terminal portion of HI0017 as determined by NMR spectroscopy. The structure consists of a five-stranded antiparallel β-sheet and two short α-helices. It is similar to the C-terminal domain of Diphtheria toxin repressor (DtxR). The C-terminal portion of HI0017 has an amino acid sequence that closely resembles pyruvate formate-lyase - an enzyme that converts pyruvate and CoA into acetyl-CoA and formate by a radical mechanism. Based on structural and sequence comparisons, we propose that the C-terminus of HI0017 functions as an enzyme with a glycyl radical mechanism, while the N-terminus participates in protein/protein interactions involving an activase (iron-sulfur protein) and/or the substrate

  1. The Acinetobacter baumannii Two-Component System AdeRS Regulates Genes Required for Multidrug Efflux, Biofilm Formation, and Virulence in a Strain-Specific Manner

    Directory of Open Access Journals (Sweden)

    Grace E. Richmond

    2016-04-01

    Full Text Available The opportunistic pathogen Acinetobacter baumannii is able to persist in the environment and is often multidrug resistant (MDR, causing difficulties in the treatment of infections. Here, we show that the two-component system AdeRS, which regulates the production of the AdeABC multidrug resistance efflux pump, is required for the formation of a protective biofilm in an ex vivo porcine mucosal model, which mimics a natural infection of the human epithelium. Interestingly, deletion of adeB impacted only on the ability of strain AYE to form a biofilm on plastic and only on the virulence of strain Singapore 1 for Galleria mellonella. RNA-Seq revealed that loss of AdeRS or AdeB significantly altered the transcriptional landscape, resulting in the changed expression of many genes, notably those associated with antimicrobial resistance and virulence interactions. For example, A. baumannii lacking AdeRS displayed decreased expression of adeABC, pil genes, com genes, and a pgaC-like gene, whereas loss of AdeB resulted in increased expression of pil and com genes and decreased expression of ferric acinetobactin transport system genes. These data define the scope of AdeRS-mediated regulation, show that changes in the production of AdeABC mediate important phenotypes controlled by AdeRS, and suggest that AdeABC is a viable target for antimicrobial drug and antibiofilm discovery.

  2. Relationship between secretion of the Anton blood group antigen in saliva and adherence of Haemophilus influenzae to oropharynx epithelial cells

    NARCIS (Netherlands)

    van Alphen, L.; van Ham, M.; Geelen-van den Broek, L.; Pieters, T.

    1989-01-01

    Inhibition of adherence of bacteria to epithelial cells contributes to a reduction of infections by these bacteria. We have shown that the Anton blood group antigen, the erythrocyte receptor for Haemophilus influenzae (van Alphen et al. 1986, FEMS Microbiol. Lett. 37, 69-71), occurs in saliva, that

  3. Risk of Febrile Seizures and Epilepsy After Vaccination With Diphtheria, Tetanus, Acellular Pertussis, Inactivated Poliovirus, and Haemophilus Influenzae Type b

    DEFF Research Database (Denmark)

    Sun, Yuelian; Christensen, Jakob Christensen; Hviid, Anders

    2012-01-01

    -acellular pertussis–inactivated poliovirus– Haemophilus influenzae type b (DTaP-IPV-Hib) vaccine since September 2002. Objective To estimate the risk of febrile seizures and epilepsy after DTaP-IPV-Hib vaccination given at 3, 5, and 12 months. Design, Setting, and Participants A population-based cohort study of 378...

  4. Identification of a group of Haemophilus influenzae penicillin-binding proteins that may have complementary physiological roles

    International Nuclear Information System (INIS)

    Malouin, F.; Parr, T.R. Jr.; Bryan, L.E.

    1990-01-01

    [35S]penicillin bound to different Haemophilus influenzae proteins in assays performed at 20, 37, or 42 degrees C. Penicillin-binding proteins 3a, 3b, 4, and 4' formed a group characterized by their affinity for moxalactam, cefotaxime, and piperacillin. Penicillin-binding protein 4' showed specific properties that may reflect its complementary role in septation

  5. In Vitro Capability of Faropenem To Select for Resistant Mutants of Streptococcus pneumoniae and Haemophilus influenzae▿ †

    Science.gov (United States)

    Kosowska-Shick, Klaudia; Clark, Catherine; Credito, Kim; Dewasse, Bonifacio; Beachel, Linda; Ednie, Lois; Appelbaum, Peter C.

    2008-01-01

    When tested against nine strains of pneumococci and six of Haemophilus influenzae of various resistotypes, faropenem failed to select for resistant mutants after 50 days of consecutive subculture in subinhibitory concentrations. Faropenem also yielded low rates of spontaneous mutations against all organisms of both species. By comparison, resistant clones were obtained with macrolides, ketolides, and quinolones. PMID:18086853

  6. Influence of prevaccination immunity on the human B-lymphocyte response to a Haemophilus influenzae type b conjugate vaccine

    DEFF Research Database (Denmark)

    Barington, T; Kristensen, K; Henrichsen, J

    1991-01-01

    of Haemophilus influenzae type b capsular polysaccharide (PRP) and diphtheria toxoid (DT), and the response was related to the prevaccination levels of PRP and DT antibodies. Positive correlations were found between increases in plasma PRP (median, 32.0 micrograms/ml) and DT (1.14 IU/ml) antibodies and numbers...

  7. Towards a sustainable, quality and affordable Haemophilus influenzae type b vaccine for every child in the world

    NARCIS (Netherlands)

    Hamidi, A.

    2016-01-01

    Haemophilus influenzae type b (Hib) conjugate vaccine is a safe and effective vaccine that can prevent meningitis and pneumonia caused by Hib disease. Hib vaccine is recommended for all children under 5 years. Despite the availability of safe and effective Hib vaccines since early 1987, Gambia was

  8. The KDM5 family is required for activation of pro-proliferative cell cycle genes during adipocyte differentiation

    DEFF Research Database (Denmark)

    Brier, Ann-Sofie B; Loft, Anne; Madsen, Jesper G S

    2017-01-01

    The KDM5 family of histone demethylases removes the H3K4 tri-methylation (H3K4me3) mark frequently found at promoter regions of actively transcribed genes and is therefore generally considered to contribute to corepression. In this study, we show that knockdown (KD) of all expressed members...... of the KDM5 family in white and brown preadipocytes leads to deregulated gene expression and blocks differentiation to mature adipocytes. KDM5 KD leads to a considerable increase in H3K4me3 at promoter regions; however, these changes in H3K4me3 have a limited effect on gene expression per se. By contrast......, genome-wide analyses demonstrate that KDM5A is strongly enriched at KDM5-activated promoters, which generally have high levels of H3K4me3 and are associated with highly expressed genes. We show that KDM5-activated genes include a large set of cell cycle regulators and that the KDM5s are necessary...

  9. The L locus, one of complementary genes required for anthocyanin production in onions (Allium cepa), encodes anthocyanidin synthase.

    Science.gov (United States)

    Kim, Sunggil; Jones, Rick; Yoo, Kil-Sun; Pike, Leonard M

    2005-06-01

    Bulb color in onions (Allium cepa) is an important trait, but its complex, unclear mechanism of inheritance has been a limiting factor in onion cultivar improvement. The identity of the L locus, which is involved in the color difference between Brazilian yellow and red onions, is revealed in this study. A cross was made between a US-type yellow breeding line and a Brazilian yellow cultivar. The segregation ratio of nine red to seven yellow onions in the F(2) population supports the involvement of two complementary genes in anthocyanin production in the F(1) hybrids. The high-performance liquid chromatography (HPLC) and reverse-transcriptase (RT)-PCR analysis of the Brazilian yellow onions indicated that the genes are involved late in the anthocyanin synthesis pathway. The genomic sequence of the anthocyanidin synthase (ANS) gene in Brazilian yellow onions showed a point mutation, which results in an amino acid change of a glycine to an arginine at residue 229. Because this residue is located adjacent to a highly conserved iron-binding active site, this mutation is likely responsible for the inactivation of the ANS gene in Brazilian yellow onions. Following the isolation of the promoter sequence of the mutant allele, a PCR-based marker for allelic selection of the ANS gene was designed. This assay is based on an insertion (larger than 3 kb) mutation. The marker perfectly co-segregated with the color phenotypes in the F(2) populations, thereby indicating that the L locus encodes ANS.

  10. The AtCAO gene, encoding chlorophyll a oxygenase, is required for chlorophyll b synthesis in Arabidopsis thaliana

    Science.gov (United States)

    Espineda, Cromwell E.; Linford, Alicia S.; Devine, Domenica; Brusslan, Judy A.

    1999-01-01

    Chlorophyll b is synthesized from chlorophyll a and is found in the light-harvesting complexes of prochlorophytes, green algae, and both nonvascular and vascular plants. We have used conserved motifs from the chlorophyll a oxygenase (CAO) gene from Chlamydomonas reinhardtii to isolate a homologue from Arabidopsis thaliana. This gene, AtCAO, is mutated in both leaky and null chlorina1 alleles, and DNA sequence changes cosegregate with the mutant phenotype. AtCAO mRNA levels are higher in three different mutants that have reduced levels of chlorophyll b, suggesting that plants that do not have sufficient chlorophyll b up-regulate AtCAO gene expression. Additionally, AtCAO mRNA levels decrease in plants that are grown under dim-light conditions. We have also found that the six major Lhcb proteins do not accumulate in the null ch1-3 allele. PMID:10468639

  11. Sexual and asexual oogenesis require the expression of unique and shared sets of genes in the insect Acyrthosiphon pisum

    Directory of Open Access Journals (Sweden)

    Gallot Aurore

    2012-02-01

    Full Text Available Abstract Background Although sexual reproduction is dominant within eukaryotes, asexual reproduction is widespread and has evolved independently as a derived trait in almost all major taxa. How asexuality evolved in sexual organisms is unclear. Aphids, such as Acyrthosiphon pisum, alternate between asexual and sexual reproductive means, as the production of parthenogenetic viviparous females or sexual oviparous females and males varies in response to seasonal photoperiodism. Consequently, sexual and asexual development in aphids can be analyzed simultaneously in genetically identical individuals. Results We compared the transcriptomes of aphid embryos in the stages of development during which the trajectory of oogenesis is determined for producing sexual or asexual gametes. This study design aimed at identifying genes involved in the onset of the divergent mechanisms that result in the sexual or asexual phenotype. We detected 33 genes that were differentially transcribed in sexual and asexual embryos. Functional annotation by gene ontology (GO showed a biological signature of oogenesis, cell cycle regulation, epigenetic regulation and RNA maturation. In situ hybridizations demonstrated that 16 of the differentially-transcribed genes were specifically expressed in germ cells and/or oocytes of asexual and/or sexual ovaries, and therefore may contribute to aphid oogenesis. We categorized these 16 genes by their transcription patterns in the two types of ovaries; they were: i expressed during sexual and asexual oogenesis; ii expressed during sexual and asexual oogenesis but with different localizations; or iii expressed only during sexual or asexual oogenesis. Conclusions Our results show that asexual and sexual oogenesis in aphids share common genetic programs but diverge by adapting specificities in their respective gene expression profiles in germ cells and oocytes.

  12. Hox genes require homothorax and extradenticle for body wall identity specification but not for appendage identity specification during metamorphosis of Tribolium castaneum.

    Science.gov (United States)

    Smith, Frank W; Jockusch, Elizabeth L

    2014-11-01

    The establishment of segment identity is a key developmental process that allows for divergence along the anteroposterior body axis in arthropods. In Drosophila, the identity of a segment is determined by the complement of Hox genes it expresses. In many contexts, Hox transcription factors require the protein products of extradenticle (exd) and homothorax (hth) as cofactors to perform their identity specification functions. In holometabolous insects, segment identity may be specified twice, during embryogenesis and metamorphosis. To glean insight into the relationship between embryonic and metamorphic segmental identity specification, we have compared these processes in the flour beetle Tribolium castaneum, which develops ventral appendages during embryogenesis that later metamorphose into adult appendages with distinct morphologies. At metamorphosis, comparisons of RNAi phenotypes indicate that Hox genes function jointly with Tc-hth and Tc-exd to specify several region-specific aspects of the adult body wall. On the other hand, Hox genes specify appendage identities along the anteroposterior axis independently of Tc-hth/Tc-exd and Tc-hth/Tc-exd specify proximal vs. distal identity within appendages independently of Hox genes during this stage. During embryogenesis, Tc-hth and Tc-exd play a broad role in the segmentation process and are required for specification of body wall identities in the thorax; however, contrasting with results from other species, we did not obtain homeotic transformations of embryonic appendages in response to Tc-hth or Tc-exd RNAi. In general, the homeotic effects of interference with the function of Hox genes and Tc-hth/Tc-exd during metamorphosis did not match predictions based on embryonic roles of these genes. Comparing metamorphic patterning in T. castaneum to embryonic and post-embryonic development in hemimetabolous insects suggests that holometabolous metamorphosis combines patterning processes of both late embryogenesis and

  13. A genetic screen for modifiers of UFO meristem activity identifies three novel FUSED FLORAL ORGANS genes required for early flower development in Arabidopsis.

    Science.gov (United States)

    Levin, J Z; Fletcher, J C; Chen, X; Meyerowitz, E M

    1998-06-01

    In a screen to identify novel genes required for early Arabidopsis flower development, we isolated four independent mutations that enhance the Ufo phenotype toward the production of filamentous structures in place of flowers. The mutants fall into three complementation groups, which we have termed FUSED FLORAL ORGANS (FFO) loci. ffo mutants have specific defects in floral organ separation and/or positioning; thus, the FFO genes identify components of a boundary formation mechanism(s) acting between developing floral organ primordia. FFO1 and FFO3 have specific functions in cauline leaf/stem separation and in first- and third-whorl floral organ separation, with FFO3 likely acting to establish and FFO1 to maintain floral organ boundaries. FFO2 acts at early floral stages to regulate floral organ number and positioning and to control organ separation within and between whorls. Plants doubly mutant for two ffo alleles display additive phenotypes, indicating that the FFO genes may act in separate pathways. Plants doubly mutant for an ffo gene and for ufo, lfy, or clv3 reveal that the FFO genes play roles related to those of UFO and LFY in floral meristem initiation and that FFO2 and FFO3 may act to control cell proliferation late in inflorescence development.

  14. Identification of a cis-regulatory region of a gene in Arabidopsis thaliana whose induction by dehydration is mediated by abscisic acid and requires protein synthesis.

    Science.gov (United States)

    Iwasaki, T; Yamaguchi-Shinozaki, K; Shinozaki, K

    1995-05-20

    In Arabidopsis thaliana, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA) but the gene does not include any sequence corresponding to the consensus ABA-responsive element (ABRE), RYACGTGGYR, in its promoter region. The cis-regulatory region of the rd22 promoter was identified by monitoring the expression of beta-glucuronidase (GUS) activity in leaves of transgenic tobacco plants transformed with chimeric gene fusions constructed between 5'-deleted promoters of rd22 and the coding region of the GUS reporter gene. A 67-bp nucleotide fragment corresponding to positions -207 to -141 of the rd22 promoter conferred responsiveness to dehydration and ABA on a non-responsive promoter. The 67-bp fragment contains the sequences of the recognition sites for some transcription factors, such as MYC, MYB, and GT-1. The fact that accumulation of rd22 mRNA requires protein synthesis raises the possibility that the expression of rd22 might be regulated by one of these trans-acting protein factors whose de novo synthesis is induced by dehydration or ABA. Although the structure of the RD22 protein is very similar to that of a non-storage seed protein, USP, of Vicia faba, the expression of the GUS gene driven by the rd22 promoter in non-stressed transgenic Arabidopsis plants was found mainly in flowers and bolted stems rather than in seeds.

  15. Requirements for chromatin reassembly during transcriptional downregulation of a heat shock gene in em>S. cerevisiaeem>

    DEFF Research Database (Denmark)

    Jensen, Mette Moesgaard; Christensen, Marianne Skovgaard; Bonven, Bjarne Juul

    2008-01-01

    Heat shock genes respond to moderate heat stress by a wave of transcription. The induction phase is accompanied by massive eviction of histones, which later reassemble with DNA during the ensuing phase of transcription downregulation. Here, we identify determinants of this reassembly throughout...

  16. The MP65 gene is required for cell wall integrity, adherence to epithelial cells and biofilm formation in Candida albicans

    Directory of Open Access Journals (Sweden)

    Girolamo Antonietta

    2011-05-01

    Full Text Available Abstract Background The MP65 gene of Candida albicans (orf19.1779 encodes a putative β-glucanase mannoprotein of 65 kDa, which plays a main role in a host-fungus relationship, morphogenesis and pathogenicity. In this study, we performed an extensive analysis of a mp65Δ mutant to assess the role of this protein in cell wall integrity, adherence to epithelial cells and biofilm formation. Results The mp65Δ mutant showed a high sensitivity to a range of cell wall-perturbing and degrading agents, especially Congo red, which induced morphological changes such as swelling, clumping and formation of hyphae. The mp65Δ mutant showed an activation of two MAPKs (Mkc1p and Cek1p, a high level of expression of two stress-related genes (DDR48 and SOD5, and a modulated expression of β-glucan epitopes, but no gross changes in cell wall polysaccharide composition. Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type. All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion. Conclusions We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus.

  17. The AVR2-SIX5 gene pair is required to activate I-2-mediated immunity in tomato

    NARCIS (Netherlands)

    Ma, L.; Houterman, P.M.; Gawehns, F.; Cao, L.; Sillo, F.; Richter, H.; Clavijo-Ortiz, M.J.; Schmidt, S.M.; Boeren, S.; Vervoort, J.; Cornelissen, B.J.C.; Rep, M.; Takken, F.L.W.

    2015-01-01

    Plant-invading microbes betray their presence to a plant by exposure of antigenic molecules such as small, secreted proteins called 'effectors'. In Fusarium oxysporum f. sp. lycopersici (Fol) we identified a pair of effector gene candidates, AVR2-SIX5, whose expression is controlled by a shared

  18. The AVR2–SIX5 gene pair is required to activate I-2-mediated immunity in tomato

    NARCIS (Netherlands)

    Ma, L.; Houterman, P.M.; Gawehns, F.; Cao, L.; Sillo, F.; Richter, H.; Clavijo-Ortiz, M.J.; Schmidt, S.M.; Boeren, J.A.; Vervoort, J.J.M.; Cornelissen, B.J.C.; Rep, M.; Takken, F.L.W.

    2015-01-01

    •Plant-invading microbes betray their presence to a plant by exposure of antigenic molecules such as small, secreted proteins called ‘effectors’. In Fusarium oxysporum f. sp. lycopersici (Fol) we identified a pair of effector gene candidates, AVR2-SIX5, whose expression is controlled by a shared

  19. Identification of Salmonella typhimurium Genes Required for Colonization of the Chicken Alimentary Tract and for Virulence in Newly Hatched Chicks

    Science.gov (United States)

    Turner, Arthur K.; Lovell, Margaret A.; Hulme, Scott D.; Zhang-Barber, Li; Barrow, Paul A.

    1998-01-01

    From a collection of 2,800 Tn5-TC1 transposon mutants of Salmonella typhimurium F98, 18 that showed reduced intestinal colonization of 3-week-old chicks were identified. The sites of transposon insertion were determined for most of the mutants and included insertions in the lipopolysaccharide biosynthesis genes rfaK, rfaY, rfbK, and rfbB and the genes dksA, clpB, hupA, and sipC. In addition, identification was made of an insertion into a novel gene that encodes a protein showing similarity to the IIC component of the mannose class of phosphoenolpyruvate-carbohydrate phosphotransferase systems, which we putatively called ptsC. Transduction of most of the transposon mutations to a fresh S. typhimurium F98 genetic background and construction of defined mutations in the rfbK, dksA, hupA, sipC, and ptsC genes of S. typhimurium F98 supported the role in colonization of all but the pts locus. The virulence of the rfbK, dksA, hupA, sipC, and ptsC defined mutants and clpB and rfaY transductants in 1-day-old chicks was tested. All but the ptsC and rfaY mutants were attenuated for virulence. A number of other phenotypes associated with some of the mutations are described. PMID:9573095

  20. Imipenem heteroresistance in nontypeable Haemophilus influenzae is linked to a combination of altered PBP3, slow drug influx and direct efflux regulation.

    Science.gov (United States)

    Cherkaoui, A; Diene, S M; Renzoni, A; Emonet, S; Renzi, G; François, P; Schrenzel, J

    2017-02-01

    To investigate the potential roles of PBPs, efflux pumps and slow drug influx for imipenem heteroresistance in nontypeable Haemophilus influenzae (NTHi). Fifty-nine NTHi clinical isolates examined in this study were collected at Geneva University Hospitals between 2009 and 2014. Alterations in PBPs were investigated by gene sequencing. To evaluate the affinities of the PBPs to imipenem, steady-state concentration-response experiments were carried out using imipenem in a competition assay with Bocillin-FL. The effect of the carbonyl cyanide m-chlorophenylhydrazone (CCCP) on imipenem susceptibility was assessed using broth dilution and viable cell counting. Using whole-genome sequencing, we explored the potential roles of outer membrane protein P2 (OmpP2), LytM proteins and the dcw gene cluster in imipenem heteroresistance. All 46 imipenem-heteroresistant isolates (IMI hR ) harboured amino acid substitutions in the ftsI gene, which encodes PBP3, corresponding to 25 different mutation patterns that varied from the ftsI gene mutation patterns found in imipenem-susceptible isolates. Among all PBPs, the highest affinity to imipenem was documented for PBP3 (IC 50 , 0.004 μg/mL). Different amino acid substitutions and insertions were noted in OmpP2, suggesting a relationship with imipenem heteroresistance. The IMI hR isolates were affected by CCCP differently and displayed a higher percentage of killing by imipenem in CCCP-treated cells at concentrations ranging between 0.5 and 8 μg/mL. The present study provides robust evidence indicating that in combination with the altered PBP3, the slowed drug influx and its enhanced efflux due to the loss of regulation led to the development of imipenem heteroresistance in NTHi. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  1. Identification of the rctA Gene, Which Is Required for Repression of Conjugative Transfer of Rhizobial Symbiotic Megaplasmids†

    Science.gov (United States)

    Pérez-Mendoza, Daniel; Sepúlveda, Edgardo; Pando, Victoria; Muñoz, Socorro; Nogales, Joaquina; Olivares, José; Soto, Maria J.; Herrera-Cervera, José A.; Romero, David; Brom, Susana; Sanjuán, Juan

    2005-01-01

    An analysis of the conjugative transfer of pRetCFN42d, the symbiotic plasmid (pSym) of Rhizobium etli, has revealed a novel gene, rctA, as an essential element of a regulatory system for silencing the conjugative transfer of R. etli pSym by repressing the transcription of conjugal transfer genes in standard laboratory media. The rctA gene product lacks sequence conservation with other proteins of known function but may belong to the winged-helix DNA-binding subfamily of transcriptional regulators. Similar to that of many transcriptional repressors, rctA transcription seems to be positively autoregulated. rctA expression is greatly reduced upon overexpression of another gene, rctB, previously identified as a putative activator of R. etli pSym conjugal transfer. Thus, rctB seems to counteract the repressive action of rctA. rctA homologs are present in at least three other bacterial genomes within the order Rhizobiales, where they are invariably located adjacent to and divergently transcribed from putative virB-like operons. We show that similar to that of R. etli pSym, conjugative transfer of the 1.35-Mb symbiotic megaplasmid A of Sinorhizobium meliloti is also subjected to the inhibitory action of rctA. Our data provide strong evidence that the R. etli and S. meliloti pSym plasmids are indeed self-conjugative plasmids and that this property would only be expressed under optimal, as yet unknown conditions that entail inactivation of the rctA function. The rctA gene seems to represent novel but probably widespread regulatory systems controlling the transfer of conjugative elements within the order Rhizobiales. PMID:16237017

  2. PTTG1, A novel androgen responsive gene is required for androgen-induced prostate cancer cell growth and invasion

    International Nuclear Information System (INIS)

    Zhang, Zheng; Jin, Bo; Jin, Yaqiong; Huang, Shengquan; Niu, Xiaohua; Mao, Zebin; Xin, Dianqi

    2017-01-01

    Androgens (AR) play an important role in initiation and progression of prostate cancer. It has been shown that AR exert their effects mainly through the androgen-activated AR which binds to androgen response elements (AREs) in the regulatory regions of target genes to regulate the transcription of androgen-responsive genes, thus, identification of AR downstream target gene is critical to understand androgen function in prostate cancer. In this study, our results showed that androgen treatment of LNCaP cells induced PTTG1 expression, which was blocked by the androgen receptor antagonist, Casodex. Bioinformatics analysis and experiments using PTTG1 promoter deletion mutants showed that the PTTG1 promoter contains a putative androgen response element (ARE), which localizes in the −851 to −836 region of the promoter. Androgen activated androgen receptor (AR) binding to this ARE was confirmed by Chromatin immunoprecipitation (ChIP) assay. Furthermore, Knockdown of PTTG1 expression using short hairpin RNA significantly reduced androgen-induced LNCaP cell growth and invasion. In addition, we showed PTTG1 is highly expressed in metastasis prostate cancer tissue. These results suggest that PTTG1 is a novel downstream target gene of androgen receptor and take part in prostate cancer proliferation and metastasis. - Highlights: • Androgen treatment of LNCaP cells induced PTTG1 expression. • Knockdown of PTTG1 expression significantly reduced androgen-induced LNCaP cell growth and invasion. • PTTG1 is highly expressed in metastasis prostate cancer tissue. • PTTG1 is a novel downstream target gene of androgen receptor.

  3. Airway inflammation in nonobstructive and obstructive chronic bronchitis with chronic haemophilus influenzae airway infection. Comparison with noninfected patients with chronic obstructive pulmonary disease

    NARCIS (Netherlands)

    Bresser, P.; Out, T. A.; van Alphen, L.; Jansen, H. M.; Lutter, R.

    2000-01-01

    Nonencapsulated Haemophilus influenzae often causes chronic infections of the lower respiratory tract in both nonobstructive and obstructive chronic bronchitis. We assessed airway inflammation in clinically stable, chronically H. influenzae-infected patients with nonobstructive (CB-HI, n = 10) and

  4. Killing curve activity of ciprofloxacin is comparable to synergistic effect of beta-lactam-tobramycin combinations against Haemophilus species endocarditis strains

    DEFF Research Database (Denmark)

    Westh, H; Frimodt-Møller, N; Gutschik, E

    1992-01-01

    Nine Haemophilus species strains, all beta-lactamase negative, isolated from patients with endocarditis were tested in killing curve experiments. Antibiotics used were penicillin, amoxicillin, aztreonam alone and in combination with tobramycin, as well as ciprofloxacin alone. Synergism between beta...

  5. Multicentre in-vitro evaluation of the susceptibility of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis to ciprofloxacin, clarithromycin, co-amoxiclav and sparfloxacin

    NARCIS (Netherlands)

    HoogkampKorstanje, JAA; DirksGo, SIS; Kabel, P; Manson, WL; Stobberingh, EE; Vreede, RW; Davies, BI

    Seven laboratories, including a reference laboratory, tested the susceptibility of Moraxella catarrhalis, Streptococcus pneumoniae and Haemophilus influenzae strains to ciprofloxacin, clarithromycin, co-amoxiclav and sparfloxacin with the Etest. A total of 976 strains were collected. The results

  6. Endogenous and exogenous reinfections by Haemophilus influenzae in patients with chronic obstructive pulmonary disease: the effect of antibiotic treatment on persistence

    NARCIS (Netherlands)

    Groeneveld, K.; van Alphen, L.; Eijk, P. P.; Visschers, G.; Jansen, H. M.; Zanen, H. C.

    1990-01-01

    To analyze whether exacerbations in chronic obstructive pulmonary disease (COPD) coincide with reinfection by Haemophilus influenzae, 16 COPD patients were studied longitudinally for 3 years. Exacerbations coincided with reinfection by H. influenzae, either endogenous, by a strain with a DNA

  7. cDNA sequence of human transforming gene hst and identification of the coding sequence required for transforming activity

    International Nuclear Information System (INIS)

    Taira, M.; Yoshida, T.; Miyagawa, K.; Sakamoto, H.; Terada, M.; Sugimura, T.

    1987-01-01

    The hst gene was originally identified as a transforming gene in DNAs from human stomach cancers and from a noncancerous portion of stomach mucosa by DNA-mediated transfection assay using NIH3T3 cells. cDNA clones of hst were isolated from the cDNA library constructed from poly(A) + RNA of a secondary transformant induced by the DNA from a stomach cancer. The sequence analysis of the hst cDNA revealed the presence of two open reading frames. When this cDNA was inserted into an expression vector containing the simian virus 40 promoter, it efficiently induced the transformation of NIH3T3 cells upon transfection. It was found that one of the reading frames, which coded for 206 amino acids, was responsible for the transforming activity

  8. Identification of Streptococcus sanguinis Genes Required for Biofilm Formation and Examination of Their Role in Endocarditis Virulence▿

    OpenAIRE

    Ge, Xiuchun; Kitten, Todd; Chen, Zhenming; Lee, Sehmi P.; Munro, Cindy L.; Xu, Ping

    2008-01-01

    Streptococcus sanguinis is one of the pioneers in the bacterial colonization of teeth and is one of the most abundant species in the oral biofilm called dental plaque. S. sanguinis is also the most common viridans group streptococcal species implicated in infective endocarditis. To investigate the association of biofilm and endocarditis, we established a biofilm assay and examined biofilm formation with a signature-tagged mutagenesis library of S. sanguinis. Four genes that have not previousl...

  9. A CHROMATIN MODIFYING ENZYME, SDG8, IS REQUIRED FOR MORPHOLOGICAL, GENE EXPRESSION, AND EPIGENETIC RESPONSES TO MECHANICAL STIMULATION

    OpenAIRE

    Christopher Ian Cazzonelli; Nazia eNisar; Andrea C Roberts; Kevin eMurray; Justin O Borevitz; Barry James Pogson

    2014-01-01

    Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzy...

  10. A transposon mutant library of Bacillus cereus ATCC 10987 reveals novel genes required for biofilm formation and implicates motility as an important factor for pellicle-biofilm formation.

    Science.gov (United States)

    Okshevsky, Mira; Louw, Matilde Greve; Lamela, Elena Otero; Nilsson, Martin; Tolker-Nielsen, Tim; Meyer, Rikke Louise

    2018-04-01

    Bacillus cereus is one of the most common opportunistic pathogens causing foodborne illness, as well as a common source of contamination in the dairy industry. B. cereus can form robust biofilms on food processing surfaces, resulting in food contamination due to shedding of cells and spores. Despite the medical and industrial relevance of this species, the genetic basis of biofilm formation in B. cereus is not well studied. In order to identify genes required for biofilm formation in this bacterium, we created a library of 5000 +  transposon mutants of the biofilm-forming strain B. cereusATCC 10987, using an unbiased mariner transposon approach. The mutant library was screened for the ability to form a pellicle biofilm at the air-media interface, as well as a submerged biofilm at the solid-media interface. A total of 91 genes were identified as essential for biofilm formation. These genes encode functions such as chemotaxis, amino acid metabolism and cellular repair mechanisms, and include numerous genes not previously known to be required for biofilm formation. Although the majority of disrupted genes are not directly responsible for motility, further investigations revealed that the vast majority of the biofilm-deficient mutants were also motility impaired. This observation implicates motility as a pivotal factor in the formation of a biofilm by B. cereus. These results expand our knowledge of the fundamental molecular mechanisms of biofilm formation by B. cereus. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  11. Unique C-terminal region of Hap3 is required for methanol-regulated gene expression in the methylotrophic yeast Candida boidinii.

    Science.gov (United States)

    Oda, Saori; Yurimoto, Hiroya; Nitta, Nobuhisa; Sakai, Yasuyoshi

    2016-05-01

    The Hap complex of the methylotrophic yeast Candida boidinii was found to be required for methanol-regulated gene expression. In this study, we performed functional characterization of CbHap3p, one of the Hap complex components in C. boidinii. Sequence alignment of Hap3 proteins revealed the presence of a unique extended C-terminal region, which is not present in Hap3p from Saccharomyces cerevisiae (ScHap3p), but is found in Hap3p proteins of methylotrophic yeasts. Deletion of the C-terminal region of CbHap3p (Δ256-292 or Δ107-237) diminished activation of methanol-regulated genes and abolished the ability to grow on methanol, but did not affect nuclear localization or DNA-binding ability. However, deletion of the N-terminal region of CbHap3p (Δ1-20) led to not only a growth defect on methanol and a decreased level of methanol-regulated gene expression, but also impaired nuclear localization and binding to methanol-regulated gene promoters. We also revealed that CbHap3p could complement the growth defect of the Schap3Δ strain on glycerol, although ScHap3p could not complement the growth defect of a Cbhap3Δ strain on methanol. We conclude that the unique C-terminal region of CbHap3p contributes to maximum activation of methanol-regulated genes, whilst the N-terminal region is required for nuclear localization and binding to DNA.

  12. Genome-wide transposon mutagenesis of Proteus mirabilis: Essential genes, fitness factors for catheter-associated urinary tract infection, and the impact of polymicrobial infection on fitness requirements

    Science.gov (United States)

    Smith, Sara N.; Zhao, Lili; Wu, Weisheng

    2017-01-01

    The Gram-negative bacterium Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Numerous prior studies have uncovered virulence factors for P. mirabilis pathogenicity in a murine model of ascending UTI, but little is known concerning pathogenesis during CAUTI or polymicrobial infection. In this study, we utilized five pools of 10,000 transposon mutants each and transposon insertion-site sequencing (Tn-Seq) to identify the full arsenal of P. mirabilis HI4320 fitness factors for single-species versus polymicrobial CAUTI with Providencia stuartii BE2467. 436 genes in the input pools lacked transposon insertions and were therefore concluded to be essential for P. mirabilis growth in rich medium. 629 genes were identified as P. mirabilis fitness factors during single-species CAUTI. Tn-Seq from coinfection with P. stuartii revealed 217/629 (35%) of the same genes as identified by single-species Tn-Seq, and 1353 additional factors that specifically contribute to colonization during coinfection. Mutants were constructed in eight genes of interest to validate the initial screen: 7/8 (88%) mutants exhibited the expected phenotypes for single-species CAUTI, and 3/3 (100%) validated the expected phenotypes for polymicrobial CAUTI. This approach provided validation of numerous previously described P. mirabilis fitness determinants from an ascending model of UTI, the discovery of novel fitness determinants specifically for CAUTI, and a stringent assessment of how polymicrobial infection influences fitness requirements. For instance, we describe a requirement for branched-chain amino acid biosynthesis by P. mirabilis during coinfection due to high-affinity import of leucine by P. stuartii. Further investigation of genes and pathways that provide a competitive advantage during both single-species and polymicrobial CAUTI will likely provide robust targets for therapeutic intervention to reduce P. mirabilis

  13. Genome-wide transposon mutagenesis of Proteus mirabilis: Essential genes, fitness factors for catheter-associated urinary tract infection, and the impact of polymicrobial infection on fitness requirements.

    Science.gov (United States)

    Armbruster, Chelsie E; Forsyth-DeOrnellas, Valerie; Johnson, Alexandra O; Smith, Sara N; Zhao, Lili; Wu, Weisheng; Mobley, Harry L T

    2017-06-01

    The Gram-negative bacterium Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Numerous prior studies have uncovered virulence factors for P. mirabilis pathogenicity in a murine model of ascending UTI, but little is known concerning pathogenesis during CAUTI or polymicrobial infection. In this study, we utilized five pools of 10,000 transposon mutants each and transposon insertion-site sequencing (Tn-Seq) to identify the full arsenal of P. mirabilis HI4320 fitness factors for single-species versus polymicrobial CAUTI with Providencia stuartii BE2467. 436 genes in the input pools lacked transposon insertions and were therefore concluded to be essential for P. mirabilis growth in rich medium. 629 genes were identified as P. mirabilis fitness factors during single-species CAUTI. Tn-Seq from coinfection with P. stuartii revealed 217/629 (35%) of the same genes as identified by single-species Tn-Seq, and 1353 additional factors that specifically contribute to colonization during coinfection. Mutants were constructed in eight genes of interest to validate the initial screen: 7/8 (88%) mutants exhibited the expected phenotypes for single-species CAUTI, and 3/3 (100%) validated the expected phenotypes for polymicrobial CAUTI. This approach provided validation of numerous previously described P. mirabilis fitness determinants from an ascending model of UTI, the discovery of novel fitness determinants specifically for CAUTI, and a stringent assessment of how polymicrobial infection influences fitness requirements. For instance, we describe a requirement for branched-chain amino acid biosynthesis by P. mirabilis during coinfection due to high-affinity import of leucine by P. stuartii. Further investigation of genes and pathways that provide a competitive advantage during both single-species and polymicrobial CAUTI will likely provide robust targets for therapeutic intervention to reduce P. mirabilis

  14. Incidence of invasive Haemophilus influenzae type b disease in Italian children

    International Nuclear Information System (INIS)

    Tozzi, Alberto E.; Salmaso, Stefania; Atti, Marta L. Ciofi degli; Panei, Pietro; Anemona, Alessandra; Scuderi, Gabriella; Wassilak, Steven G.F.

    1997-01-01

    To estimate the incidence of Haemophilus influenzae type b (Hib) invasive disease in Italian infants we performed a prospective study in a cohort of newborns enrolled for a randomized trial on safety and efficacy of three pertussis vaccines and followed for onset of serious disease or pertussis. The overall cumulative incidence observed in 15,601 children was 51.3/100,000 for all invasive Hib infections and 38.4/100,000 for Hib meningitis, over 27 months of observation. The incidence density of all invasive Hib diseases was 28.7/100,000 person-years, while meningitis occurred with an incidence of 21.5/100,000 person-years. Among the eight cases detected, six were meningitis, one sepsis, and one cellulitis. The child with sepsis died. The incidence and epidemiology of invasive Hib disease in Italy are comparable to those reported from other European countries. Cost-benefit analyses are needed for planning Italian vaccination policy

  15. Vaccination for the control of childhood bacterial pneumonia - Haemophilus influenzae type b and pneumococcal vaccines

    Directory of Open Access Journals (Sweden)

    Diana C Otczyk

    2013-01-01

    Full Text Available Pneumonia in childhood is endemic in large parts of the world and in particular, in developing countries, as well as in many indigenous communities within developed nations. Haemophilus influenzae type b and Streptococcus pneumoniae conjugate vaccines are currently available against the leading bacterial causes of pneumonia.  The use of the vaccines in both industrialised and developing countries have shown a dramatic reduction in the burden of pneumonia and invasive disease in children.  However, the greatest threat facing pneumococcal conjugate vaccine effectiveness is serotype replacement.  The current vaccines provide serotype-specific, antibody–mediated protection against only a few of the 90+ capsule serotypes.  Therefore, there has been a focus in recent years to rapidly advance technologies that will result in broader disease coverage and more affordable vaccines that can be used in developing countries.  The next generation of pneumococcal vaccines have advanced to clinical trials.

  16. Susceptibility of Haemophilus equigenitalis, the causal agent of contagious equine metritis, to 31 antimicrobial agents.

    Science.gov (United States)

    Sugimoto, C; Isayama, Y; Kashiwazaki, M; Mitani, K

    1981-01-01

    The minimal inhibitory concentrations of 31 antimicrobial agents were determined for 99 isolates of Haemophilus equigenitalis by the agar dilution method. All the isolates showed good susceptibility to 26 antimicrobial agents tests, minimal inhibitory concentrations of which were less than 3.13 micrograms/ml for more than 90% of the isolates. Of these agents, 4 macrolides (erythromycin, oleandomycin, kitasamycin, tylosin), 3 tetracyclines (tetracycline, chlortetracycline, oxytetracycline), 1 peptide (colistin), 1 penicillin (ampicillin) and 1 pleuromutilin (tiamulin) were the most active agents, showing a minimal inhibitory concentration of less than 0.39 micrograms/ml for more than 90% of the isolates. The growth of more than 90% of the isolates was not inhibited by 800 micrograms/ml of streptomycin.

  17. Development of an improved species specific PCR test for detection of Haemophilus parasuis

    DEFF Research Database (Denmark)

    Angen, Øystein; Oliveira, Simone; Ahrens, Peter

    2007-01-01

    , the present PCR test was found to be 100% species specific for H. parasuis, in contrast to the PCR test of Oliveira et al., which also tested positive for strains belonging to A. indolicus, A. porcinus, and A. minor, species commonly occurring in the upper respiratory tract. However, when the PCR test...... with representatives of H. parasuis. The test was further evaluated on 55 clinical samples from 16 Danish pigs suspected for being infected with H. parasuis, showing polyserositis or septicemia at autopsy as well as on 492 nasal swabs. The test was compared with the performance of a PCR test earlier published...... by Oliveira et al. [Oliveira, S., Galina, L., Pijoan, C., 2001. Development of a PCR test to diagnose Haemophilus parasuis infections. J. Vet. Diagn. Invest. 13, 495-501]. The sensitivity of the present PCR test was found to be slightly lower when applied on clinical samples from diseased pigs and 10-fold...

  18. Clinical characteristics of Haemophilus influenzae meningitis in Denmark in the post-vaccination era

    DEFF Research Database (Denmark)

    Pedersen, T.I.; Howitz, Michael Frantz; Andersen, Christian Østergaard

    2010-01-01

    P>The introduction of Haemophilus influenzae type b (Hib) vaccine into the Danish childhood vaccination programme in 1993 may have influenced the epidemiology of H. influenzae meningitis (i.e. increasing frequency of other non-vaccine types; presentation in other age groups). Based on nationwide...... infected with Hib, two cases (13%) were identified as true vaccine failures. Six patients (9%) died; one premature infant infected with serotype f and five adults (age 83-96 years) with non-typeable H. influenzae. Hearing loss was reported in 16% of the surviving children and in 10% of the surviving adults....... The presence of a lung focus was an independent prognostic factor for an unfavourable outcome (p 0.03). In conclusion, meningitis caused by Hib has been infrequent in Denmark after introduction of the Hib vaccine in the childhood vaccination programme, and no increase in meningitis cases due to non-b type H...

  19. Haemophilus influenzae type B genital infection and septicemia in pregnant woman: a case report

    Directory of Open Access Journals (Sweden)

    Hosuru Subramanya Supram

    2014-06-01

    Full Text Available Haemophilus influenzae (H. influenzae type B a non-motile, aerobic, gram negative cocobacillus is a commensal of upper respiratory tract. Genitourinary infection due to H. influenzae has been reported but bacteremia associated with such infection appears to be rare. We report a case of 19 years young primigravida with complaints of amenorrhea of 32 weeks and 5 days, pyrexia, abdominal pain and blood stained discharge per vaginum. H. influenzae type B was recovered from the genital tract as well as blood of the mother indicating maternal septicemia. Septicemia caused by H. influenzae type B in pregnant women following vaginal colonization and infection is rare. It has been reported in many parts of world over the years; to the best of our knowledge this is the first reported case from Nepal. H. influenzae should be considered as a potential maternal, fetal, and neonatal pathogen.

  20. Haemophilus influenzae vulvovaginitis associated with rhinitis caused by the same clone in a prepubertal girl.

    Science.gov (United States)

    Chen, Xiao; Chen, Lifeng; Zeng, Wenjie; Zhao, Xiaofeng

    2017-06-01

    Vulvovaginitis caused by upper respiratory flora is generally considered to be the most common gynecological problem in prepubertal girls. To date, however, no direct evidence has been obtained for the underlying mechanism of transmission. This report describes a case of non-capsulate Haemophilus influenzae vulvovaginitis in a 6-year-old girl with a history of foreign bodies (cotton wool) in her vagina. Moreover, this girl had recurrent rhinitis for approximately 3 years. On Pulsed Field Gel Electrophoresis (PFGE) analysis the H. influenzae strain isolated from vaginal secretions and the H. influenzae strain isolated from nasal secretions were derived from the same clone. The patient was successfully treated with appropriate antibiotics. The present case might provide the first direct evidence of the nose-hand-vagina method of transmission. © 2017 Japan Society of Obstetrics and Gynecology.

  1. A review of the role of Haemophilus influenzae in community-acquired pneumonia

    Directory of Open Access Journals (Sweden)

    Mary PE Slack

    2015-06-01

    Full Text Available In an era when Haemophilus influenzae type b (Hib conjugate vaccine is widely used, the incidence of Hib as a cause of community-acquired pneumonia (CAP has dramatically declined. Non-typeable H. influenzae (NTHi strains and, occasionally, other encapsulated serotypes of H. influenzae are now the cause of the majority of invasive H. influenzae infections, including bacteraemic CAP. NTHi have long been recognised as an important cause of lower respiratory tract infection, including pneumonia, in adults, especially those with underlying diseases. The role of NTHi as a cause of non-bacteraemic CAP in children is less clear. In this review the evidence for the role of NTHi and capsulated strains of H. influenzae will be examined.

  2. Clinical characteristics of Haemophilus influenzae meningitis in Denmark in the post-vaccination era

    DEFF Research Database (Denmark)

    Pedersen, T.I.; Howitz, M.; Andersen, Christian Østergaard

    2010-01-01

    P>The introduction of Haemophilus influenzae type b (Hib) vaccine into the Danish childhood vaccination programme in 1993 may have influenced the epidemiology of H. influenzae meningitis (i.e. increasing frequency of other non-vaccine types; presentation in other age groups). Based on nationwide...... registration, clinical information and laboratory findings were collected from all 65 confirmed cases of H. influenzae meningitis during the period 1994-2005. Twenty-nine patients (45%) were 24 years old [median 62 years (range 25...... infected with Hib, two cases (13%) were identified as true vaccine failures. Six patients (9%) died; one premature infant infected with serotype f and five adults (age 83-96 years) with non-typeable H. influenzae. Hearing loss was reported in 16% of the surviving children and in 10% of the surviving adults...

  3. Serious systemic infection caused by non-encapsulated Haemophilus influenzae biotype III in an adult

    DEFF Research Database (Denmark)

    Lester, Anne; Pedersen, P B

    1991-01-01

    Haemophilus influenzae is the aetiological agent in less than 1% of septic arthritis cases in adults and most often serotype b is involved. We report here a case of severe systemic infection due to non-encapsulated H. influenzae biotype III in a 40-year-old man, previously healthy although alcohol...... abuser. Cholangitis and acute alcoholic hepatitis were diagnosed simultaneously. The organism was grown from blood and from synovial fluid of the left knee, but several other joints were also affected. The close relationship between H. influenzae biotype III and H. aegyptius is mentioned in view...... of recent reports of fatal childhood illness caused by a special clone of H. aegyptius and the importance of reporting both serotype and biotype in severe H. influenzae induced disease is emphasized....

  4. Recurrent Posttraumatic Meningitis due to Nontypable Haemophilus influenzae: Case Report and Review of the Literature

    DEFF Research Database (Denmark)

    Kunze, W; Müller, L; Kilian, Mogens

    2007-01-01

    We report a case of relapsing Haemophilus influenzae meningitis in a boy at the age of nearly 3 years and 4.2 years who had been successfully vaccinated against H. influenzae serotype b (Hib). The pathogen was a nonencapsulated (nontypable) H. influenzae strain of biotypes III and VI, respectively....... A rhinobasal impalement injury with development of a posttraumatic encephalocele is considered to be the predisposing condition. Review of the literature reveals that in patients systemically infected by nonencapsulated H. influenzae strains predisposing factors such as cerebrospinal fluid-shunts, implants...... and traumas are often found. To obtain further information on potential new disease patterns H. influenzae isolates from cerebrospinal fluid should be examined for capsule production and, if relevant, further characterized by capsular typing....

  5. Transcription factor VdCmr1 is required for pigment production, protection from UV irradiation, and regulates expression of melanin biosynthetic genes in Verticillium dahliae.

    Science.gov (United States)

    Wang, Yonglin; Hu, Xiaoping; Fang, Yulin; Anchieta, Amy; Goldman, Polly H; Hernandez, Gustavo; Klosterman, Steven J

    2018-04-01

    Verticillium dahliae is a soilborne fungus that causes vascular wilt diseases on numerous plant species worldwide. The production of darkly melanized microsclerotia is crucial in the disease cycle of V. dahliae, as these structures allow for long-term survival in soil. Previously, transcriptomic and genomic analysis identified a cluster of genes in V. dahliae that encodes some dihydroxynaphthalene (DHN) melanin biosynthetic pathway homologues found in related fungi. In this study, we explored the roles of cluster-specific transcription factor VdCmr1, as well as two other genes within the cluster encoding a polyketide synthase (VdPKS1) and a laccase (VdLac1), enzymes at initial and endpoint steps in DHN melanin production. The results revealed that VdCmr1 and VdPKS1 are required for melanin production, but neither is required for microsclerotia production. None of the three genes were required for pathogenesis on tobacco and lettuce. Exposure of ΔVdCmr1 and wild-type strains to UV irradiation, or to high temperature (40 °C), revealed an approx. 50 % reduction of survival in the ΔVdCmr1 strain, relative to the wild-type strain, in response to either condition. Expression profiles revealed that expression of some melanin biosynthetic genes are in part dependent on VdCmr1. Combined data indicate VdCmr1 is a key regulator of melanin biosynthesis, and that via regulation of melanogenesis, VdCmr1 affects survival of V. dahliae in response to abiotic threats. We conclude with a model showing regulation of VdCmr1 by a high osmolarity glycerol response (Hog)-type MAP kinase pathway.

  6. Haemophilus influenzae Sepsis and Placental Abruption in an Unvaccinated Immigrant

    Directory of Open Access Journals (Sweden)

    Paul A. Calner

    2012-04-01

    Full Text Available Background: Haemophilus influenzae infections have declined dramatically in the United States sinceimplementation of the conjugate vaccine. However, in countries where widespread immunization is notroutine, H influenzae remains a significant cause of morbidity and mortality. We report a case of apreviously unvaccinated immigrant with confirmed H influenzae sepsis and placental abruption leadingto spontaneous abortion.Objectives: To alert emergency medicine practitioners that H influenzae should be recognized as amaternal, fetal, and neonatal pathogen. Clinicians should consider this diagnosis in immigrants presentingwith uncertain vaccination history, as H influenzae can cause significant morbidity and mortality.Case Presentation: A 36-year-old female was referred to our emergency department (ED with lowerabdominal pain with some vaginal spotting. The patient had an initial visit with normal laboratoryinvestigations and normal imaging results, with complete resolution of symptoms. The patient returned tothe ED with sudden onset of vaginal bleeding and abdominal pain. She presented at this time with sepsis,which progressed to septic shock, causing placental abruption and ultimately, spontaneous abortion. Thepatient was treated with pressors and antibiotics and was admitted to the medical intensive care unitwhere she received ampicillin, gentamycin, and clindamycin for suspected chorioamnionitis. The patient’sblood cultures came back positive after 1 day for H influenzae. The patient did well and was dischargedfrom the hospital 4 days later.Conclusion: Haemophilus influenzae should be recognized as a neonatal and maternal pathogen.Clinicians should consider this diagnosis in immigrants presenting with uncertain vaccination history,especially in pregnant females, as H influenzae can cause significant morbidity and mortality. [West JEmerg Med. 2012;13(1:133–135.

  7. Frequency of Haemophilus spp. in urinary and and genital tract samples

    Directory of Open Access Journals (Sweden)

    Tatjana Marijan,

    2010-02-01

    Full Text Available Aim To determine the prevalence and antibiotic susceptibility of Haemophilus influenzae and H. parainfluenzae isolated from the urinary and genital tracts. Methods Identification of strains bacteria Haemophilus spp. was carried out by using API NH identifi-cation system, and antibiotic susceptibility was performed by Kirby-Bauer disk diffusion method. Results A total number of 50 (0,03% H. influenzae and 14 (0,01% H. parainfluenzae (out of 180, 415 samples were isolated from genitourinary tract. From urine samples of the girls under 15 years of age these bacteria were isolated in 13 (0,88% and two (0,13% cases, respectively, and only in one case(0,11% of the UTI in boys (H. influenzae. In persons of fertile age, it was only H. influenzae bacteria that was found in urine samples of the five women (0,04% and in three men (0,22%. As a cause of vulvovaginitis, H. influenzae was isolated in four (5,63%, and H. parainfluenzae in two (2,82% girls. In persons of fertile age, H. influenzae was isolated from 10 (0,49% smears of the cervix, and in nine (1,74% male samples. H. parainfluenzae was isolated from seven (1,36% male samples. (p<0.01. Susceptibility testing of H. influenzae and H. parainfluenzae revealed that both pathogens were signifi- cantly resistant to cotrimoxasol only (26.0% and 42.9%, respectively. Conclusion In the etiology of genitourinary infections of girls during childhood, genital infections of women in fertile age (especially in pregnant women, and men with cases of epididimytis and/or orchitis,it is important to think about this rare and demanding bacteria in terms of cultivation.

  8. Structure-based functional annotation of putative conserved proteins having lyase activity from Haemophilus influenzae.

    Science.gov (United States)

    Shahbaaz, Mohd; Ahmad, Faizan; Imtaiyaz Hassan, Md

    2015-06-01

    Haemophilus influenzae is a small pleomorphic Gram-negative bacteria which causes several chronic diseases, including bacteremia, meningitis, cellulitis, epiglottitis, septic arthritis, pneumonia, and empyema. Here we extensively analyzed the sequenced genome of H. influenzae strain Rd KW20 using protein family databases, protein structure prediction, pathways and genome context methods to assign a precise function to proteins whose functions are unknown. These proteins are termed as hypothetical proteins (HPs), for which no experimental information is available. Function prediction of these proteins would surely be supportive to precisely understand the biochemical pathways and mechanism of pathogenesis of Haemophilus influenzae. During the extensive analysis of H. influenzae genome, we found the presence of eight HPs showing lyase activity. Subsequently, we modeled and analyzed three-dimensional structure of all these HPs to determine their functions more precisely. We found these HPs possess cystathionine-β-synthase, cyclase, carboxymuconolactone decarboxylase, pseudouridine synthase A and C, D-tagatose-1,6-bisphosphate aldolase and aminodeoxychorismate lyase-like features, indicating their corresponding functions in the H. influenzae. Lyases are actively involved in the regulation of biosynthesis of various hormones, metabolic pathways, signal transduction, and DNA repair. Lyases are also considered as a key player for various biological processes. These enzymes are critically essential for the survival and pathogenesis of H. influenzae and, therefore, these enzymes may be considered as a potential target for structure-based rational drug design. Our structure-function relationship analysis will be useful to search and design potential lead molecules based on the structure of these lyases, for drug design and discovery.

  9. Development of a diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples.

    LENUS (Irish Health Repository)

    Meyler, Kenneth L

    2012-12-01

    Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies\\/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and\\/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF.

  10. Host Polymorphisms in TLR9 and IL10 Are Associated With the Outcomes of Experimental Haemophilus ducreyi Infection in Human Volunteers.

    Science.gov (United States)

    Singer, Martin; Li, Wei; Morré, Servaas A; Ouburg, Sander; Spinola, Stanley M

    2016-08-01

    In humans inoculated with Haemophilus ducreyi, there are host effects on the possible clinical outcomes-pustule formation versus spontaneous resolution of infection. However, the immunogenetic factors that influence these outcomes are unknown. Here we examined the role of 14 single-nucleotide polymorphisms (SNPs) in 7 selected pathogen-recognition pathways and cytokine genes on the gradated outcomes of experimental infection. DNAs from 105 volunteers infected with H. ducreyi at 3 sites were genotyped for SNPs, using real-time polymerase chain reaction. The participants were classified into 2 cohorts, by race, and into 4 groups, based on whether they formed 0, 1, 2, or 3 pustules. χ(2) tests for trend and logistic regression analyses were performed on the data. In European Americans, the most significant findings were a protective association of the TLR9 +2848 GG genotype and a risk-enhancing association of the TLR9 TA haplotype with pustule formation; logistic regression showed a trend toward protection for the TLR9 +2848 GG genotype. In African Americans, logistic regression showed a protective effect for the IL10 -2849 AA genotype and a risk-enhancing effect for the IL10 AAC haplotype. Variations in TLR9 and IL10 are associated with the outcome of H. ducreyi infection. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  11. Development of a diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples.

    Science.gov (United States)

    Meyler, Kenneth L; Meehan, Mary; Bennett, Desiree; Cunney, Robert; Cafferkey, Mary

    2012-12-01

    Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Characterization of dapB, a gene required by Pseudomonas syringae pv. tabaci BR2.024 for lysine and tabtoxinine-beta-lactam biosynthesis.

    Science.gov (United States)

    Liu, L; Shaw, P D

    1997-01-01

    The dapB gene, which encodes L-2,3-dihydrodipicolinate reductase, the second enzyme of the lysine branch of the aspartic amino acid family, was cloned and sequenced from a tabtoxin-producing bacterium, Pseudomonas syringae pv. tabaci BR2.024. The deduced amino acid sequence shared 60 to 90% identity to known dapB gene products from gram-negative bacteria and 19 to 21% identity to the dapB products from gram-positive bacteria. The consensus sequence for the NAD(P)H binding site [(V/I)(A/G)(V/I)XGXXGXXG)] and the proposed substrate binding site (HHRHK) were conserved in the polypeptide. A BR2.024 dapB mutant is a diaminopimelate auxotroph and tabtoxin negative. The addition of a mixture of L-,L-, D,D-, and meso-diaminopimelate to defined media restored growth but not tabtoxin production. Cloned DNA fragments containing the parental dapB gene restored the ability to grow in defined media and tabtoxin production to the dapB mutant. These results indicate that the dapB gene is required for both lysine and tabtoxin biosynthesis, thus providing the first genetic evidence that the biosynthesis of tabtoxin proceeds in part along the lysine biosynthetic pathway. These data also suggest that L-2,3,4,5-tetrahydrodipicolinate is a common intermediate for both lysine and tabtoxin biosynthesis. PMID:8990304

  13. Expression of multiple slow myosin heavy chain genes reveals a diversity of zebrafish slow twitch muscle fibres with differing requirements for Hedgehog and Prdm1 activity.

    Science.gov (United States)

    Elworthy, Stone; Hargrave, Murray; Knight, Robert; Mebus, Katharina; Ingham, Philip W

    2008-06-01

    The zebrafish embryo develops a series of anatomically distinct slow twitch muscle fibres that characteristically express genes encoding lineage-specific isoforms of sarcomeric proteins such as MyHC and troponin. We show here that different subsets of these slow fibres express distinct members of a tandem array of slow MyHC genes. The first slow twitch muscle fibres to differentiate, which are specified by the activity of the transcription factor Prdm1 (also called Ubo or Blimp1) in response to Hedgehog (Hh) signalling, express the smyhc1 gene. Subsequently, secondary slow twitch fibres differentiate in most cases independently of Hh activity. We find that although some of these later-forming fibres also express smyhc1, others express smyhc2 or smyhc3. We show that the smyhc1-positive fibres express the ubo (prdm1) gene and adopt fast twitch fibre characteristics in the absence of Prdm1 activity, whereas those that do not express smyhc1 can differentiate independently of Prdm1 function. Conversely, some smyhc2-expressing fibres, although independent of Prdm1 function, require Hh activity to form. The adult trunk slow fibres express smyhc2 and smyhc3, but lack smyhc1 expression. The different slow fibres in the craniofacial muscles variously express smyhc1, smyhc2 and smyhc3, and all differentiate independently of Prdm1.

  14. Bcıı--RFLP profiles for serum amiloid A1 and mutated MEFV gene prevalence in chronic renal failure patients requiring long-term hemodialysis.

    Science.gov (United States)

    Ozdemir, Ozturk; Kayatas, Mansur; Cetinkaya, Selma; Yildirim, Malik Ejder; Silan, Fatma; Kurtulgan, Hande Kucuk; Koksal, Binnur; Urfali, Mine; Candan, Ferhan

    2015-03-01

    There is an increased mortality risk in long-term hemodialysis patients of renal failure due to the chronic inflammation. The relationship between the chronic renal failure (CRF) and the role of familial genetic markers remains incompletely understood. In the current study, it was aimed to find out the prevalence of common MEFV gene mutations and BcII polymorphism in serum amyloid A1 (SAA1) gene in chronic renal patients (CRF) who require long-term hemodialysis. Current cohort includes 242 CRF patients and 245 healthy individuals from the same population. Total genomic DNA was isolated from peripheral blood-EDTA samples and genotyping of target MEFV gene was carried out by reverse hybridization Strip Assay and real-time techniques. The SAA1 gene was genotyped by the BclI-RFLP method. Increased mutated MEFV genotypes were found in current CRF patients when compared with the control group from the same ethnicity and the difference was statistically significant (Table 2) (OR: 4.9401, 95% CI: 3.0694-7.9509), pchronic inflammation.

  15. Ampicillin-Resistant Non-β-Lactamase-Producing Haemophilus influenzae in Spain: Recent Emergence of Clonal Isolates with Increased Resistance to Cefotaxime and Cefixime▿

    Science.gov (United States)

    García-Cobos, Silvia; Campos, José; Lázaro, Edurne; Román, Federico; Cercenado, Emilia; García-Rey, César; Pérez-Vázquez, María; Oteo, Jesús; de Abajo, Francisco

    2007-01-01

    The sequence of the ftsI gene encoding the transpeptidase domain of penicillin-binding protein 3 (PBP 3) was determined for 354 nonconsecutive Haemophilus influenzae isolates from Spain; 17.8% of them were ampicillin susceptible, 56% were β-lactamase nonproducing ampicillin resistant (BLNAR), 15.8% were β-lactamase producers and ampicillin resistant, and 10.4% displayed both resistance mechanisms. The ftsI gene sequences had 28 different mutation patterns and amino acid substitutions at 23 positions. Some 93.2% of the BLNAR strains had amino acid substitutions at the Lys-Thr-Gly (KTG) motif, the two most common being Asn526 to Lys (83.9%) and Arg517 to His (9.3%). Amino acid substitutions at positions 377, 385, and 389, which conferred cefotaxime and cefixime MICs 10 to 60 times higher than those of susceptible strains, were found for the first time in Europe. In 72 isolates for which the repressor acrR gene of the AcrAB efflux pump was sequenced, numerous amino acid substitutions were found. Eight isolates with ampicillin MICs of 0.25 to 2 μg/ml showed changes that predicted the early termination of the acrR reading frame. Pulsed-field gel electrophoresis analysis demonstrated that most BLNAR strains were genetically diverse, although clonal dissemination was detected in a group of isolates presenting with increased resistance to cefotaxime and cefixime. Background antibiotic use at the community level revealed a marked trend toward increased amoxicillin-clavulanic acid consumption. BLNAR H. influenzae strains have arisen by vertical and horizontal spread and have evolved to adapt rapidly to the increased selective pressures posed by the use of oral penicillins and cephalosporins. PMID:17470649

  16. Sequencing analysis reveals a unique gene organization in the gyrB region of Mycoplasma hominis

    DEFF Research Database (Denmark)

    Ladefoged, Søren; Christiansen, Gunna

    1994-01-01

    of which showed similarity to that which encodes the LicA protein of Haemophilus influenzae. The organization of the genes in the region showed no resemblance to that in the corresponding regions of other bacteria sequenced so far. The gyrA gene was mapped 35 kb downstream from the gyrB gene.......The homolog of the gyrB gene, which has been reported to be present in the vicinity of the initiation site of replication in bacteria, was mapped on the Mycoplasma hominis genome, and the region was subsequently sequenced. Five open reading frames were identified flanking the gyrB gene, one...

  17. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    International Nuclear Information System (INIS)

    Woloschak, G.E.

    1994-01-01

    Experiments were designed to examine the effects Of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements (γ- and β-actin and α-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either α-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide revealed that cycloheximide repressed accumulation of α-tubulin following exposure to high dose-rate neutrons or γ rays; this did not occur following similar low dose-rate exposure. (2) Cycloheximide did not affect accumulation of MRNA for actin genes; and that cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to γ rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of α-tubulin and fibronectin MRNA accumulation following exposure to ionizing radiation. in addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons

  18. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    International Nuclear Information System (INIS)

    Woloschak, G.E.; Felcher, P.; Chang-Liu, Chin-Mei

    1992-01-01

    Experiments were designed to examine the effects of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements (γ- and β-actin and α-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either α-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide, however, revealed several interesting and novel findings: (1) Cycloheximide repressed accumulation of α-tubulin following exposure to high dose-rate neutrons or γ rays; this did not occur following similar low dose-rate exposure (2) Cycloheximide did not affect accumulation of mRNA for actin genes. Cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to γ rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of α-tubulin and fibronectin mRNA accumulation following exposure to ionizing radiation. In addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons

  19. Functional dissection of a napin gene promoter: identification of promoter elements required for embryo and endosperm-specific transcription.

    Science.gov (United States)

    Ellerström, M; Stålberg, K; Ezcurra, I; Rask, L

    1996-12-01

    The promoter region (-309 to +44) of the Brassica napus storage protein gene napA was studied in transgenic tobacco by successive 5' as well as internal deletions fused to the reporter gene GUS (beta-glucuronidase). The expression in the two main tissues of the seed, the endosperm and the embryo, was shown to be differentially regulated. This tissue-specific regulation within the seed was found to affect the developmental expression during seed development. The region between -309 to -152, which has a large effect on quantitative expression, was shown to harbour four elements regulating embryo and one regulating endosperm expression. This region also displayed enhancer activity. Deletion of eight bp from position -152 to position -144 totally abolished the activity of the napA promoter. This deletion disrupted a cis element with similarity to an ABA-responsive element (ABRE) overlapping with an E-box, demonstrating its crucial importance for quantitative expression. An internal deletion of the region -133 to -120, resulted in increased activity in both leaves and endosperm and a decreased activity in the embryo. Within this region, a cis element similar to the (CA)n element, found in other storage protein promoters, was identified. This suggest that the (CA)n element is important for conferring seed specificity by serving both as an activator and a repressor element.

  20. Aspectos epidemiológicos da infecção por Haemophilus influenzae tipo b Epidemiologic aspects of Haemophilus influenzae type b infection

    Directory of Open Access Journals (Sweden)

    Maria Angela Loguercio Bouskela

    2000-05-01

    Full Text Available O objetivo do presente trabalho foi revisar o papel do Haemophilus influenzae tipo b (Hib como um dos patógenos mais importantes implicados em doenças infecciosas invasivas, especialmente nos 2 primeiros anos de vida. Nos países em desenvolvimento, o H. Influenzae chega a causar 30% dos casos de pneumonia com cultura positiva e de 20 a 60% dos casos de meningite bacteriana. No presente estudo, dados epidemiológicos do Brasil foram comparados com dados internacionais obtidos em bancos de dados (Medline, 1966 a 1995; LILACS, 1982 a 1995; Thesis databank, 1980 a 1995; e Dissertation abstracts, 1988 a 1994. Analisámos o coeficiente de incidência do Hib no Brasil por estado e por faixa etária, com estratificação inclusive para o 1° ano de vida. A meningite foi utilizada como marcador do coeficiente de incidência devido às dificuldades para obter material adequado para a identificação do microrganismo nos outros quadros, como pneumonia, osteomielite, epiglotite ou endocardite. Nossa análise revelou que os dados nacionais mascaram a incidência e a letalidade regionais do H. influenzae: por exemplo, em 1991, a incidência no Brasil foi de 18,4 em 100 000 crianças menores de 1 ano; no mesmo período, a incidência no Distrito Federal foi de 175 em 100 000 crianças entre 4 e 6 meses. Além disso, a letalidade na região Norte foi de 35% em 1987, contra 22% para o Brasil como um todo. Nosso estudo abre a discussão sobre aspectos epidemiológicos relevantes das infecções por Hib e sobre o custo-benefício da profilaxia e vacinação nas faixas etárias de maior risco.This paper reviews the role of Haemophilus influenzae type b (Hib as one of the most important pathogens causing invasive infectious diseases, especially in the first 2 years of life. In developing countries H. influenzae is responsible for 30% of all pneumonia cases with positive cultures and for 20% to 60% of all bacterial meningitis cases. In this study we compared

  1. Passive immunization with a polyclonal antiserum to the hemoglobin receptor of Haemophilus ducreyi confers protection against a homologous challenge in the experimental swine model of chancroid.

    Science.gov (United States)

    Leduc, Isabelle; Fusco, William G; Choudhary, Neelima; Routh, Patty A; Cholon, Deborah M; Hobbs, Marcia M; Almond, Glen W; Orndorff, Paul E; Elkins, Christopher

    2011-08-01

    Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme.

  2. Passive Immunization with a Polyclonal Antiserum to the Hemoglobin Receptor of Haemophilus ducreyi Confers Protection against a Homologous Challenge in the Experimental Swine Model of Chancroid▿

    Science.gov (United States)

    Leduc, Isabelle; Fusco, William G.; Choudhary, Neelima; Routh, Patty A.; Cholon, Deborah M.; Hobbs, Marcia M.; Almond, Glen W.; Orndorff, Paul E.; Elkins, Christopher

    2011-01-01

    Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme. PMID:21646451

  3. Genome-wide screening of the genes required for tolerance to vanillin, which is a potential inhibitor of bioethanol fermentation, in Saccharomyces cerevisiae.

    Science.gov (United States)

    Endo, Ayako; Nakamura, Toshihide; Ando, Akira; Tokuyasu, Ken; Shima, Jun

    2008-04-15

    Lignocellulosic materials are abundant and among the most important potential sources for bioethanol production. Although the pretreatment of lignocellulose is necessary for efficient saccharification and fermentation, numerous by-products, including furan derivatives, weak acids, and phenolic compounds, are generated in the pretreatment step. Many of these components inhibit the growth and fermentation of yeast. In particular, vanillin is one of the most effective inhibitors in lignocellulose hydrolysates because it inhibits fermentation at very low concentrations. To identify the genes required for tolerance to vanillin, we screened a set of diploid yeast deletion mutants, which are powerful tools for clarifying the function of particular genes. Seventy-six deletion mutants were identified as vanillin-sensitive mutants. The numerous deleted genes in the vanillin-sensitive mutants were classified under the functional categories for 'chromatin remodeling' and 'vesicle transport', suggesting that these functions are important for vanillin tolerance. The cross-sensitivity of the vanillin-sensitive mutants to furan derivatives, weak acids, and phenolic compounds was also examined. Genes for ergosterol biosynthesis were required for tolerance to all inhibitory compounds tested, suggesting that ergosterol is a key component of tolerance to various inhibitors. Our analysis predicts that vanillin tolerance in Saccharomyces cerevisiae is affected by various complicated processes that take place on both the molecular and the cellular level. In addition, the ergosterol biosynthetic process is important for achieving a tolerance to various inhibitors. Our findings provide a biotechnological basis for the molecular engineering as well as for screening of more robust yeast strains that may potentially be useful in bioethanol fermentation.

  4. Genome-wide screening of the genes required for tolerance to vanillin, which is a potential inhibitor of bioethanol fermentation, in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Tokuyasu Ken

    2008-04-01

    Full Text Available Abstract Background Lignocellulosic materials are abundant and among the most important potential sources for bioethanol production. Although the pretreatment of lignocellulose is necessary for efficient saccharification and fermentation, numerous by-products, including furan derivatives, weak acids, and phenolic compounds, are generated in the pretreatment step. Many of these components inhibit the growth and fermentation of yeast. In particular, vanillin is one of the most effective inhibitors in lignocellulose hydrolysates because it inhibits fermentation at very low concentrations. To identify the genes required for tolerance to vanillin, we screened a set of diploid yeast deletion mutants, which are powerful tools for clarifying the function of particular genes. Results Seventy-six deletion mutants were identified as vanillin-sensitive mutants. The numerous deleted genes in the vanillin-sensitive mutants were classified under the functional categories for 'chromatin remodeling' and 'vesicle transport', suggesting that these functions are important for vanillin tolerance. The cross-sensitivity of the vanillin-sensitive mutants to furan derivatives, weak acids, and phenolic compounds was also examined. Genes for ergosterol biosynthesis were required for tolerance to all inhibitory compounds tested, suggesting that ergosterol is a key component of tolerance to various inhibitors. Conclusion Our analysis predicts that vanillin tolerance in Saccharomyces cerevisiae is affected by various complicated processes that take place on both the molecular and the cellular level. In addition, the ergosterol biosynthetic process is important for achieving a tolerance to various inhibitors. Our findings provide a biotechnological basis for the molecular engineering as well as for screening of more robust yeast strains that may potentially be useful in bioethanol fermentation.

  5. Radioimmunoassay of capsular polysaccaride antigens of groups A and C meningococci and Haemophilus influenza type b in cerebrospinal fluid

    International Nuclear Information System (INIS)

    Kaeyhty, H.; Maekelae, P.H.; Ruoslahti, E.

    1977-01-01

    Sensitive radioimmunoassays capable of measuring 0.5 ng/ml of the Haemophilus influenza type b polysaccharide and 2 ng/ml of the groups A and C meningococcal polysaccharides were developed and used to detect these substances in cerebrospinal fluid (CSF). Polysaccharide of the causative agent was detected in the CSF of 14 out of 15 patients with Haemophilus influenza type b meningitis, in 18 out of 23 patients with group A, and in two out of four patients with group C meningococcal meningitis. In some cases the antigen could be detected even after three days of antibacterial treatment. No false positive reactions were seen. The assay procedure could be shortened to approximately three hours. These assays could be useful in routine diagnostic work and epidemiological investigations. (author)

  6. Brain abscess caused by Haemophilus para phrophilus following a dental treatment in a girl. Report of a case

    Directory of Open Access Journals (Sweden)

    Vázquez Toledo María Eugenia

    2014-07-01

    Full Text Available We report the case of a six year old girl who developed three brain abscesses following a dental intervention (extraction of a tooth two weeks before. The etiologic agent was identified as Haemophilus paraprophilus, a gramnegative microorganism bacillus native mi- crobiota of the oropharynx nasopharynx and the gastrointestinal tract. 1 The patient was given ceftriaxone due to the sensitivity of the microorganism and metronidaole -because we couldn’t discard etiology by anaerobic- during six weeks. Surgical drainage of the abscesses was performed successfully. To date the patient has minimal neurologic sequelae. The importance of this case is that is the second report in the international literature of brain abscess secondary to Haemophilus paraprophilus in a pediatric patient and without associated heart disease.

  7. The Babesia bovis hap2 gene is not required for blood stage replication, but expressed upon in vitro sexual stage induction.

    Directory of Open Access Journals (Sweden)

    Hala E Hussein

    2017-10-01

    Full Text Available Babesia bovis, is a tick borne apicomplexan parasite responsible for important cattle losses globally. Babesia parasites have a complex life cycle including asexual replication in the mammalian host and sexual reproduction in the tick vector. Novel control strategies aimed at limiting transmission of the parasite are needed, but transmission blocking vaccine candidates remain undefined. Expression of HAP2 has been recognized as critical for the fertilization of parasites in the Babesia-related Plasmodium, and is a leading candidate for a transmission blocking vaccine against malaria. Hereby we identified the B. bovis hap2 gene and demonstrated that it is widely conserved and differentially transcribed during development within the tick midgut, but not by blood stage parasites. The hap2 gene was disrupted by transfecting B. bovis with a plasmid containing the flanking regions of the hap2 gene and the GPF-BSD gene under the control of the ef-1α-B promoter. Comparison of in vitro growth between a hap2-KO B. bovis clonal line and its parental wild type strain showed that HAP2 is not required for the development of B. bovis in erythrocytes. However, xanthurenic acid-in vitro induction experiments of sexual stages of parasites recovered after tick transmission resulted in surface expression of HAP2 exclusively in sexual stage induced parasites. In addition, hap2-KO parasites were not able to develop such sexual stages as defined both by morphology and by expression of the B. bovis sexual marker genes 6-Cys A and B. Together, the data strongly suggests that tick midgut stage differential expression of hap2 is associated with the development of B. bovis sexual forms. Overall these studies are consistent with a role of HAP2 in tick stages of the parasite and suggest that HAP2 is a potential candidate for a transmission blocking vaccine against bovine babesiosis.

  8. The Babesia bovis hap2 gene is not required for blood stage replication, but expressed upon in vitro sexual stage induction

    Science.gov (United States)

    Hussein, Hala E.; Bastos, Reginaldo G.; Schneider, David A.; Johnson, Wendell C.; Adham, Fatma K.; Davis, William C.; Laughery, Jacob M.; Herndon, David R.; Alzan, Heba F.

    2017-01-01

    Babesia bovis, is a tick borne apicomplexan parasite responsible for important cattle losses globally. Babesia parasites have a complex life cycle including asexual replication in the mammalian host and sexual reproduction in the tick vector. Novel control strategies aimed at limiting transmission of the parasite are needed, but transmission blocking vaccine candidates remain undefined. Expression of HAP2 has been recognized as critical for the fertilization of parasites in the Babesia-related Plasmodium, and is a leading candidate for a transmission blocking vaccine against malaria. Hereby we identified the B. bovis hap2 gene and demonstrated that it is widely conserved and differentially transcribed during development within the tick midgut, but not by blood stage parasites. The hap2 gene was disrupted by transfecting B. bovis with a plasmid containing the flanking regions of the hap2 gene and the GPF-BSD gene under the control of the ef-1α-B promoter. Comparison of in vitro growth between a hap2-KO B. bovis clonal line and its parental wild type strain showed that HAP2 is not required for the development of B. bovis in erythrocytes. However, xanthurenic acid-in vitro induction experiments of sexual stages of parasites recovered after tick transmission resulted in surface expression of HAP2 exclusively in sexual stage induced parasites. In addition, hap2-KO parasites were not able to develop such sexual stages as defined both by morphology and by expression of the B. bovis sexual marker genes 6-Cys A and B. Together, the data strongly suggests that tick midgut stage differential expression of hap2 is associated with the development of B. bovis sexual forms. Overall these studies are consistent with a role of HAP2 in tick stages of the parasite and suggest that HAP2 is a potential candidate for a transmission blocking vaccine against bovine babesiosis. PMID:28985216

  9. Transcriptional control of the tissue-specific, developmentally regulated osteocalcin gene requires a binding motif for the Msx family of homeodomain proteins.

    Science.gov (United States)

    Hoffmann, H M; Catron, K M; van Wijnen, A J; McCabe, L R; Lian, J B; Stein, G S; Stein, J L

    1994-12-20

    The OC box of the rat osteocalcin promoter (nt -99 to -76) is the principal proximal regulatory element contributing to both tissue-specific and developmental control of osteocalcin gene expression. The central motif of the OC box includes a perfect consensus DNA binding site for certain homeodomain proteins. Homeodomain proteins are transcription factors that direct proper development by regulating specific temporal and spatial patterns of gene expression. We therefore addressed the role of the homeodomain binding motif in the activity of the OC promoter. In this study, by the combined application of mutagenesis and site-specific protein recognition analysis, we examined interactions of ROS 17/2.8 osteosarcoma cell nuclear proteins and purified Msx-1 homeodomain protein with the OC box. We detected a series of related specific protein-DNA interactions, a subset of which were inhibited by antibodies directed against the Msx-1 homeodomain but which also recognize the Msx-2 homeodomain. Our results show that the sequence requirements for binding the Msx-1 or Msx-2 homeodomain closely parallel those necessary for osteocalcin gene promoter activity in vivo. This functional relationship was demonstrated by transient expression in ROS 17/2.8 osteosarcoma cells of a series of osteocalcin promoter (nt -1097 to +24)-reporter gene constructs containing mutations within and flanking the homeodomain binding site of the OC box. Northern blot analysis of several bone-related cell types showed that all of the cells expressed msx-1, whereas msx-2 expression was restricted to cells transcribing osteocalcin. Taken together, our results suggest a role for Msx-1 and -2 or related homeodomain proteins in transcription of the osteocalcin gene.

  10. TaCPK2-A, a calcium-dependent protein kinase gene that is required for wheat powdery mildew resistance enhances bacterial blight resistance in transgenic rice.

    Science.gov (United States)

    Geng, Shuaifeng; Li, Aili; Tang, Lichuan; Yin, Lingjie; Wu, Liang; Lei, Cailin; Guo, Xiuping; Zhang, Xin; Jiang, Guanghuai; Zhai, Wenxue; Wei, Yuming; Zheng, Youliang; Lan, Xiujin; Mao, Long

    2013-08-01

    Calcium-dependent protein kinases (CPKs) are important Ca2+ signalling components involved in complex immune and stress signalling networks; but the knowledge of CPK gene functions in the hexaploid wheat is limited. Previously, TaCPK2 was shown to be inducible by powdery mildew (Blumeria graminis tritici, Bgt) infection in wheat. Here, its functions in disease resistance are characterized further. This study shows the presence of defence-response and cold-response cis-elements on the promoters of the A subgenome homoeologue (TaCPK2-A) and D subgenome homoeologue (TaCPK2-D), respectively. Their expression patterns were then confirmed by quantitative real-time PCR (qRT-PCR) using genome-specific primers, where TaCPK2-A was induced by Bgt treatment while TaCPK2-D mainly responded to cold treatment. Downregulation of TaCPK2-A by virus-induced gene silencing (VIGS) causes loss of resistance to Bgt in resistant wheat lines, indicating that TaCPK2-A is required for powdery mildew resistance. Furthermore, overexpression of TaCPK2-A in rice enhanced bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo) resistance. qRT-PCR analysis showed that overexpression of TaCPK2-A in rice promoted the expression of OsWRKY45-1, a transcription factor involved in both fungal and bacterial resistance by regulating jasmonic acid and salicylic acid signalling genes. The opposite effect was found in wheat TaCPK2-A VIGS plants, where the homologue of OsWRKY45-1 was significantly repressed. These data suggest that modulation of WRKY45-1 and associated defence-response genes by CPK2 genes may be the common mechanism for multiple disease resistance in grass species, which may have undergone subfunctionalization in promoters before the formation of hexaploid wheat.

  11. The Aspergillus nidulans acuL gene encodes a mitochondrial carrier required for the utilization of carbon sources that are metabolized via the TCA cycle.

    Science.gov (United States)

    Flipphi, Michel; Oestreicher, Nathalie; Nicolas, Valérie; Guitton, Audrey; Vélot, Christian

    2014-07-01

    In Aspergillus nidulans, the utilization of acetate as sole carbon source requires several genes (acu). Most of them are also required for the utilization of fatty acids. This is the case for acuD and acuE, which encode the two glyoxylate cycle-specific enzymes, isocitrate lyase and malate synthase, respectively, but also for acuL that we have identified as AN7287, and characterized in this study. Deletion of acuL resulted in the same phenotype as the original acuL217 mutant. acuL encodes a 322-amino acid protein which displays all the structural features of a mitochondrial membrane carrier, and shares 60% identity with the Saccharomyces cerevisiae succinate/fumarate mitochondrial antiporter Sfc1p (also named Acr1p). Consistently, the AcuL protein was shown to localize in mitochondria, and partial cross-complementation was observed between the S. cerevisiae and A. nidulans homologues. Extensive phenotypic characterization suggested that the acuL gene is involved in the utilization of carbon sources that are catabolized via the TCA cycle, and therefore require gluconeogenesis. In addition, acuL proves to be co-regulated with acuD and acuE. Overall, our data suggest that AcuL could link the glyoxylate cycle to gluconeogenesis by exchanging cytoplasmic succinate for mitochondrial fumarate. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals.

    Science.gov (United States)

    Fagard, M; Boutet, S; Morel, J B; Bellini, C; Vaucheret, H

    2000-10-10

    Introduction of transgene DNA may lead to specific degradation of RNAs that are homologous to the transgene transcribed sequence through phenomena named post-transcriptional gene silencing (PTGS) in plants, quelling in fungi, and RNA interference (RNAi) in animals. It was shown previously that PTGS, quelling, and RNAi require a set of related proteins (SGS2, QDE-1, and EGO-1, respectively). Here we report the isolation of Arabidopsis mutants impaired in PTGS which are affected at the Argonaute1 (AGO1) locus. AGO1 is similar to QDE-2 required for quelling and RDE-1 required for RNAi. Sequencing of ago1 mutants revealed one amino acid essential for PTGS that is also present in QDE-2 and RDE-1 in a highly conserved motif. Taken together, these results confirm the hypothesis that these processes derive from a common ancestral mechanism that controls expression of invading nucleic acid molecules at the post-transcriptional level. As opposed to rde-1 and qde-2 mutants, which are viable, ago1 mutants display several developmental abnormalities, including sterility. These results raise the possibility that PTGS, or at least some of its elements, could participate in the regulation of gene expression during development in plants.

  13. The nociception genes painless and Piezo are required for the cellular immune response of Drosophila larvae to wasp parasitization.

    Science.gov (United States)

    Tokusumi, Yumiko; Tokusumi, Tsuyoshi; Schulz, Robert A

    2017-05-13

    In vertebrates, interaction between the nervous system and immune system is important to protect a challenged host from stress inputs from external sources. In this study, we demonstrate that sensory neurons are involved in the cellular immune response elicited by wasp infestation of Drosophila larvae. Multidendritic class IV neurons sense contacts from external stimuli and induce avoidance behaviors for host defense. Our findings show that inactivation of these sensory neurons impairs the cellular response against wasp parasitization. We also demonstrate that the nociception genes encoding the mechanosensory receptors Painless and Piezo, both expressed in class IV neurons, are essential for the normal cellular immune response to parasite challenge. Copyright © 2017. Published by Elsevier Inc.

  14. SPDEF is required for mouse pulmonary goblet cell differentiation and regulates a network of genes associated with mucus production.

    Science.gov (United States)

    Chen, Gang; Korfhagen, Thomas R; Xu, Yan; Kitzmiller, Joseph; Wert, Susan E; Maeda, Yutaka; Gregorieff, Alexander; Clevers, Hans; Whitsett, Jeffrey A

    2009-10-01

    Various acute and chronic inflammatory stimuli increase the number and activity of pulmonary mucus-producing goblet cells, and goblet cell hyperplasia and excess mucus production are central to the pathogenesis of chronic pulmonary diseases. However, little is known about the transcriptional programs that regulate goblet cell differentiation. Here, we show that SAM-pointed domain-containing Ets-like factor (SPDEF) controls a transcriptional program critical for pulmonary goblet cell differentiation in mice. Initial cell-lineage-tracing analysis identified nonciliated secretory epithelial cells, known as Clara cells, as the progenitors of goblet cells induced by pulmonary allergen exposure in vivo. Furthermore, in vivo expression of SPDEF in Clara cells caused rapid and reversible goblet cell differentiation in the absence of cell proliferation. This was associated with enhanced expression of genes regulating goblet cell differentiation and protein glycosylation, including forkhead box A3 (Foxa3), anterior gradient 2 (Agr2), and glucosaminyl (N-acetyl) transferase 3, mucin type (Gcnt3). Consistent with these findings, levels of SPDEF and FOXA3 were increased in mouse goblet cells after sensitization with pulmonary allergen, and the proteins were colocalized in goblet cells lining the airways of patients with chronic lung diseases. Deletion of the mouse Spdef gene resulted in the absence of goblet cells in tracheal/laryngeal submucosal glands and in the conducting airway epithelium after pulmonary allergen exposure in vivo. These data show that SPDEF plays a critical role in regulating a transcriptional network mediating the goblet cell differentiation and mucus hyperproduction associated with chronic pulmonary disorders.

  15. The mechanosensory structure of the hair cell requires clarin-1, a protein encoded by Usher syndrome III causative gene.

    Science.gov (United States)

    Geng, Ruishuang; Melki, Sami; Chen, Daniel H-C; Tian, Guilian; Furness, David N; Oshima-Takago, Tomoko; Neef, Jakob; Moser, Tobias; Askew, Charles; Horwitz, Geoff; Holt, Jeffrey R; Imanishi, Yoshikazu; Alagramam, Kumar N

    2012-07-11

    Mutation in the clarin-1 gene (Clrn1) results in loss of hearing and vision in humans (Usher syndrome III), but the role of clarin-1 in the sensory hair cells is unknown. Clarin-1 is predicted to be a four transmembrane domain protein similar to members of the tetraspanin family. Mice carrying null mutation in the clarin-1 gene (Clrn1(-/-)) show loss of hair cell function and a possible defect in ribbon synapse. We investigated the role of clarin-1 using various in vitro and in vivo approaches. We show by immunohistochemistry and patch-clamp recordings of Ca(2+) currents and membrane capacitance from inner hair cells that clarin-1 is not essential for formation or function of ribbon synapse. However, reduced cochlear microphonic potentials, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] loading, and transduction currents pointed to diminished cochlear hair bundle function in Clrn1(-/-) mice. Electron microscopy of cochlear hair cells revealed loss of some tall stereocilia and gaps in the v-shaped bundle, although tip links and staircase arrangement of stereocilia were not primarily affected by Clrn1(-/-) mutation. Human clarin-1 protein expressed in transfected mouse cochlear hair cells localized to the bundle; however, the pathogenic variant p.N48K failed to localize to the bundle. The mouse model generated to study the in vivo consequence of p.N48K in clarin-1 (Clrn1(N48K)) supports our in vitro and Clrn1(-/-) mouse data and the conclusion that CLRN1 is an essential hair bundle protein. Furthermore, the ear phenotype in the Clrn1(N48K) mouse suggests that it is a valuable model for ear disease in CLRN1(N48K), the most prevalent Usher syndrome III mutation in North America.

  16. The Mechanosensory Structure of the Hair Cell Requires Clarin-1, a Protein Encoded by Usher Syndrome III Causative Gene

    Science.gov (United States)

    Geng, Ruishuang; Melki, Sami; Chen, Daniel H.-C.; Tian, Guilian; Furness, David; Oshima-Takago, Tomoko; Neef, Jakob; Moser, Tobias; Askew, Charles; Horwitz, Geoff; Holt, Jeffrey; Imanishi, Yoshikazu; Alagramam, Kumar N.

    2012-01-01

    Mutation in the clarin-1 gene results in loss of hearing and vision in humans (Usher syndrome III), but the role of clarin-1 in the sensory hair cells is unknown. Clarin-1 is predicted to be a four transmembrane domain protein similar to members of the tetraspanin family. Mice carrying null mutation in the clarin-1 (Clrn1−/−) gene show loss of hair cell function and a possible defect in ribbon synapse. We investigated the role of clarin-1 using various in vitro and in vivo approaches. We show by immunohistochemistry and patch-clamp recordings of Ca2+ currents and membrane capacitance from IHCs that clarin-1 is not essential for formation or function of ribbon synapse. However, reduced cochlear microphonic potentials, FM1-43 loading and transduction currents pointed to diminished cochlear hair bundle function in Clrn1−/− mice. Electron microscopy of cochlear hair cells revealed loss of some tall stereocilia and gaps in the v-shaped bundle, although tip-links and staircase arrangement of stereocilia were not primarily affected by Clrn1−/− mutation. Human clarin-1 protein expressed in transfected mouse cochlear hair cells localized to the bundle; however, the pathogenic variant, p.N48K, failed to localize to the bundle. The mouse model generated to study the in vivo consequence of p. N48K in clarin-1 (Clrn1N48K) supports our in vitro and Clrn1−/− mouse data and the conclusion that CLRN1 is an essential hair bundle protein. Further, the ear phenotype in the Clrn1N48K mouse suggests that it is a valuable model for ear disease in CLRN1N48K, the most prevalent Usher III mutation in North America. PMID:22787034

  17. Genomic Variability of Haemophilus influenzae Isolated from Mexican Children Determined by Using Enterobacterial Repetitive Intergenic Consensus Sequences and PCR

    OpenAIRE

    Gomez-De-Leon, Patricia; Santos, Jose I.; Caballero, Javier; Gomez, Demostenes; Espinosa, Luz E.; Moreno, Isabel; Piñero, Daniel; Cravioto, Alejandro

    2000-01-01

    Genomic fingerprints from 92 capsulated and noncapsulated strains of Haemophilus influenzae from Mexican children with different diseases and healthy carriers were generated by PCR using the enterobacterial repetitive intergenic consensus (ERIC) sequences. A cluster analysis by the unweighted pair-group method with arithmetic averages based on the overall similarity as estimated from the characteristics of the genomic fingerprints, was conducted to group the strains. A total of 69 fingerprint...

  18. The pro1(+) gene from Sordaria macrospora encodes a C6 zinc finger transcription factor required for fruiting body development.

    Science.gov (United States)

    Masloff, S; Pöggeler, S; Kück, U

    1999-05-01

    During sexual morphogenesis, the filamentous ascomycete Sordaria macrospora differentiates into multicellular fruiting bodies called perithecia. Previously it has been shown that this developmental process is under polygenic control. To further understand the molecular mechanisms involved in fruiting body formation, we generated the protoperithecia forming mutant pro1, in which the normal development of protoperithecia into perithecia has been disrupted. We succeeded in isolating a cosmid clone from an indexed cosmid library, which was able to complement the pro1(-) mutation. Deletion analysis, followed by DNA sequencing, subsequently demonstrated that fertility was restored to the pro1 mutant by an open reading frame encoding a 689-amino-acid polypeptide, which we named PRO1. A region from this polypeptide shares significant homology with the DNA-binding domains found in fungal C6 zinc finger transcription factors, such as the GAL4 protein from yeast. However, other typical regions of C6 zinc finger proteins, such as dimerization elements, are absent in PRO1. The involvement of the pro1(+) gene in fruiting body development was further confirmed by trying to complement the mutant phenotype with in vitro mutagenized and truncated versions of the pro1 open reading frame. Southern hybridization experiments also indicated that pro1(+) homologues are present in other sexually propagating filamentous ascomycetes.

  19. The FBPase Encoding Gene glpX Is Required for Gluconeogenesis, Bacterial Proliferation and Division In Vivo of Mycobacterium marinum.

    Science.gov (United States)

    Tong, Jingfeng; Meng, Lu; Wang, Xinwei; Liu, Lixia; Lyu, Liangdong; Wang, Chuan; Li, Yang; Gao, Qian; Yang, Chen; Niu, Chen

    2016-01-01

    Lipids have been identified as important carbon sources for Mycobacterium tuberculosis (Mtb) to utilize in vivo. Thus gluconeogenesis bears a key role for Mtb to survive and replicate in host. A rate-limiting enzyme of gluconeogenesis, fructose 1, 6-bisphosphatase (FBPase) is encoded by the gene glpX. The functions of glpX were studied in M. marinum, a closely related species to Mtb. The glpX deletion strain (ΔglpX) displayed altered gluconeogenesis, attenuated virulence, and altered bacterial proliferation. Metabolic profiles indicate an accumulation of the FBPase substrate, fructose 1, 6-bisphosphate (FBP) and altered gluconeogenic flux when ΔglpX is cultivated in a gluconeogenic carbon substrate, acetate. In both macrophages and zebrafish, the proliferation of ΔglpX was halted, resulting in dramatically attenuated virulence. Intracellular ΔglpX exhibited an elongated morphology, which was also observed when ΔglpX was grown in a gluconeogenic carbon source. This elongated morphology is also supported by the observation of unseparated multi-nucleoid cell, indicating that a complete mycobacterial division in vivo is correlated with intact gluconeogenesis. Together, our results indicate that glpX has essential functions in gluconeogenesis, and plays an indispensable role in bacterial proliferation in vivo and virulence of M. marinum.

  20. Cortical Development Requires Mesodermal Expression of Tbx1, a Gene Haploinsufficient in 22q11.2 Deletion Syndrome.

    Science.gov (United States)

    Flore, Gemma; Cioffi, Sara; Bilio, Marchesa; Illingworth, Elizabeth

    2017-03-01

    In mammals, proper temporal control of neurogenesis and neural migration during embryonic development ensures correct formation of the cerebral cortex. Changes in the distribution of cortical projection neurons and interneurons are associated with behavioral disorders and psychiatric diseases, including schizophrenia and autism, suggesting that disrupted cortical connectivity contributes to the brain pathology. TBX1 is the major candidate gene for 22q11.2 deletion syndrome (22q11.2DS), a chromosomal deletion disorder characterized by a greatly increased risk for schizophrenia. We have previously shown that Tbx1 heterozygous mice have reduced prepulse inhibition, a behavioral abnormality that is associated with 22q11.2DS and nonsyndromic schizophrenia. Here, we show that loss of Tbx1 disrupts corticogenesis in mice by promoting premature neuronal differentiation in the medio-lateral embryonic cortex, which gives rise to the somatosensory cortex (S1). In addition, we found altered polarity in both radially migrating excitatory neurons and tangentially migrating inhibitory interneurons. Together, these abnormalities lead to altered lamination in the S1 at the terminal stages of corticogenesis in Tbx1 null mice and similar anomalies in Tbx1 heterozygous adult mice. Finally, we show that mesoderm-specific inactivation of Tbx1 is sufficient to recapitulate the brain phenotype indicating that Tbx1 exerts a cell nonautonomous role in cortical development from the mesoderm. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Genes Required for Survival in Microgravity Revealed by Genome-Wide Yeast Deletion Collections Cultured during Spaceflight

    Directory of Open Access Journals (Sweden)

    Corey Nislow

    2015-01-01

    Full Text Available Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However, the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic, unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen, in parallel, the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes yeast deletion strains, each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground, as well as plus and minus hyperosmolar sodium chloride, providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness.

  2. Identification of Streptococcus sanguinis Genes Required for Biofilm Formation and Examination of Their Role in Endocarditis Virulence▿

    Science.gov (United States)

    Ge, Xiuchun; Kitten, Todd; Chen, Zhenming; Lee, Sehmi P.; Munro, Cindy L.; Xu, Ping

    2008-01-01

    Streptococcus sanguinis is one of the pioneers in the bacterial colonization of teeth and is one of the most abundant species in the oral biofilm called dental plaque. S. sanguinis is also the most common viridans group streptococcal species implicated in infective endocarditis. To investigate the association of biofilm and endocarditis, we established a biofilm assay and examined biofilm formation with a signature-tagged mutagenesis library of S. sanguinis. Four genes that have not previously been associated with biofilm formation in any other bacterium, purB, purL, thrB, and pyrE, were putatively identified as contributing to in vitro biofilm formation in S. sanguinis. By examining 800 mutants for attenuation in the rabbit endocarditis model and for reduction in biofilm formation in vitro, we found some mutants that were both biofilm defective and attenuated for endocarditis. However, we also identified mutants with only reduced biofilm formation or with only attenuation in the endocarditis model. This result indicates that the ability to form biofilms in vitro is not associated with endocarditis virulence in vivo in S. sanguinis. PMID:18390999

  3. Identification of Streptococcus sanguinis genes required for biofilm formation and examination of their role in endocarditis virulence.

    Science.gov (United States)

    Ge, Xiuchun; Kitten, Todd; Chen, Zhenming; Lee, Sehmi P; Munro, Cindy L; Xu, Ping

    2008-06-01

    Streptococcus sanguinis is one of the pioneers in the bacterial colonization of teeth and is one of the most abundant species in the oral biofilm called dental plaque. S. sanguinis is also the most common viridans group streptococcal species implicated in infective endocarditis. To investigate the association of biofilm and endocarditis, we established a biofilm assay and examined biofilm formation with a signature-tagged mutagenesis library of S. sanguinis. Four genes that have not previously been associated with biofilm formation in any other bacterium, purB, purL, thrB, and pyrE, were putatively identified as contributing to in vitro biofilm formation in S. sanguinis. By examining 800 mutants for attenuation in the rabbit endocarditis model and for reduction in biofilm formation in vitro, we found some mutants that were both biofilm defective and attenuated for endocarditis. However, we also identified mutants with only reduced biofilm formation or with only attenuation in the endocarditis model. This result indicates that the ability to form biofilms in vitro is not associated with endocarditis virulence in vivo in S. sanguinis.

  4. Multiple Taf subunits of TFIID interact with Ino2 activation domains and contribute to expression of genes required for yeast phospholipid biosynthesis.

    Science.gov (United States)

    Hintze, Stefan; Engelhardt, Maike; van Diepen, Laura; Witt, Eric; Schüller, Hans-Joachim

    2017-12-01

    Expression of phospholipid biosynthetic genes in yeast requires activator protein Ino2 which can bind to the UAS element inositol/choline-responsive element (ICRE) and trigger activation of target genes, using two separate transcriptional activation domains, TAD1 and TAD2. However, it is still unknown which cofactors mediate activation by TADs of Ino2. Here, we show that multiple subunits of basal transcription factor TFIID (TBP-associated factors Taf1, Taf4, Taf6, Taf10 and Taf12) are able to interact in vitro with activation domains of Ino2. Interaction was no longer observed with activation-defective variants of TAD1. We were able to identify two nonoverlapping regions in the N-terminus of Taf1 (aa 1-100 and aa 182-250) each of which could interact with TAD1 of Ino2 as well as with TAD4 of activator Adr1. Specific missense mutations within Taf1 domain aa 182-250 affecting basic and hydrophobic residues prevented interaction with wild-type TAD1 and caused reduced expression of INO1. Using chromatin immunoprecipitation we demonstrated Ino2-dependent recruitment of Taf1 and Taf6 to ICRE-containing promoters INO1 and CHO2. Transcriptional derepression of INO1 was no longer possible with temperature-sensitive taf1 and taf6 mutants cultivated under nonpermissive conditions. This result supports the hypothesis of Taf-dependent expression of structural genes activated by Ino2. © 2017 John Wiley & Sons Ltd.

  5. HU participates in expression of a specific set of genes required for growth and survival at acidic pH in Escherichia coli.

    Science.gov (United States)

    Bi, Hongkai; Sun, Lianle; Fukamachi, Toshihiko; Saito, Hiromi; Kobayashi, Hiroshi

    2009-05-01

    The major histone-like Escherichia coli protein, HU, is composed of alpha and beta subunits respectively encoded by hupA and hupB in Escherichia coli. A mutant deficient in both hupA and hupB grew at a slightly slower rate than the wild type at pH 7.5. Growth of the mutant diminished with a decrease in pH, and no growth was observed at pH 4.6. Mutants of either hupA or hupB grew at all pH levels tested. The arginine-dependent survival at pH 2.5 was diminished approximately 60-fold by the deletion of both hupA and hupB, whereas the survival was slightly affected by the deletion of either hupA or hupB. The mRNA levels of adiA and adiC, which respectively encode arginine decarboxylase and arginine/agmatine antiporter, were low in the mutant deficient in both hupA and hupB. The deletion of both hupA and hupB had little effect on survival at pH 2.5 in the presence of glutamate or lysine, and expression of the genes for glutamate and lysine decarboxylases was not impaired by the deletion of the HU genes. These results suggest that HU regulates expression of the specific set of genes required for growth and survival in acidic environments.

  6. Machine Learning Analysis Identifies Drosophila Grunge/Atrophin as an Important Learning and Memory Gene Required for Memory Retention and Social Learning.

    Science.gov (United States)

    Kacsoh, Balint Z; Greene, Casey S; Bosco, Giovanni

    2017-11-06

    High-throughput experiments are becoming increasingly common, and scientists must balance hypothesis-driven experiments with genome-wide data acquisition. We sought to predict novel genes involved in Drosophila learning and long-term memory from existing public high-throughput data. We performed an analysis using PILGRM, which analyzes public gene expression compendia using machine learning. We evaluated the top prediction alongside genes involved in learning and memory in IMP, an interface for functional relationship networks. We identified Grunge/Atrophin ( Gug/Atro ), a transcriptional repressor, histone deacetylase, as our top candidate. We find, through multiple, distinct assays, that Gug has an active role as a modulator of memory retention in the fly and its function is required in the adult mushroom body. Depletion of Gug specifically in neurons of the adult mushroom body, after cell division and neuronal development is complete, suggests that Gug function is important for memory retention through regulation of neuronal activity, and not by altering neurodevelopment. Our study provides a previously uncharacterized role for Gug as a possible regulator of neuronal plasticity at the interface of memory retention and memory extinction. Copyright © 2017 Kacsoh et al.

  7. The Vaccinium corymbosum FLOWERING LOCUS T-like gene (VcFT): a flowering activator reverses photoperiodic and chilling requirements in blueberry.

    Science.gov (United States)

    Song, Guo-qing; Walworth, Aaron; Zhao, Dongyan; Jiang, Ning; Hancock, James F

    2013-11-01

    The blueberry FLOWERING LOCUS T ( FT )-like gene ( VcFT ) cloned from the cDNA of a tetraploid, northern highbush blueberry ( Vaccinium corymbosum L.) is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering. Blueberry is a woody perennial bush with a longer juvenile period than annual crops, requiring vernalization to flower normally. Few studies have been reported on the molecular mechanism of flowering in blueberry or other woody plants. Because FLOWERING LOCUS T (FT) from Arabidopsis thaliana plays a multifaceted role in generating mobile molecular signals to regulate plant flowering time, isolation and functional analysis of the blueberry (Vaccinium corymbosum L.) FT-like gene (VcFT) will facilitate the elucidation of molecular mechanisms of flowering in woody plants. Based on EST sequences, a 525-bpVcFT was identified and cloned from the cDNA of a tetraploid, northern highbush blueberry cultivar, Bluecrop. Ectopic expression of 35S:VcFT in tobacco induced flowering an average of 28 days earlier than wild-type plants. Expression of the 35S:VcFT in the blueberry cultivar Aurora resulted in an extremely early flowering phenotype, which flowered not only during in vitro culture, a growth stage when nontransgenic shoots had not yet flowered, but also in 6-10-week old, soil-grown transgenic plants, in contrast to the fact that at least 1 year and 800 chilling hours are required for the appearance of the first flower of both nontransgenic 'Aurora' and transgenic controls with the gusA. These results demonstrate that the VcFT is a functional floral activator and overexpression of the VcFT is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering.

  8. Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

    Science.gov (United States)

    Lu, Chenggang; Fuller, Margaret T

    2015-12-01

    Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s). In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC), a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs), spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages.

  9. Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

    Directory of Open Access Journals (Sweden)

    Chenggang Lu

    2015-12-01

    Full Text Available Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s. In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC, a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs, spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages.

  10. The genetic interaction network of CCW12, a Saccharomyces cerevisiae gene required for cell wall integrity during budding and formation of mating projections

    Science.gov (United States)

    2011-01-01

    Background Mannoproteins construct the outer cover of the fungal cell wall. The covalently linked cell wall protein Ccw12p is an abundant mannoprotein. It is considered as crucial structural cell wall component since in baker's yeast the lack of CCW12 results in severe cell wall damage and reduced mating efficiency. Results In order to explore the function of CCW12, we performed a Synthetic Genetic Analysis (SGA) and identified genes that are essential in the absence of CCW12. The resulting interaction network identified 21 genes involved in cell wall integrity, chitin synthesis, cell polarity, vesicular transport and endocytosis. Among those are PFD1, WHI3, SRN2, PAC10, FEN1 and YDR417C, which have not been related to cell wall integrity before. We correlated our results with genetic interaction networks of genes involved in glucan and chitin synthesis. A core of genes essential to maintain cell integrity in response to cell wall stress was identified. In addition, we performed a large-scale transcriptional analysis and compared the transcriptional changes observed in mutant ccw12Δ with transcriptomes from studies investigating responses to constitutive or acute cell wall damage. We identified a set of genes that are highly induced in the majority of the mutants/conditions and are directly related to the cell wall integrity pathway and cell wall compensatory responses. Among those are BCK1, CHS3, EDE1, PFD1, SLT2 and SLA1 that were also identified in the SGA. In contrast, a specific feature of mutant ccw12Δ is the transcriptional repression of genes involved in mating. Physiological experiments substantiate this finding. Further, we demonstrate that Ccw12p is present at the cell periphery and highly concentrated at the presumptive budding site, around the bud, at the septum and at the tip of the mating projection. Conclusions The combination of high throughput screenings, phenotypic analyses and localization studies provides new insight into the function of Ccw

  11. Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function.

    Science.gov (United States)

    Chen, B; Han, B H; Sun, X H; Lim, R W

    1997-01-15

    We have examined the role of an Id-like protein, Id3 (also known as HLH462), in the regulation of muscle-specific gene expression. Id proteins are believed to block expression of muscle-specific genes by preventing the dimerization between ubiquitous bHLH proteins (E proteins) and myogenic bHLH proteins such as MyoD. Consistent with its putative role as an inhibitor of differentiation, Id3 mRNA was detected in proliferating skeletal muscle cells, was further induced by basic fibroblast growth factor (bFGF) and was down-regulated in differentiated muscle cultures. Overexpression of Id3 efficiently inhibited the MyoD-mediated activation of the muscle-specific creatine kinase (MCK) reporter gene. Deletion analysis indicated that the C-terminal 15 amino acids of Id3 are critical for the full inhibitory activity while deleting up to 42 residues from the C-terminus of the related protein, Id2, did not affect its ability to inhibit the MCK reporter gene. Chimeric protein containing the N-terminal region of Id3 and the C-terminus of Id2 was also non-functional in transfected cells. In contrast, wild-type Id3, the C-terminal mutants, and the Id3/Id2 chimera could all interact with the E-protein E47in vitro. Additional studies indicated that truncation of the Id3 C-terminus might have adversely affected the expression level of the mutant proteins but the Id3/Id2 chimera was stably expressed. Taken together, our results revealed a more complex requirement for the expression and proper function of the Id family proteins than was hitherto expected.

  12. The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N5-oxygenase, is required for virulence.

    Science.gov (United States)

    Hissen, Anna H T; Wan, Adrian N C; Warwas, Mark L; Pinto, Linda J; Moore, Margo M

    2005-09-01

    Aspergillus fumigatus is the leading cause of invasive mold infection and is a serious problem in immunocompromised populations worldwide. We have previously shown that survival of A. fumigatus in serum may be related to secretion of siderophores. In this study, we identified and characterized the sidA gene of A. fumigatus, which encodes l-ornithine N(5)-oxygenase, the first committed step in hydroxamate siderophore biosynthesis. A. fumigatus sidA codes for a protein of 501 amino acids with significant homology to other fungal l-ornithine N(5)-oxygenases. A stable DeltasidA strain was created by deletion of A. fumigatus sidA. This strain was unable to synthesize the siderophores N',N",N'''-triacetylfusarinine C (TAF) and ferricrocin. Growth of the DeltasidA strain was the same as that of the wild type in rich media; however, the DeltasidA strain was unable to grow in low-iron defined media or media containing 10% human serum unless supplemented with TAF or ferricrocin. No significant differences in ferric reduction activities were observed between the parental strain and the DeltasidA strain, indicating that blocking siderophore secretion did not result in upregulation of this pathway. Unlike the parental strain, the DeltasidA strain was unable to remove iron from human transferrin. A rescued strain (DeltasidA + sidA) was constructed; it produced siderophores and had the same growth as the wild type on iron-limited media. Unlike the wild-type and rescued strains, the DeltasidA strain was avirulent in a mouse model of invasive aspergillosis, indicating that sidA is necessary for A. fumigatus virulence.

  13. The baculovirus core gene ac83 is required for nucleocapsid assembly and per os infectivity of Autographa californica nucleopolyhedrovirus.

    Science.gov (United States)

    Zhu, Shimao; Wang, Wei; Wang, Yan; Yuan, Meijin; Yang, Kai

    2013-10-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac83 is a baculovirus core gene whose function in the AcMNPV life cycle is unknown. In the present study, an ac83-knockout AcMNPV (vAc83KO) was constructed to investigate the function of ac83 through homologous recombination in Escherichia coli. No budded virions were produced in vAc83KO-transfected Sf9 cells, although viral DNA replication was unaffected. Electron microscopy revealed that nucleocapsid assembly was aborted due to the ac83 deletion. Domain-mapping studies revealed that the expression of Ac83 amino acid residues 451 to 600 partially rescued the ability of AcMNPV to produce infectious budded virions. Bioassays indicated that deletion of the chitin-binding domain of Ac83 resulted in the failure of oral infection of Trichoplusia ni larvae by AcMNPV, but AcMNPV remained infectious following intrahemocoelic injection, suggesting that the domain is involved in the binding of occlusion-derived virions to the peritrophic membrane and/or to other chitin-containing insect tissues. It has been demonstrated that Ac83 is the only component with a chitin-binding domain in the per os infectivity factor complex on the occlusion-derived virion envelope. Interestingly, a functional inner nuclear membrane sorting motif, which may facilitate the localization of Ac83 to the envelopes of occlusion-derived virions, was identified by immunofluorescence analysis. Taken together, these results demonstrate that Ac83 plays an important role in nucleocapsid assembly and the establishment of oral infection.

  14. The milkweed pod1 Gene Encodes a KANADI Protein That Is Required for Abaxial/Adaxial Patterning in Maize Leaves[W

    Science.gov (United States)

    Candela, Héctor; Johnston, Robyn; Gerhold, Abigail; Foster, Toshi; Hake, Sarah

    2008-01-01

    Leaf primordia initiate from the shoot apical meristem with inherent polarity; the adaxial side faces the meristem, while the abaxial side faces away from the meristem. Adaxial/abaxial polarity is thought to be necessary for laminar growth of leaves, as mutants lacking either adaxial or abaxial cell types often develop radially symmetric lateral organs. The milkweed pod1 (mwp1) mutant of maize (Zea mays) has adaxialized sectors in the sheath, the proximal part of the leaf. Ectopic leaf flaps develop where adaxial and abaxial cell types juxtapose. Ectopic expression of the HD-ZIPIII gene rolled leaf1 (rld1) correlates with the adaxialized regions. Cloning of mwp1 showed that it encodes a KANADI transcription factor. Double mutants of mwp1-R with a microRNA-resistant allele of rld1, Rld1-N1990, show a synergistic phenotype with polarity defects in sheath and blade and a failure to differentiate vascular and photosynthetic cell types in the adaxialized sectors. The sectored phenotype and timing of the defect suggest that mwp1 is required late in leaf development to maintain abaxial cell fate. The phenotype of mwp1; Rld1 double mutants shows that both genes are also required early in leaf development to delineate leaf margins as well as to initiate vascular and photosynthetic tissues. PMID:18757553

  15. Hydrolysis of N-succinyl-L,L-diaminopimelic acid by the Haemophilus influenzae dapE-encoded desuccinylase: metal activation, solvent isotope effects, and kinetic mechanism.

    Science.gov (United States)

    Born, T L; Zheng, R; Blanchard, J S

    1998-07-21

    Hydrolysis of N-succinyl-L,L-diaminopimelic acid by the dapE-encoded desuccinylase is required for the bacterial synthesis of lysine and meso-diaminopimelic acid. We have investigated the catalytic mechanism of the recombinant enzyme from Haemophilus influenzae. The desuccinylase was overexpressed in Escherichia coli and purified to homogeneity. Steady-state kinetic experiments verified that the enzyme is metal-dependent, with a Km for N-succinyl-L,L-diaminopimelic acid of 1.3 mM and a turnover number of 200 s-1 in the presence of zinc. The maximal velocity was independent of pH above 7 but decreased with a slope of 1 below pH 7. The pH dependence of V/K was bell-shaped with apparent pKs of 6.5 and 8.3. Both L,L- and D,L-diaminopimelic acid were competitive inhibitors of the substrate, but d,d-diaminopimelic acid was not. Solvent kinetic isotope effect studies yielded inverse isotope effects, with values for D2OV/K of 0.62 and D2OV of 0.78. Determination of metal stoichiometry by ICP-AES indicated one tightly bound metal ion, while sequence homologies suggest the presence of two metal binding sites. On the basis of these observations, we propose a chemical mechanism for this metalloenzyme, which has a number of important structurally defined homologues.

  16. Somatostatin is required for masculinization of growth hormone–regulated hepatic gene expression but not of somatic growth

    Science.gov (United States)

    Low, Malcolm J.; Otero-Corchon, Veronica; Parlow, Albert F.; Ramirez, Jose L.; Kumar, Ujendra; Patel, Yogesh C.; Rubinstein, Marcelo

    2001-01-01

    Pulsatile growth hormone (GH) secretion differs between males and females and regulates the sex-specific expression of cytochrome P450s in liver. Sex steroids influence the secretory dynamics of GH, but the neuroendocrine mechanisms have not been conclusively established. Because periventricular hypothalamic somatostatin (SST) expression is greater in males than in females, we generated knockout (Smst–/–) mice to investigate whether SST peptides are necessary for sexually differentiated GH secretion and action. Despite marked increases in nadir and median plasma GH levels in both sexes of Smst–/– compared with Smst+/+ mice, the mutant mice had growth curves identical to their sibling controls and retained a normal sexual dimorphism in weight and length. In contrast, the liver of male Smst–/– mice was feminized, resulting in an identical profile of GH-regulated hepatic mRNAs between male and female mutants. Male Smst-/- mice show higher expression of two SST receptors in the hypothalamus and pituitary than do females. These data indicate that SST is required to masculinize the ultradian GH rhythm by suppressing interpulse GH levels. In the absence of SST, male and female mice exhibit similarly altered plasma GH profiles that eliminate sexually dimorphic liver function but do not affect dimorphic growth. PMID:11413165

  17. Development of a subset of forelimb muscles and their attachment sites requires the ulnar-mammary syndrome gene Tbx3

    Directory of Open Access Journals (Sweden)

    Mary P. Colasanto

    2016-11-01

    Full Text Available In the vertebrate limb over 40 muscles are arranged in a precise pattern of attachment via muscle connective tissue and tendon to bone and provide an extensive range of motion. How the development of somite-derived muscle is coordinated with the development of lateral plate-derived muscle connective tissue, tendon and bone to assemble a functional limb musculoskeletal system is a long-standing question. Mutations in the T-box transcription factor, TBX3, have previously been identified as the genetic cause of ulnar-mammary syndrome (UMS, characterized by distinctive defects in posterior forelimb bones. Using conditional mutagenesis in mice, we now show that TBX3 has a broader role in limb musculoskeletal development. TBX3 is not only required for development of posterior forelimb bones (ulna and digits 4 and 5, but also for a subset of posterior muscles (lateral triceps and brachialis and their bone eminence attachment sites. TBX3 specification of origin and insertion sites appears to be tightly linked with whether these particular muscles develop and may represent a newly discovered mechanism for specification of anatomical muscles. Re-examination of an individual with UMS reveals similar previously unrecognized muscle and bone eminence defects and indicates a conserved role for TBX3 in regulating musculoskeletal development.

  18. The Aspergillus flavus Spermidine Synthase (spds Gene, Is Required for Normal Development, Aflatoxin Production, and Pathogenesis During Infection of Maize Kernels

    Directory of Open Access Journals (Sweden)

    Rajtilak Majumdar

    2018-03-01

    Full Text Available Aspergillus flavus is a soil-borne saprophyte and an opportunistic pathogen of both humans and plants. This fungus not only causes disease in important food and feed crops such as maize, peanut, cottonseed, and tree nuts but also produces the toxic and carcinogenic secondary metabolites (SMs known as aflatoxins. Polyamines (PAs are ubiquitous polycations that influence normal growth, development, and stress responses in living organisms and have been shown to play a significant role in fungal pathogenesis. Biosynthesis of spermidine (Spd is critical for cell growth as it is required for hypusination-mediated activation of eukaryotic translation initiation factor 5A (eIF5A, and other biochemical functions. The tri-amine Spd is synthesized from the diamine putrescine (Put by the enzyme spermidine synthase (Spds. Inactivation of spds resulted in a total loss of growth and sporulation in vitro which could be partially restored by addition of exogenous Spd. Complementation of the Δspds mutant with a wild type (WT A. flavus spds gene restored the WT phenotype. In WT A. flavus, exogenous supply of Spd (in vitro significantly increased the production of sclerotia and SMs. Infection of maize kernels with the Δspds mutant resulted in a significant reduction in fungal growth, sporulation, and aflatoxin production compared to controls. Quantitative PCR of Δspds mutant infected seeds showed down-regulation of aflatoxin biosynthetic genes in the mutant compared to WT A. flavus infected seeds. Expression analyses of PA metabolism/transport genes during A. flavus-maize interaction showed significant increase in the expression of arginine decarboxylase (Adc and S-adenosylmethionine decarboxylase (Samdc genes in the maize host and PA uptake transporters in the fungus. The results presented here demonstrate that Spd biosynthesis is critical for normal development and pathogenesis of A. flavus and pre-treatment of a Δspds mutant with Spd or Spd uptake from the

  19. Biofilm-specific extracellular matrix proteins of non-typeable Haemophilus influenzae

    Science.gov (United States)

    Wu, Siva; Baum, Marc M.; Kerwin, James; Guerrero-Given, Debbie; Webster, Simon; Schaudinn, Christoph; VanderVelde, David; Webster, Paul

    2014-01-01

    Non-typeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24 hr and 96 hr NTHi biofilms contained polysaccharides and proteinaceous components as detected by NMR and FTIR spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24 hr biofilms, two were found only in 96 hr biofilms, and fifteen were present in the ECM of both 24 hr and 96 hr NTHi biofilms. All proteins identified were either associated with bacterial membranes or were cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation. PMID:24942343

  20. Mutation of Haemophilus influenzae transforming DNA in vitro with near-ultraviolet radiation: action spectrum

    Energy Technology Data Exchange (ETDEWEB)

    Cabrera-Juarez, E; Setlow, J K [Escuela Nacional de Ciencias Biologicas, Mexico City. Dept. de Bioquimica; Oak Ridge National Lab., Tenn. (USA). Biology Div.)

    1976-05-01

    Mutations were produced in purified transforming DNA from Haemophilus influenzae by near UV radiation and were assayed as mutants among cells transformed with irradiated DNA. The maximum efficiency of mutation induction was at around 334 nm, and the efficiency dropped off steeply at lower and higher wavelengths. The difference between the action spectrum for mutation and that for the oxygen-independent inactivation of transforming DNA, which had a shoulder at 365 nm, indicates that there are different lesions involved in the inactivating and mutagenic effects of near-UV. The presence of histidine during irradiation enhanced the mutagenic effect at 334 and 365 nm, although it protected against inactivation at 365 nm. The effective near-UV wavelengths for in vitro mutation are to some extent the same as the effective wavelengths for mutation in vivo reported previously. These findings indicate that mutations are produced in vivo by near-UV with DNA as the primary target molecule rather than by a secondary non-photochemical reaction between DNA and some other cell component.

  1. [Optimized isolation and purification of non-typeable Haemophilus influenzae Haps protein].

    Science.gov (United States)

    Li, Wan-yi; Kuang, Yu; Li, Ming-yuan; Yang, Yuan; Jiang, Zhong-hua; Yao, Feng; Chen, Chang-chun

    2007-12-01

    To optimize the isolation and purification conditions for Hap(s) protein of non-typeable Haemophilus influenzae. Hap(s) protein was purified by ammonium sulfate precipitation, dialysis desalting and Hitrap weak cation exchange columns of CM Sepharose Fast Flow. The condition of the elution was optimized for pH and ionic strength, the absorbance at 280 nm of the elution samples were detected, and the targeted protein band in the collected samples was observed by SDS-PAGE electrophoresis. The Hitrap ion exchange column was eluted with buffer 1, which resulted in a baseline distribution of absorbance at 280 nm. Buffer 2 elution of the column resulted in the presence of peak absorbance with trails, which was identified to be constituted by some low molecular weight bands by subsequent SDS-PAGE. In serial column elution with buffer 3 with different ionic strength, a peak absorbance was observed with the ionic strength of 100 mmol/L NaCl, and SDS-PAGE confirmed that the peak was generated by the target protein. No obvious peaks or bands in SDS-PAGE occurred with the other ionic strengths. The pH of the buffer only affect the elution of the irrelevant proteins rather than the Hap(s) protein, and elution with the buffer containing 100 mmol/L NaCl can be optimal for eluting the Hap(s) protein.

  2. Structural determinants of the interaction between the Haemophilus influenzae Hap autotransporter and fibronectin.

    Science.gov (United States)

    Spahich, Nicole A; Kenjale, Roma; McCann, Jessica; Meng, Guoyu; Ohashi, Tomoo; Erickson, Harold P; St Geme, Joseph W

    2014-06-01

    Haemophilus influenzae is a Gram-negative cocco-bacillus that initiates infection by colonizing the upper respiratory tract. Hap is an H. influenzae serine protease autotransporter protein that mediates adherence, invasion and microcolony formation in assays with human epithelial cells and is presumed to facilitate the process of colonization. Additionally, Hap mediates adherence to fibronectin, laminin and collagen IV, extracellular matrix (ECM) proteins that are present in the respiratory tract and are probably important targets for H. influenzae colonization. The region of Hap responsible for adherence to ECM proteins has been localized to the C-terminal 511 aa of the Hap passenger domain (HapS). In this study, we characterized the structural determinants of the interaction between HapS and fibronectin. Using defined fibronectin fragments, we established that Hap interacts with the fibronectin repeat fragment called FNIII(1-2). Using site-directed mutagenesis, we found a series of motifs in the C-terminal region of HapS that contribute to the interaction with fibronectin. Most of these motifs are located on the F1 and F3 faces of the HapS structure, suggesting that the F1 and F3 faces may be responsible for the HapS-fibronectin interaction. © 2014 The Authors.

  3. Antimicrobial susceptibility patterns of Haemophilus parasuis from pigs in the United Kingdom and Spain.

    Science.gov (United States)

    de la Fuente, A J Martín; Tucker, A W; Navas, J; Blanco, M; Morris, S J; Gutiérrez-Martín, C B

    2007-02-25

    A total of 30 British and 30 Spanish Haemophilus parasuis isolates were tested for their susceptibility to 19 of the antimicrobials currently used in swine practice with a broth microdilution method in order to know the emergence of resistance against these compounds in this porcine pathogen. All the British isolates were susceptible to penicillin, ceftiofur, erythromycin, tilmicosin, enrofloxacin, and florfenicol, and most of them were susceptible to the remaining antimicrobials (the highest resistance rate found was of 20% to neomycin). In contrast, all the Spanish isolates were susceptible exclusively to florfenicol, and high proportions of resistance were encountered for penicillin, ampicillin, oxytetracycline, erythromycin, tilmicosin, tiamulin and trimethoprim+sulphamethoxazole; in addition, a bimodal or multimodal distribution, or tailing of Spanish isolates over the MIC range was observed for clindamycin, sulphonamides and tylosine tartrate, suggesting the development of acquired resistance. In addition, several multiresistance patterns were found among the Spanish isolates, 23.3% of them being resistant to at least eight antimicrobials, the same rate as that encountered for those being susceptible to all antimicrobials tested. This study showed that in general British H. parasuis isolates are susceptible to antimicrobial agents routinely used for treatment of porcine respiratory diseases; however, the Spanish isolates need a more continuous surveillance of their susceptibility patterns.

  4. Nontypeable Haemophilus influenzae in chronic obstructive pulmonary disease and lung cancer

    Directory of Open Access Journals (Sweden)

    Seyed Javad Moghaddam

    2011-01-01

    Full Text Available Seyed Javad Moghaddam1, Cesar E Ochoa1,2, Sanjay Sethi3, Burton F Dickey1,41Department of Pulmonary Medicine, the University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2Tecnológico de Monterrey School of Medicine, Monterrey, Nuevo León, Mexico; 3Department of Medicine, University at Buffalo, State University of New York, Buffalo, NY, USA; 4Center for Inflammation and Infection, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX, USAAbstract: Chronic obstructive pulmonary disease (COPD is predicted to become the third leading cause of death in the world by 2020. It is characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lungs to noxious particles and gases, most commonly cigarette smoke. Among smokers with COPD, even following withdrawal of cigarette smoke, inflammation persists and lung function continues to deteriorate. One possible explanation is that bacterial colonization of smoke-damaged airways, most commonly with nontypeable Haemophilus influenzae (NTHi, perpetuates airway injury and inflammation. Furthermore, COPD has also been identified as an independent risk factor for lung cancer irrespective of concomitant cigarette smoke exposure. In this article, we review the role of NTHi in airway inflammation that may lead to COPD progression and lung cancer promotion.Keywords: COPD, NTHi, inflammation

  5. Limited Interactions between Streptococcus Suis and Haemophilus Parasuis in In Vitro Co-Infection Studies

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    Annabelle Mathieu-Denoncourt

    2018-01-01

    Full Text Available Streptococcus suis and Haemophilus parasuis are normal inhabitants of the porcine upper respiratory tract but are also among the most frequent causes of disease in weaned piglets worldwide, causing inflammatory diseases such as septicemia, meningitis and pneumonia. Using an in vitro model of infection with tracheal epithelial cells or primary alveolar macrophages (PAMs, it was possible to determine the interaction between S. suis serotype 2 and H. parasuis strains with different level of virulence. Within H. parasuis strains, the low-virulence F9 strain showed higher adhesion levels to respiratory epithelial cells and greater association levels to PAMs than the high-virulence Nagasaki strain. Accordingly, the low-virulence F9 strain induced, in general, higher levels of pro-inflammatory cytokines than the virulent Nagasaki strain from both cell types. In general, S. suis adhesion levels to respiratory epithelial cells were similar to H. parasuis Nagasaki strain. Yet, S. suis strains induced a significantly lower level of pro-inflammatory cytokine expression from epithelial cells and PAMs than those observed with both H. parasuis strains. Finally, this study has shown that, overall and under the conditions used in the present study, S. suis and H. parasuis have limited in vitro interactions between them and use probably different host receptors, regardless to their level of virulence.

  6. Reliability of Haemophilus influenzae biofilm measurement via static method, and determinants of in vitro biofilm production.

    Science.gov (United States)

    Obaid, Najla A; Tristram, Stephen; Narkowicz, Christian K; Jacobson, Glenn A

    2016-12-01

    Information is lacking regarding the precision of microtitre plate (MTP) assays used to measure biofilm. This study investigated the precision of an MTP assay to measure biofilm production by nontypeable Haemophilus influenzae (NTHi) and the effects of frozen storage and inoculation technique on biofilm production. The density of bacterial final growth was determined by absorbance after 18-20 h incubation, and biofilm production was then measured by absorbance after crystal violet staining. Biofilm formation was categorised as high and low for each strain. For the high biofilm producing strains of NTHi, interday reproducibility of NTHi biofilm formation measured using the MTP assay was excellent and met the acceptance criteria, but higher variability was observed in low biofilm producers. Method of inoculum preparation was a determinant of biofilm formation with inoculum prepared directly from solid media showing increased biofilm production for at least one of the high producing strains. In general, storage of NTHi cultures at -80 °C for up to 48 weeks did not have any major effect on their ability to produce biofilm.

  7. Evidence for covalent attachment of phospholipid to the capsular polysaccharide of Haemophilus influenzae type b

    International Nuclear Information System (INIS)

    Kuo, J.S.; Doelling, V.W.; Graveline, J.F.; McCoy, D.W.

    1985-01-01

    Cells of Haemophilus influenzae type b were grown in a liquid medium containing [ 3 H]palmitate or [ 14 C]ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( [ 3 H]palmitate and [ 14 C]ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( [ 3 H]palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. Only after acid, alkaline, or phospholipase A2 treatment of PRP labeled with [ 3 H]palmitate or [ 3 H]palmitate and [ 14 C]ribose followed by chloroform-methanol extraction could most of the 3 H-radioactivity be recovered in the organic phase. The chloroform-soluble acid-hydrolyzed or phospholipase A2-treated product was identified as palmitic acid after thin-layer chromatography. These results strongly suggest that a phospholipid moiety is covalently associated with the H. influenzae type b polysaccharide PRP

  8. Multilocus sequence typing and virulence analysis of Haemophilus parasuis strains isolated in five provinces of China.

    Science.gov (United States)

    Wang, Liyan; Ma, Lina; Liu, Yongan; Gao, Pengcheng; Li, Youquan; Li, Xuerui; Liu, Yongsheng

    2016-10-01

    Haemophilus parasuis is the etiological agent of Glässers disease, which causes high morbidity and mortality in swine herds. Although H. parasuis strains can be classified into 15 serovars with the Kielstein-Rapp-Gabrielson serotyping scheme, a large number of isolates cannot be classified and have been designated 'nontypeable' strains. In this study, multilocus sequence typing (MLST) of H. parasuis was used to analyze 48 H. parasuis field strains isolated in China and two strains from Australia. Twenty-six new alleles and 29 new sequence types (STs) were detected, enriching the H. parasuis MLST databases. A BURST analysis indicated that H. parasuis lacks stable population structure and is highly heterogeneous, and that there is no association between STs and geographic area. When an UPGMA dendrogram was constructed, two major clades, clade A and clade B, were defined. Animal experiments, in which guinea pigs were challenged intraperitoneally with the bacterial isolates, supported the hypothesis that the H. parasuis STs in clade A are generally avirulent or weakly virulent, whereas the STs in clade B tend to be virulent. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Cost-benefit analysis of a Haemophilus influenzae type b meningitis prevention programme in The Philippines.

    Science.gov (United States)

    Limcangco, M R; Armour, C L; Salole, E G; Taylor, S J

    2001-01-01

    Haemophilus influenzae type b (Hib) meningitis is associated with high mortality and serious sequelae in children under 5 years of age. Vaccines which can prevent this infection are available. To evaluate the costs and benefits of a 3-dose immunisation schedule in Manila, Philippines. Government and societal perspectives. A cost-benefit analysis based on a birth cohort of 100,000 children. The state of health of the cohort with and without a Hib immunisation programme was modelled over a 5-year period. A survey of medical records of patients with Hib in Manila provided data on the extent and cost of sequelae following infection. A 3-dose Hib vaccination programme given at ages 2, 3 and 4 months. The model predicted that vaccinating children against Hib meningitis would prevent 553 cases per year in a birth cohort of 100,000, at a cost of 56,200 Philippine pesos (PHP) [$US1,605; 1998 exchange rate] per case (base case assumptions of 90% vaccine efficacy rate, 95 per 100,000 Hib incidence rate, 85% vaccination coverage). Results from the cost-benefit analyses indicated that the saving to the government would be around PHP39 million ($US1.11 million), and the saving to society would be PHP255 million ($US7.28 million). There would be a positive economic benefit for the Philippine government and for the Filipino society if a Hib vaccination programme was introduced in Manila.

  10. Limited Interactions between Streptococcus Suis and Haemophilus Parasuis in In Vitro Co-Infection Studies

    Science.gov (United States)

    Mathieu-Denoncourt, Annabelle; Letendre, Corinne; Auger, Jean-Philippe; Segura, Mariela; Aragon, Virginia; Lacouture, Sonia; Gottschalk, Marcelo

    2018-01-01

    Streptococcus suis and Haemophilus parasuis are normal inhabitants of the porcine upper respiratory tract but are also among the most frequent causes of disease in weaned piglets worldwide, causing inflammatory diseases such as septicemia, meningitis and pneumonia. Using an in vitro model of infection with tracheal epithelial cells or primary alveolar macrophages (PAMs), it was possible to determine the interaction between S. suis serotype 2 and H. parasuis strains with different level of virulence. Within H. parasuis strains, the low-virulence F9 strain showed higher adhesion levels to respiratory epithelial cells and greater association levels to PAMs than the high-virulence Nagasaki strain. Accordingly, the low-virulence F9 strain induced, in general, higher levels of pro-inflammatory cytokines than the virulent Nagasaki strain from both cell types. In general, S. suis adhesion levels to respiratory epithelial cells were similar to H. parasuis Nagasaki strain. Yet, S. suis strains induced a significantly lower level of pro-inflammatory cytokine expression from epithelial cells and PAMs than those observed with both H. parasuis strains. Finally, this study has shown that, overall and under the conditions used in the present study, S. suis and H. parasuis have limited in vitro interactions between them and use probably different host receptors, regardless to their level of virulence. PMID:29316613

  11. A biphasic epigenetic switch controls immunoevasion, virulence and niche adaptation in non-typeable Haemophilus influenzae.

    Science.gov (United States)

    Atack, John M; Srikhanta, Yogitha N; Fox, Kate L; Jurcisek, Joseph A; Brockman, Kenneth L; Clark, Tyson A; Boitano, Matthew; Power, Peter M; Jen, Freda E-C; McEwan, Alastair G; Grimmond, Sean M; Smith, Arnold L; Barenkamp, Stephen J; Korlach, Jonas; Bakaletz, Lauren O; Jennings, Michael P

    2015-07-28

    Non-typeable Haemophilus influenzae contains an N(6)-adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression (random ON/OFF switching). Five modA alleles, modA2, modA4, modA5, modA9 and modA10, account for over two-thirds of clinical otitis media isolates surveyed. Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all five of these modA alleles. Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biofilm formation (modA2) and immunoevasion (modA4). Analyses of a modA2 strain in the chinchilla model of otitis media show a clear selection for ON switching of modA2 in the middle ear. Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.

  12. Nontypeable Haemophilus influenzae induces sustained lung oxidative stress and protease expression.

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    Paul T King

    Full Text Available Nontypeable Haemophilus influenzae (NTHi is a prevalent bacterium found in a variety of chronic respiratory diseases. The role of this bacterium in the pathogenesis of lung inflammation is not well defined. In this study we examined the effect of NTHi on two important lung inflammatory processes 1, oxidative stress and 2, protease expression. Bronchoalveolar macrophages were obtained from 121 human subjects, blood neutrophils from 15 subjects, and human-lung fibroblast and epithelial cell lines from 16 subjects. Cells were stimulated with NTHi to measure the effect on reactive oxygen species (ROS production and extracellular trap formation. We also measured the production of the oxidant, 3-nitrotyrosine (3-NT in the lungs of mice infected with this bacterium. NTHi induced widespread production of 3-NT in mouse lungs. This bacterium induced significantly increased ROS production in human fibroblasts, epithelial cells, macrophages and neutrophils; with the highest levels in the phagocytic cells. In human macrophages NTHi caused a sustained, extracellular production of ROS that increased over time. The production of ROS was associated with the formation of macrophage extracellular trap-like structures which co-expressed the protease metalloproteinase-12. The formation of the macrophage extracellular trap-like structures was markedly inhibited by the addition of DNase. In this study we have demonstrated that NTHi induces lung oxidative stress with macrophage extracellular trap formation and associated protease expression. DNase inhibited the formation of extracellular traps.

  13. Carbon storage regulator A contributes to the virulence of Haemophilus ducreyi in humans by multiple mechanisms.

    Science.gov (United States)

    Gangaiah, Dharanesh; Li, Wei; Fortney, Kate R; Janowicz, Diane M; Ellinger, Sheila; Zwickl, Beth; Katz, Barry P; Spinola, Stanley M

    2013-02-01

    The carbon storage regulator A (CsrA) controls a wide variety of bacterial processes, including metabolism, adherence, stress responses, and virulence. Haemophilus ducreyi, the causative agent of chancroid, harbors a homolog of csrA. Here, we generated an unmarked, in-frame deletion mutant of csrA to assess its contribution to H. ducreyi pathogenesis. In human inoculation experiments, the csrA mutant was partially attenuated for pustule formation compared to its parent. Deletion of csrA resulted in decreased adherence of H. ducreyi to human foreskin fibroblasts (HFF); Flp1 and Flp2, the determinants of H. ducreyi adherence to HFF cells, were downregulated in the csrA mutant. Compared to its parent, the csrA mutant had a significantly reduced ability to tolerate oxidative stress and heat shock. The enhanced sensitivity of the mutant to oxidative stress was more pronounced in bacteria grown to stationary phase compared to that in bacteria grown to mid-log phase. The csrA mutant also had a significant survival defect within human macrophages when the bacteria were grown to stationary phase but not to mid-log phase. Complementation in trans partially or fully restored the mutant phenotypes. These data suggest that CsrA contributes to virulence by multiple mechanisms and that these contributions may be more profound in bacterial cell populations that are not rapidly dividing in the human host.

  14. QseC Mediates Osmotic Stress Resistance and Biofilm Formation in Haemophilus parasuis

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    Lvqin He

    2018-02-01

    Full Text Available Haemophilus parasuis is known as a commensal organism discovered in the upper respiratory tract of swine where the pathogenic bacteria survive in various adverse environmental stress. QseC, a histidine protein kinase of the two-component regulatory systems CheY/QseC, is involved in the environmental adaptation in bacteria. To investigate the role of QseC in coping with the adverse environment stresses and survive in the host, we constructed a qseC mutant of H. parasuis serovar 13 strain (ΔqseC, MY1902. In this study, we found that QseC was involved in stress tolerance of H. parasuis, by the ΔqseC exhibited a decreased resistance to osmotic pressure, oxidative stress, and heat shock. Moreover, the ΔqseC weakened the ability to take up iron and biofilm formation. We also found that the QseC participate in sensing the epinephrine in environment to regulate the density of H. parasuis.

  15. Methodology optimization and diversification for the investigation of virulence potential in Haemophilus influenzae clinical strains.

    Science.gov (United States)

    Giucă, Mihaela Cristina; Străuţ, Monica; Surdeanu, Maria; Nica, Maria; Ungureanu, Vasilica; Mihăescu, Grigore

    2011-01-01

    Ten Haemophilus influenzae strains were isolated from patients aged between 1.6 - 24 years, with various diagnoses (acute meningitis, acute upper respiratory infection, otitis media and acute sinusitis). Identification was based on phenotypic and molecular characteristics; antibiotic susceptibility testing was performed by diffusion method according to CLSI standards 2011 for seven antibiotics. The results of molecular testing showed that all the studied strains produced an amplicon of 1000 bp with ompP2 primers indicating that all strains were H. influenzae. For six strains, the PCR amplicon obtained with bexA specific primers, proving that the strains were capsulated. The results of phenotypic testing showed that four strains were ampicillin nonsusceptible and (beta-lactamase-positive. The virulence potential of H. influenzae clinical strains was investigated by phenotypic methods, including the assessment of the soluble virulence factors on specific media containing the biochemical substratum for the investigated enzymatic factor, as well as the adherence and invasion capacity to HeLa cells monolayer using Cravioto modified method. The studied strains exhibited mainly a diffuse adherence pattern and different adherence indexes. Interestingly, two strains isolated from the same pacient (blood and CSF) showed a different degree of invasiveness, the strain isolated from blood being 20 times more invasive than the one isolated from CSF.

  16. In vitro selection of resistance in haemophilus influenzae by 4 quinolones and 5 beta-lactams.

    Science.gov (United States)

    Clark, Catherine; Kosowska, Klaudia; Bozdogan, Bülent; Credito, Kim; Dewasse, Bonifacio; McGhee, Pamela; Jacobs, Michael R; Appelbaum, Peter C

    2004-05-01

    We tested abilities of ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, amoxicillin, amoxicillin/clavulanate, cefixime, cefpodoxime, and cefdinir to select resistant mutants in 5 beta-lactamase positive and 5 beta-lactamase negative Haemophilus influenzae strains by single and multistep methodology. In multistep tests, amoxicillin, amoxicillin/clavulanate and cefpodoxime exposure did not cause >4-fold minimum inhibitory concentration (MIC) increase after 50 days. One mutant selected by cefdinir had one amino acid substitution (Gly490Glu) in PBP3 and became resistant to cefdinir. Cefixime exposure caused 8-fold MIC-increase in 1 strain with TEM but the mutant remained cefixime susceptible and had no alteration in PBP3 or TEM. Among 10 strains tested, ciprofloxacin, moxifloxacin, gatifloxacin, levofloxacin caused >4-fold MIC increase in 6, 6, 5, and 2 strain, respectively. Despite the increases in quinolone MICs, none of the mutants became resistant to quinolones by established criteria. Quinolone selected mutants had quindone resistance-determining region (QRDR) alterations in GyrA, GyrB, ParC, ParE. Four quinolone mutants had no QRDR alterations. Among beta-lactams cefdinir and cefixime selected one mutant each with higher MICs however amoxicillin, amoxicillin/clavulanate, and cefpodoxime exposure did not select resistant mutants.

  17. Haemophilus influenzae type b disease in Auckland children during the Hib vaccination era: 1995-2009.

    Science.gov (United States)

    Leung, Bonnie; Taylor, Susan; Drinkovic, Dragana; Roberts, Sally; Carter, Phil; Best, Emma

    2012-11-09

    To characterise Haemophilus influenzae type b (Hib) invasive disease in the era of Hib vaccination, in children of the greater Auckland region of New Zealand. Identification of sterile site culture positive Hib via the Auckland hospital laboratories databases and national laboratory surveillance database in the time period; 1995 to 2009. There were a total of 26 cases in the Auckland Region. Over the 15-year period, the annual incidence of invasive Hib disease was 0.61 per 100,000 (95% CI: 0.4-0.9) for children aged under 15 years and 1.65 per 100,000 (95% CI: 1.1-2.5) for children aged under 5 years. Ninety-two percent were under 5 years and 54% were under 1 year. Sixty percent of the children were of Maori and Pacific ethnicity. The predominant diagnosis was meningitis, accounting for 15 cases (60%). There were no fatalities. Forty-eight percent of affected children were completely unimmunised with the Hib vaccine which has been fully funded on the National Immunisation Schedule since 1994. Since the introduction of the Hib vaccine, the disease rates have greatly reduced in the Auckland region. Although ethnic disparities have improved amongst the cases that occur, immunisation rates in cases are low and infants remain most at risk. Current emphasis on intensifying immunisation programmes to achieve higher vaccination rates and timeliness of delivery will help in efforts to achieve elimination of the disease in New Zealand.

  18. Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase

    Science.gov (United States)

    Broussard, Tyler C.; Pakhomova, Svetlana; Neau, David B.; Bonnot, Ross; Waldrop, Grover L.

    2015-01-01

    Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1′-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1′-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO2 from the carboxyphosphate intermediate to biotin. PMID:26020841

  19. Antimicrobial susceptibility of Pasteurella multocida and Haemophilus parasuis isolates associated with porcine pneumonia

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    Kateřina Nedbalcová

    2013-01-01

    Full Text Available Pasteurella multocida and Haemophilus parasuis pig isolates obtained in the Czech Republic were tested for their susceptibility against selected antimicrobial agents by broth microdilution method between 2008 and 2011. A low degree of resistance was observed for ampicillin, amoxicillin/clavulanic acid, ceftiofur, tulathromycin, tilmicosin, florfenicol and enrofloxacin in 20 (6.0%, 15 (4.5 %, 2 (0.6%, 8 (2.4%, 13 (3.9%, 5 (1.5% and 5 (1.5% P. multocida isolates as well as for tiamulin, gentamicin, tulathromycin, tilmicosin and ampicillin in 2 (2.4%, 2 (2.4%, 3 (3.6%, 3 (3.6% and 6 (7.2% H. parasuis isolates. In addition, moderate level of resistance to tiamulin was found in 60 (18.1% P. multocida isolates and high level of resistance for tetracycline was detected in 107 (32.2 % P. multocida isolates and in 23 (27.7 % H. parasuis isolates. Differences between resistance rates of P. multocida and H. parasuis were significant (P ≤ 0.5 only for tiamulin. These data confirmed that antimicrobial resistance is not very widespread among current porcine P. multocida and H. parasuis isolates in the Czech Republic.

  20. β-1,6-glucan synthesis-associated genes are required for proper spore wall formation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Pan, Hua-Ping; Wang, Ning; Tachikawa, Hiroyuki; Nakanishi, Hideki; Gao, Xiao-Dong

    2017-11-01

    The yeast spore wall is an excellent model to study the assembly of an extracellular macromolecule structure. In the present study, mutants defective in β-1,6-glucan synthesis, including kre1∆, kre6∆, kre9∆ and big1∆, were sporulated to analyse the effect of β-1,6-glucan defects on the spore wall. Except for kre6∆, these mutant spores were sensitive to treatment with ether, suggesting that the mutations perturb the integrity of the spore wall. Morphologically, the mutant spores were indistinguishable from wild-type spores. They lacked significant sporulation defects partly because the chitosan layer, which covers the glucan layer, compensated for the damage. The proof for this model was obtained from the effect of the additional deletion of CHS3 that resulted in the absence of the chitosan layer. Among the double mutants, the most severe spore wall deficiency was observed in big1∆ spores. The majority of the big1∆chs3∆ mutants failed to form visible spores at a higher temperature. Given that the big1∆ mutation caused a failure to attach a GPI-anchored reporter, Cwp2-GFP, to the spore wall, β-1,6-glucan is involved in tethering of GPI-anchored proteins in the spore wall as well as in the vegetative cell wall. Thus, β-1,6-glucan is required for proper organization of the spore wall. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  1. Characterization of nifB, nifS, and nifU genes in the cyanobacterium Anabaena variabilis: NifB is required for the vanadium-dependent nitrogenase.

    OpenAIRE

    Lyons, E M; Thiel, T

    1995-01-01

    Anabaena variabilis ATCC 29413 is a heterotrophic, nitrogen-fixing cyanobacterium containing both a Mo-dependent nitrogenase encoded by the nif genes and V-dependent nitrogenase encoded by the vnf genes. The nifB, nifS, and nifU genes of A. variabilis were cloned, mapped, and partially sequenced. The fdxN gene was between nifB and nifS. Growth and acetylene reduction assays using wild-type and mutant strains indicated that the nifB product (NifB) was required for nitrogen fixation not only by...

  2. Of the Nine Cytidine Deaminase-Like Genes in Arabidopsis, Eight Are Pseudogenes and Only One Is Required to Maintain Pyrimidine Homeostasis in Vivo.

    Science.gov (United States)

    Chen, Mingjia; Herde, Marco; Witte, Claus-Peter

    2016-06-01

    CYTIDINE DEAMINASE (CDA) catalyzes the deamination of cytidine to uridine and ammonia in the catabolic route of C nucleotides. The Arabidopsis (Arabidopsis thaliana) CDA gene family comprises nine members, one of which (AtCDA) was shown previously in vitro to encode an active CDA. A possible role in C-to-U RNA editing or in antiviral defense has been discussed for other members. A comprehensive bioinformatic analysis of plant CDA sequences, combined with biochemical functionality tests, strongly suggests that all Arabidopsis CDA family members except AtCDA are pseudogenes and that most plants only require a single CDA gene. Soybean (Glycine max) possesses three CDA genes, but only two encode functional enzymes and just one has very high catalytic efficiency. AtCDA and soybean CDAs are located in the cytosol. The functionality of AtCDA in vivo was demonstrated with loss-of-function mutants accumulating high amounts of cytidine but also CMP, cytosine, and some uridine in seeds. Cytidine hydrolysis in cda mutants is likely caused by NUCLEOSIDE HYDROLASE1 (NSH1) because cytosine accumulation is strongly reduced in a cda nsh1 double mutant. Altered responses of the cda mutants to fluorocytidine and fluorouridine indicate that a dual specific nucleoside kinase is involved in cytidine as well as uridine salvage. CDA mutants display a reduction in rosette size and have fewer leaves compared with the wild type, which is probably not caused by defective pyrimidine catabolism but by the accumulation of pyrimidine catabolism intermediates reaching toxic concentrations. © 2016 American Society of Plant Biologists. All Rights Reserved.

  3. Rice gene SDL/RNRS1, encoding the small subunit of ribonucleotide reductase, is required for chlorophyll synthesis and plant growth development.

    Science.gov (United States)

    Qin, Ran; Zeng, Dongdong; Liang, Rong; Yang, Chengcong; Akhter, Delara; Alamin, Md; Jin, Xiaoli; Shi, Chunhai

    2017-09-05

    A new mutant named sdl (stripe and drooping leaf) was characterized from indica cultivar Zhenong 34 by ethylmethane sulfonate (EMS) mutagenesis. The mutant sdl exhibited development defects including stripe and drooping leaf, dwarfism and deformed floral organs. The gene SDL was found allelic to RNRS1 by map-based cloning, which was homologous to Arabidopsis TSO2 encoding the small subunit of ribonucleotide reductase. The gDNA sequencing results of sdl in mutant showed that there was a repetitive sequence insertion of 138-bp at the 475 th bp in the exon. The redundant sequence was conserved in SDL homologous proteins, which contained the active site (tyrosine), as well as two amino acids glutamate and histidine involved in the binding of iron. There were fewer chloroplasts and grana lamellas in sdl leaf compared with those of wild-type. Additionally, the stripe leaves of sdl seedlings were highly sensitive to temperature, since the chlorophyll content was increased with the temperature rising. The drooping leaf of sdl might be resulted from the disappearance of vascular bundles and mesophyll cells in both leaf midrib and lateral veins. Fittingly to the phenotypes of mutant sdl, the expression levels of genes associated with photosynthesis and chlorophyll synthesis were found to be down- or up-regulated at different temperatures in mutant sdl. Also, the transcriptional levels of genes related to plant height and floral organ formation showed obvious differences between wild-type and sdl. The "SDL/RNRS1" was, hence, required for the chlorophyll biosynthesis and also played pleiotropic roles in the regulation of plant development. Copyright © 2017. Published by Elsevier B.V.

  4. Tipificación capsular mediante PCR de aislamientos de Haemophilus influenzae no tipificables por aglutinación PCR-based capsular typing of Haemophilus influenzae isolates non-typeable by agglutination

    Directory of Open Access Journals (Sweden)

    G. Weltman

    2005-12-01

    Full Text Available Haemophilus influenzae es reconocido como un agente patógeno responsable de infecciones localizadas y sistémicas. Se han descrito 6 tipos de polisacáridos capsulares antigénicamente distintos (a, b, c, d, e, y f que se pueden identificar por aglutinación en lámina con antisueros específicos. También existen cepas no capsuladas (NC fenotípicamente no tipificables (NT. La introducción de la vacuna conjugada produjo una marcada disminución de las enfermedades invasivas causadas por H. influenzae tipo b. En este contexto, la tipificación capsular mediante PCR es el método más apropiado para distinguir las cepas no capsuladas de las mutantes b deficientes en cápsula (b- y detectar la presencia de cepas pertenecientes a otros serotipos que no puedan ser tipificables por aglutinación. Se determinó el genotipo capsular a 38 aislamientos de Haemophilus influenzae no tipificables por aglutinación, derivados al servicio de Bacteriología Clínica del INEI-ANLIS "Dr. Carlos G. Malbrán" en el período 2002-2004. El 78,9% de los aislamientos provenían de hemocultivos y la mayor parte de ellos estaban asociados a foco respiratorio. El 100% de los aislamientos fueron identificados como H. influenzae no capsulados mediante la técnica de PCR.Haemophilus influenzae is recognized as a pathogenic agent responsible of localized and systemic infections. Six antigenically different capsular polysaccharide types have been described (a, b, c, d, e, and f which can be identified by slide agglutination with specific antisera. Besides there are non capsulated strains that cannot be typed by slide agglutination. The introduction of the conjugated vaccine produced an important reduction of invasive diseases caused by H. influenzae type b. Capsular typing by PCR is the most appropriated method for distinguishing non capsulated strains from capsule deficient type b mutants (b- and for detecting strains of other serotypes that cannot be detected by slide

  5. Changes in IgA protease expression are conferred by changes in genomes during persistent infection by nontypeable Haemophilus influenzae in chronic obstructive pulmonary disease.

    Science.gov (United States)

    Gallo, Mary C; Kirkham, Charmaine; Eng, Samantha; Bebawee, Remon S; Kong, Yong; Pettigrew, Melinda M; Tettelin, Hervé; Murphy, Timothy F

    2018-05-14

    Nontypeable Haemophilus influenzae (NTHi) is an exclusively human pathobiont that plays a critical role in the course and pathogenesis of chronic obstructive pulmonary disease (COPD). NTHi causes acute exacerbations of COPD and also causes persistent infection of the lower airways. NTHi expresses four IgA protease variants (A1, A2, B1, and B2) that play different roles in virulence. Expression of IgA proteases varies among NTHi strains, but little is known about the frequency and mechanisms by which NTHi modulates IgA protease expression during infection in COPD. To assess expression of IgA protease during natural infection in COPD, we studied IgA protease expression of 101 persistent strains (median duration of persistence 161 days, range 2 to 1422) collected longitudinally from patients enrolled in a 20-year study of COPD upon initial acquisition and immediately before clearance from the host. Upon acquisition, 89 (88%) expressed IgA protease. A total of 16 of 101 (16%) strains of NTHi altered expression of IgA protease during persistence. Indels and slipped-strand mispairing of mononucleotide repeats conferred changes in expression of igaA1, igaA2, and igaB1 Strains with igaB2 underwent frequent changes in expression of IgA protease B2 during persistence, mediated by slipped-strand mispairing of a 7-nucleotide repeat, TCAAAAT, within the open reading frame of igaB2 We conclude that changes in iga gene sequences result in changes in expression of IgA proteases by NTHi during persistent infection in the respiratory tract of patients with COPD. Copyright © 2018 American Society for Microbiology.

  6. Candida albicans AGE3, the ortholog of the S. cerevisiae ARF-GAP-encoding gene GCS1, is required for hyphal growth and drug resistance.

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    Thomas Lettner

    Full Text Available BACKGROUND: Hyphal growth and multidrug resistance of C. albicans are important features for virulence and antifungal therapy of this pathogenic fungus. METHODOLOGY/PRINCIPAL FINDINGS: Here we show by phenotypic complementation analysis that the C. albicans gene AGE3 is the functional ortholog of the yeast ARF-GAP-encoding gene GCS1. The finding that the gene is required for efficient endocytosis points to an important functional role of Age3p in endosomal compartments. Most C. albicans age3Delta mutant cells which grew as cell clusters under yeast growth conditions showed defects in filamentation under different hyphal growth conditions and were almost completely disabled for invasive filamentous growth. Under hyphal growth conditions only a fraction of age3Delta cells shows a wild-type-like polarization pattern of the actin cytoskeleton and lipid rafts. Moreover, age3Delta cells were highly susceptible to several unrelated toxic compounds including antifungal azole drugs. Irrespective of the AGE3 genotype, C-terminal fusions of GFP to the drug efflux pumps Cdr1p and Mdr1p were predominantly localized in the plasma membrane. Moreover, the plasma membranes of wild-type and age3Delta mutant cells contained similar amounts of Cdr1p, Cdr2p and Mdr1p. CONCLUSIONS/SIGNIFICANCE: The results indicate that the defect in sustaining filament elongation is probably caused by the failure of age3Delta cells to polarize the actin cytoskeleton and possibly of inefficient endocytosis. The high susceptibility of age3Delta cells to azoles is not caused by inefficient transport of efflux pumps to the cell membrane. A possible role of a vacuolar defect of age3Delta cells in drug susceptibility is proposed and discussed. In conclusion, our study shows that the ARF-GAP Age3p is required for hyphal growth which is an important virulence factor of C. albicans and essential for detoxification of azole drugs which are routinely used for antifungal therapy. Thus, it

  7. The Arabidopsis NF-YA3 and NF-YA8 genes are functionally redundant and are required in early embryogenesis.

    Directory of Open Access Journals (Sweden)

    Monica Fornari

    Full Text Available Nuclear factor Y (NF-Y is a trimeric transcription factor composed of three distinct subunits called NF-YA, NF-YB and NF-YC. In Arabidopsis thaliana, NF-Y subunits are known to play roles in many processes, such as gametogenesis, embryogenesis, seed development, drought resistance, ABA signaling, flowering time, primary root elongation, Endoplasmic Reticulum (ER stress response and blue light responses. Here, we report that the closely related NF-YA3 and NF-YA8 genes control early embryogenesis. Detailed GUS and in situ analyses showed that NF-YA3 and NF-YA8 are expressed in vegetative and reproductive tissues with the highest expression being during embryo development from the globular to the torpedo embryo stage. Plants from the nf-ya3 and nf-ya8 single mutants do not display any obvious phenotypic alteration, whereas nf-ya3 nf-ya8 double mutants are embryo lethal. Morphological analyses showed that the nf-ya3 nf-ya8 embryos fail to undergo to the heart stage and develop into abnormal globular embryos with both proembryo and suspensor characterized by a disordered cell cluster with an irregular shape, suggesting defects in embryo development. The suppression of both NF-YA3 and NF-YA8 gene expression by RNAi experiments resulted in defective embryos that phenocopied the nf-ya3 nf-ya8 double mutants, whereas complementation experiments partially rescued the abnormal globular nf-ya3 nf-ya8 embryos, confirming that NF-YA3 and NF-YA8 are required in early embryogenesis. Finally, the lack of GFP expression of the auxin responsive DR5rev::GFP marker line in double mutant embryos suggested that mutations in both NF-YA3 and NF-YA8 affect auxin response in early developing embryos. Our findings indicate that NF-YA3 and NF-YA8 are functionally redundant genes required in early embryogenesis of Arabidopsis thaliana.

  8. Haemophilus influenzae pneumonia in human immunodeficiency virus-infected patients. The Grupo Andaluz para el Estudio de las Enfermedades Infecciosas.

    Science.gov (United States)

    Cordero, E; Pachón, J; Rivero, A; Girón, J A; Gómez-Mateos, J; Merino, M D; Torres-Tortosa, M; González-Serrano, M; Aliaga, L; Collado, A; Hernández-Quero, J; Barrera, A; Nuño, E

    2000-03-01

    Although Haemophilus influenzae is a common etiologic agent of pneumonia in patients infected with human immunodeficiency virus (HIV), the characteristics of this pneumonia have not been adequately assessed. We have prospectively studied features of H. influenzae pneumonia in 26 consecutive HIV-infected inpatients. Most of these patients were severely immunosuppressed; 73.1% had a CD4+ cell count caused by H. influenzae affects mainly patients with advanced HIV disease, and since its clinical and radiological features may be diverse, this etiology should be considered when pneumonia occurs in patients with advanced HIV infection. The mortality rate associated with H. influenzae pneumonia is not higher than that occurring in the general population.

  9. IgG responses to Pneumococcal and Haemophilus influenzae protein antigens are not impaired in children with a history of recurrent acute otitis media.

    Science.gov (United States)

    Wiertsema, Selma P; Corscadden, Karli J; Mowe, Eva N; Zhang, Guicheng; Vijayasekaran, Shyan; Coates, Harvey L; Mitchell, Timothy J; Thomas, Wayne R; Richmond, Peter C; Kirkham, Lea-Ann S

    2012-01-01

    Vaccines including conserved antigens from Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) have the potential to reduce the burden of acute otitis media. Little is known about the antibody response to such antigens in young children with recurrent acute otitis media, however, it has been suggested antibody production may be impaired in these children. We measured serum IgG levels against 4 pneumococcal (PspA1, PspA 2, CbpA and Ply) and 3 NTHi (P4, P6 and PD) proteins in a cross-sectional study of 172 children under 3 years of age with a history of recurrent acute otitis media (median 7 episodes, requiring ventilation tube insertion) and 63 healthy age-matched controls, using a newly developed multiplex bead assay. Children with a history of recurrent acute otitis media had significantly higher geometric mean serum IgG levels against NTHi proteins P4, P6 and PD compared with healthy controls, whereas there was no difference in antibody levels against pneumococcal protein antigens. In both children with and without a history of acute otitis media, antibody levels increased with age and were significantly higher in children colonised with S. pneumoniae or NTHi compared with children that were not colonised. Proteins from S. pneumoniae and NTHi induce serum IgG in children with a history of acute otitis media. The mechanisms in which proteins induce immunity and potential protection requires further investigation but the dogma of impaired antibody responses in children with recurrent acute otitis media should be reconsidered.

  10. IgG responses to Pneumococcal and Haemophilus influenzae protein antigens are not impaired in children with a history of recurrent acute otitis media.

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    Selma P Wiertsema

    Full Text Available BACKGROUND: Vaccines including conserved antigens from Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi have the potential to reduce the burden of acute otitis media. Little is known about the antibody response to such antigens in young children with recurrent acute otitis media, however, it has been suggested antibody production may be impaired in these children. METHODS: We measured serum IgG levels against 4 pneumococcal (PspA1, PspA 2, CbpA and Ply and 3 NTHi (P4, P6 and PD proteins in a cross-sectional study of 172 children under 3 years of age with a history of recurrent acute otitis media (median 7 episodes, requiring ventilation tube insertion and 63 healthy age-matched controls, using a newly developed multiplex bead assay. RESULTS: Children with a history of recurrent acute otitis media had significantly higher geometric mean serum IgG levels against NTHi proteins P4, P6 and PD compared with healthy controls, whereas there was no difference in antibody levels against pneumococcal protein antigens. In both children with and without a history of acute otitis media, antibody levels increased with age and were significantly higher in children colonised with S. pneumoniae or NTHi compared with children that were not colonised. CONCLUSIONS: Proteins from S. pneumoniae and NTHi induce serum IgG in children with a history of acute otitis media. The mechanisms in which proteins induce immunity and potential protection requires further investigation but the dogma of impaired antibody responses in children with recurrent acute otitis media should be reconsidered.

  11. Haemophilus ducreyi cutaneous ulcer contracted at Seram Island, Indonesia, presented in the Netherlands.

    Science.gov (United States)

    van Hattem, Jarne M; Langeveld, Tessa J C; Bruisten, Sylvia M; Kolader, Marion; Grobusch, Martin P; de Vries, Henry J C; de Bree, Godelieve J

    2018-04-01

    We describe the first case of a cutaneous ulcer caused by Haemophilus ducreyi imported from Indonesia to the Netherlands. Skin infections caused by H. ducreyi are uncommon in travellers and have been described in just a few case reports and were all contracted on the Pacific Islands. A 22-year-old healthy male visited the Center of Tropical Medicine and Travel Medicine in February 2017 with a cutaneous ulcer of the right lateral malleolus 4 weeks after returning from Indonesia (Seram and Ambon Islands). He had noticed a small skin abrasion on the right ankle after slipping on a rock during a jungle trip on Seram Island. Back in the Netherlands, a painful ulcer developed at the same body location, and despite treatment with flucloxacillin, his complaints worsened. A swab that was taken for culture showed growth of small grey colonies that were characterised as H. ducreyi with matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. Treatment with ciprofloxacin for the diagnosis of H. ducreyi cutaneous ulcer was started, and the ulcer clearly diminished, leaving only a small healing ulcer. H. ducreyi is normally the causative agent of genital ulcers but is increasingly recognised as a cause of chronic skin ulcers, e.g., in Papua New Guinea. In our patient, the infection was very likely contracted in the Maluku province of Indonesia and imported into the Netherlands. No reports of infection with H. ducreyi from Indonesia could be found in literature, but this case indicates that H. ducreyi is present in at least one of the northeastern islands of Indonesia, which is important for local healthcare. Additionally, it illustrates the role of this agent as a cause of cutaneous ulcers in previously healthy travellers.

  12. Human milk lactoferrin inactivates two putative colonization factors expressed by Haemophilus influenzae.

    Science.gov (United States)

    Qiu, J; Hendrixson, D R; Baker, E N; Murphy, T F; St Geme, J W; Plaut, A G

    1998-10-13

    Haemophilus influenzae is a major cause of otitis media and other respiratory tract disease in children. The pathogenesis of disease begins with colonization of the upper respiratory mucosa, a process that involves evasion of local immune mechanisms and adherence to epithelial cells. Several studies have demonstrated that human milk is protective against H. influenzae colonization and disease. In the present study, we examined the effect of human milk on the H. influenzae IgA1 protease and Hap adhesin, two autotransported proteins that are presumed to facilitate colonization. Our results demonstrated that human milk lactoferrin efficiently extracted the IgA1 protease preprotein from the bacterial outer membrane. In addition, lactoferrin specifically degraded the Hap adhesin and abolished Hap-mediated adherence. Extraction of IgA1 protease and degradation of Hap were localized to the N-lobe of the bilobed lactoferrin molecule and were inhibited by serine protease inhibitors, suggesting that the lactoferrin N-lobe may contain serine protease activity. Additional experiments revealed no effect of lactoferrin on the H. influenzae P2, P5, and P6 outer-membrane proteins, which are distinguished from IgA1 protease and Hap by the lack of an N-terminal passenger domain or an extracellular linker region. These results suggest that human milk lactoferrin may attenuate the pathogenic potential of H. influenzae by selectively inactivating IgA1 protease and Hap, thereby interfering with colonization. Future studies should examine the therapeutic potential of lactoferrin, perhaps as a supplement in infant formulas.

  13. Economic evaluations of Haemophilus influenzae type b (Hib) vaccine: a systematic review.

    Science.gov (United States)

    Chongmelaxme, Bunchai; Hammanee, Maythika; Phooaphirak, Wariya; Kotirum, Surachai; Hutubessy, Raymond; Chaiyakunapruk, Nathorn

    2017-10-01

    The World Health Organization (WHO) recommends the use of Haemophilus influenzae type b (Hib) conjugate vaccines, but China and Thailand have not used Hib vaccination in their national immunization programs. This systematic review aimed to update published economic evaluations of Hib vaccinations and to determine factors that potentially affected their cost-effectiveness. Searches were performed from the inception until December 2015 using 13 databases: CAB direct; CEA registry; EconLit; EMBASE; E-library; NHSEED; PAHO; POPLINE; PubMed; Redalyc project; RePEc; SciELO; and WHOLIS. Reference lists of relevant studies and grey literature were also searched. Full economic evaluations of Hib vaccination with results of costs and outcomes were included. The WHO checklist was used to evaluate the quality of the included studies. Data from eligible studies were extracted using a standardized data collection form. Out of 830 articles, 27 were included. Almost half of the studies (12/27) were conducted in high-income countries. Twelve studies (12/27) investigated the Hib vaccine as an addition to the existing vaccination program. Most studies (17/27) examined a 3-dose schedule of Hib vaccine. Nineteen studies (19/27) reported the model used, where all were decision tree models. Most of the studies (23/27) demonstrated an economic value of Hib vaccination programs, key influential parameters being incidence rates of Hib disease and vaccine price. Hib vaccination programs are mostly found to be cost-effective across geographic regions and country income levels, and Hib vaccination is recommended for inclusion into all national immunization programs. The findings are expected to support policy-makers for making decisions on allocating limited resources of the Hib vaccination program effectively.

  14. Effect of enrofloxacin on Haemophilus parasuis infection, disease and immune response.

    Science.gov (United States)

    Macedo, Nubia; Cheeran, Maxim C J; Rovira, Albert; Holtcamp, Andrew; Torremorell, Montserrat

    2017-02-01

    Haemophilus parasuis, the causative agent of Glasser's disease, is a pathogen that colonizes the upper respiratory tract (URT) of pigs, invades the bloodstream and causes polyserositis. Because antimicrobials are highly effective against H. parasuis, we hypothesized that they could have a detrimental effect on the establishment of an immune response if given at the time of URT colonization. In this study, we characterized clinical outcomes and antibody and IFN-γ responses to H. parasuis in pigs treated with enrofloxacin before or after low dose inoculation with a pathogenic H. parasuis strain. Pigs that were only inoculated with the agent (EXP group) and pigs that were treated with enrofloxacin and then inoculated (ABT/EXP group) developed signs of disease starting at 4days post inoculation (DPI), presented a significant increase in serum IgG and were protected against a subsequent homologous challenge. In contrast, pigs treated with antibiotic after inoculation (EXP/ABT group) neither showed signs of disease nor seroconverted (IgG) after low dose inoculation. EXP/ABT pigs as well as naïve control pigs [enrofloxacin only (ABT) and challenge only (CHA)] were susceptible to challenge. Variable levels of antibodies in bronchioalveolar fluid and IFN-γ in peripheral blood mononuclear cells were observed after H. parasuis inoculation, but were not associated with protection. In summary, only pigs treated before low dose H. parasuis inoculation seroconverted and were protected against subsequent challenge. Results from this study can help determine timing of antimicrobial use and contribute to our current understanding of judicious antibiotic use. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Biofilm formation in Haemophilus parasuis: relationship with antibiotic resistance, serotype and genetic typing.

    Science.gov (United States)

    Zhang, Jianmin; Xu, Chenggang; Shen, Haiyan; Li, Jingyi; Guo, Lili; Cao, Guojie; Feng, Saixiang; Liao, Ming

    2014-10-01

    Biofilms are surface-associated microbial communities, which are encased in self-synthesized extracellular environment. Biofilm formation may trigger drug resistance and inflammation, resulting in persistent infections. Haemophilus parasuis is the etiological agent of a systemic disease, Glässer's disease, characterized by fibrinous polyserositis, arthritis and meningitis in pigs. The purpose of this study was to examine the correlation between biofilm and antibiotic resistance among the clinical isolates of H. parasuis. In the present study, we tested biofilm-forming ability of 110 H. parasuis isolates from various farms using polystyrene microtiter plate assays. Seventy-three isolates of H. parasuis (66.4%) showed biofilm formation and most of them performed weak biofilm-forming ability (38/73). All isolates were tested for antimicrobial susceptibility to 18 antimicrobial agents by the broth microdilution method. H. parasuis isolates showed very high resistance (>90%) to sulfanilamide, nalidixic acid, and trimethoprim. Resistance to eight antibiotics such as penicillin (41.1% vs 8.1%), ampicillin (31.5% vs 8.1%), amoxicillin (28.8% vs 5.4%), gentamicin (46.6% vs 24.3%), cefazolin (19.2% vs 2.7%), doxycycline (19.2% vs 8.1%), cefotaxime (11% vs 2.7%), and cefaclor (13.7% vs 5.4%) was comparatively higher among biofilm producers than non-biofilm producers. Pulsed-field gel electrophoresis (PFGE) analyses could distinguish various isolates. Our data indicated that H. parasuis field isolates were able to form biofilms in vitro. In addition, biofilm positive strains had positive correlation with resistance to β-lactams antibiotics. Thus, biofilm formation may play important roles during H. parasuis infections. Copyright © 2014. Published by Elsevier Ltd.

  16. Impacto de la vacunación contra Haemophilus influenzae tipo b en Cuba

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    Dickinson Félix O.

    2001-01-01

    Full Text Available Objetivo. Determinar el impacto de la vacunación de menores de 2 años en Cuba contra Haemophilus influenzae tipo b (Hib, principal agente causal de la meningitis bacteriana en ese país. Métodos. La disponibilidad de vacunas conjugadas eficaces contra Hib motivó la vacunación nacional en 1999 de niños menores de 2 años, que alcanzó una cobertura de 97%. El impacto se evaluó mediante el Sistema Nacional de Vigilancia de Meningoencefalitis Bacterianas (SNVMEB. Resultados. La eficacia global de la vacunación se estimó en 99% y la incidencia general de la meningoencefalitis por Hib disminuyó de 1,3 a 0,6 por 100 000 habitantes (46,1%, observándose la mayor reducción en niños menores de 5 años (56,1%. En los menores de 1 año se redujo 70,5% y en el resto de los grupos de menores de 5 años disminuyó entre 25,9 y 49,6%. En el grupo diana para la vacunación, la incidencia se redujo 61,1%; entre los niños de este grupo que contrajeron la meningoencefalitis por Hib, solamente 8 (24,2% estaban vacunados, 7 de ellos con una sola dosis, aplicada 1 mes antes de enfermar. Conclusiones. Se ha demostrado que la vacunación a gran escala de los niños menores de 2 años contra Hib en Cuba a través del SNVMEB ha logrado disminuir notablemente la incidencia de meningoencefalitis por Hib.

  17. Microstructural changes of cows’ uterus in an infection caused by Haemophilus somnus

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    Danka Šťastná

    2015-09-01

    Full Text Available The reproductive form of Haemophilic infection caused by a microorganism Histophilus somni (Haemophilus somnus is in our conditions practically unknown and undiscovered disease of cow reproductive organs. The aim of this work was based on experimental work to provide a comprehensive view on the patho-histological and submicroscopic changes in the uterus of 60 cows, serological positive, crossbreeds Slovenske strakate x Holstein, aged 3.5 . 4.5 years, serological positive, which were up to 90 days after the parturition sterile. After the hormonal control of the oestrus cycle (PGF2ƒż-Oestrophan SPOFA the cows were inseminated and the non-gravid cows later killed. The reproductive organs of killed animals were pathologically . anatomically dissected with following histological and sub-microscopic study of uterus and cervix tissue. At the beginning of the experiment no clinical changes of the reproductive organs of group 1 animals with the titer of antibodies 1:25-100 were diagnosed and in group 3 with the titer 1:25. At an examination before killing were examined endometritis (EM of grade II. and vaginitis with vestibulitis in these groups. At the rest of animals appeared in every level of the titer of antibodies of EM II. and EM III. EM I. were not clinically diagnosed. Dissecting examination determined EM catarrhalis and cervicis catarrhalis in all groups. The histological examination pointed to EM catarrhalis chronica, EM interstitialis chronica, EM fibrousis and cervicis catarrhalis chronica. The leucocytes gained from a uterus lavage were in a comparison with stroma leucocytes mostly polynuclear (neutrophils and in smaller amount small forms of lymfocytes. At long-term sterility of cows, because of possible H. somnus infection, we regard as priority the serodiagnostics at which is taken into consideration any level of the titer of antibodies.

  18. Preclinical evaluation of a Haemophilus influenzae type b conjugate vaccine process intended for technology transfer.

    Science.gov (United States)

    Hamidi, Ahd; Verdijk, Pauline; Kreeftenberg, Hans

    2014-01-01

    Introduction of Haemophilus influenzae type b (Hib) vaccine in low- and middle-income countries has been limited by cost and availability of Hib conjugate vaccines for a long time. It was previously recognized by the Institute for Translational Vaccinology (Intravacc, originating from the former Vaccinology Unit of the National Institute of Public Health [RIVM] and the Netherlands Vaccine Institute [NVI]) that local production of a Hib conjugate vaccine would increase the affordability and sustainability of the vaccine and thereby help to speed up Hib introduction in these countries. A new affordable and a non-infringing production process for a Hib conjugate vaccine was developed, including relevant quality control tests, and the technology was transferred to a number of vaccine manufacturers in India, Indonesia, and China. As part of the Hib technology transfer project managed by Intravacc, a preclinical toxicity study was conducted in the Netherlands to test the safety and immunogenicity of this new Hib conjugate vaccine. The data generated by this study were used by the technology transfer partners to accelerate the clinical development of the new Hib conjugate vaccine. A repeated dose toxicity and local tolerance study in rats was performed to assess the reactogenicity and immunogenicity of a new Hib conjugate vaccine compared to a licensed vaccine. The results showed that the vaccine was well tolerated and immunogenic in rats, no major differences in both safety and immunogenicity in rats were found between the vaccine produced according to the production process developed by Intravacc and the licensed one. Rats may be useful to verify the immunogenicity of Hib conjugate vaccines and for preclinical evaluation. In general, nonclinical evaluation of the new Hib conjugate vaccine, including this proof of concept (safety and immunogenicity study in rats), made it possible for technology transfer partners, having implemented the original process with no changes

  19. The Epidemiologic and Pharmacodynamic Cutoff Values of Tilmicosin against Haemophilus parasuis

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    Haihong eHao

    2016-03-01

    Full Text Available The aim of this study was to establish antimicrobial susceptibility breakpoints for tilmicosin against Haemophilus parasuis, which is an important pathogen of respiratory tract infections. The minimum inhibitory concentrations (MICs of 103 H. parasuis isolates were determined by the agar dilution method. The wild-type (WT distribution and epidemiologic cutoff value (ECV were evaluated by statistical analysis. The new bronchoaveolar lavage (BAL was used to establish intrapulmonary pharmacokinetic (PK model in swine. The pharmacokinetic (PK parameters of tilmicosin, both in pulmonary epithelial lining fluid (PELF and in plasma, were determined using high performance liquid chromatography (HPLC method and WinNonlin software. The pharmacodynamic cutoff (COPD was calculated using Monte Carlo simulation. Our results showed that 100% of WT isolates were covered when the ECV was set at 16μg/mL. The tilmicosin had concentration-dependent activity against H. parasuis. The PK data indicated that tilmicosin concentrations in PELF was rapidly increased to high levels at 4 hours and kept stable until 48 hours after drug administration, while the tilmicosin concentration in plasma reached maximum levels at 4 hours and continued to decrease during 4-72 hours. Using Monte Carlo simulation, COPD was defined as 1 μg/mL. Conclusively, the ECV and COPD of tilmicosin against H. parasuis were established for the first time based on the MIC distribution and PK-PD analysis in the target tissue, respectively. These values are of great importance for detection of tilmicosin-resistant H. parasuis and for effective treatment of clinical intrapulmonary infection caused by H. parasuis.

  20. Comparison of radiometric and conventional culture systems in detecting Haemophilus influenzae type b bacteremia in rats

    International Nuclear Information System (INIS)

    Mitchell, M.J.; Zwahlen, A.; Elliott, H.L.; Ford, N.K.; Charache, F.P.; Moxon, E.R.

    1985-01-01

    To compare the efficiency of detecting Haemophilus influenzae type b bacteremia by the BACTEC radiometric system and a conventional Trypticase soy broth blood culture system, the authors developed an in vivo model of bacteremia in rats. After intravenous injection of 50 to 200 CFU into adult rats, there was a linear logarithmic increase in CFU per milliliter of rat blood during the first 10 h (r = 0.98), allowing accurate prediction of the level of bacteremia with time. Culture bottles were inoculated with 0.5 ml of blood obtained by cardiac puncture and processed as clinical samples in the microbiology laboratory with RS and conventional protocols. They found the following. (i) The first detection of bacteremia by RS was similar to that by TSB if a Gram stain of the TSB was done on day 1 and was superior if that smear was omitted (P less than 0.01). (ii) The detection times in both systems were comparable at different magnitudes of bacteremia (10(1) to 10(4) CFU/ml). (iii) Supplementation of inoculated bottles with 2 ml of sterile rat blood interfered with Gram stain detection in TSB but resulted in increased 14 CO 2 production in RS. (iv) No difference in detection time was found between RS and TSB for four different clinical isolates. These studies show that, in a biologically relevant model, the detection of positive blood cultures for H. influenzae type b by RS was comparable to or better than detection by TSB when blood was processed analogously to clinical specimens

  1. No evidence of increasing Haemophilus influenzae non-b infection in Australian Aboriginal children

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    Robert I. Menzies

    2013-08-01

    Full Text Available Background. High, or increasing, rates of invasive Haemophilus influenzae (Hi type a disease have been reported from North American native children from circumpolar regions, raising the question of serotype replacement being driven by vaccination against Hi type b (Hib. Indigenous Australians from remote areas had high rates of invasive Hib disease in the past, comparable to those in North American Indigenous populations. Objective. Evaluate incidence rates of invasive Hi (overall and by serotype in Indigenous Australian children over time. Design. Descriptive study of Hi incidence rates by serotype, in the Northern Territory (NT and South Australia (SA from 2001 to 2011. Comparison of NT data with a study that was conducted in the NT in 1985–1988, before Hib vaccine was introduced. Results. The average annual rate of invasive Hi type a (Hia disease in Indigenous children aged <5 years was 11/100,000 population. Although the incidence of Hi infection in Indigenous children in 2001–2003 was lower than during 2004–2011, this may be due to changes in surveillance. No other trend over time in individual serotypes or total invasive Hi disease, in Indigenous or non-Indigenous people, was identified. Compared to 1985–1988, rates in 2001–2011 were lower in all serotype groupings, by 98% for Hib, 75% for Hia, 79% for other serotypes and 67% for non-typeable Hi. Conclusions. There is no evidence of increases in invasive disease due to Hia, other specific non-b types, or non-typeable Hi in Australian Indigenous children. These data suggest that the increase in Hia some time after the introduction of Hib vaccine, as seen in the North American Arctic Region, is not common to all populations with high pre-vaccine rates of invasive Hib disease. However, small case numbers and the lack of molecular subtyping and PCR confirmation of pre-vaccine results complicate comparisons with North American epidemiology.

  2. Haemophilus influenzae Type b disease among Amish children in Pennsylvania: reasons for persistent disease.

    Science.gov (United States)

    Fry, A M; Lurie, P; Gidley, M; Schmink, S; Lingappa, J; Fischer, M; Rosenstein, N E

    2001-10-01

    To identify reservoirs of Haemophilus influenzae type b (Hib) pharyngeal carriage and assess barriers to vaccination among 2 Amish communities in Pennsylvania. We investigated recent cases, performed community surveys for Hib vaccination coverage and pharyngeal carriage, and administered a questionnaire assessing vaccination knowledge and attitudes to 298 members of 2 Amish communities (A and B) in Pennsylvania and, as a comparison group, 136 non-Amish family members who participated in state immunization clinics. From December 1999 to February 2000, 8 cases of invasive Hib disease occurred among children who were 5 years of age or younger in Pennsylvania. Six of the case-patients were from Amish communities. None of the children had been vaccinated. Among children who were 5 years of age or younger, Hib vaccine coverage was low in the 2 Amish communities: A (9 [28%] of 32) and B (3 [7%] of 41) compared with the non-Amish group (19 [95%] of 20). Hib carriage prevalence was higher in both Amish communities than in the non-Amish group (A: 3%; B: 8%; non-Amish: 0%). More households in community B had 1 or more Hib carriers than in community A (8 [28%] of 29 vs 3 [9%] of 32). Among Amish parents who did not vaccinate their children, only 25% (13 of 51) identified either religious or philosophical objections as a factor; 51% (26 of 51) reported that vaccinating was not a priority compared with other activities of daily life. Seventy-three percent (36 of 49) would vaccinate their children if vaccination were offered locally. Undervaccinated communities in the United States still exist and allow circulation of Hib strains, resulting in disease among susceptible children. Identification of undervaccinated populations, such as the Amish, and targeted education and vaccination campaigns are essential to achieving elimination of Hib disease.

  3. Defining Potential Vaccine Targets of Haemophilus ducreyi Trimeric Autotransporter Adhesin DsrA.

    Science.gov (United States)

    Fusco, William G; Choudhary, Neelima R; Stewart, Shelley M; Alam, S Munir; Sempowski, Gregory D; Elkins, Christopher; Leduc, Isabelle

    2015-04-01

    Haemophilus ducreyi is the causative agent of the sexually transmitted genital ulcer disease chancroid. Strains of H. ducreyi are grouped in two classes (I and II) based on genotypic and phenotypic differences, including those found in DsrA, an outer membrane protein belonging to the family of multifunctional trimeric autotransporter adhesins. DsrA is a key serum resistance factor of H. ducreyi that prevents binding of natural IgM at the bacterial surface and functions as an adhesin to fibronectin, fibrinogen, vitronectin, and human keratinocytes. Monoclonal antibodies (MAbs) were developed to recombinant DsrA (DsrA(I)) from prototypical class I strain 35000HP to define targets for vaccine and/or therapeutics. Two anti-DsrAI MAbs bound monomers and multimers of DsrA from genital and non-genital/cutaneous H. ducreyi strains in a Western blot and reacted to the surface of the genital strains; however, these MAbs did not recognize denatured or native DsrA from class II strains. In a modified extracellular matrix protein binding assay using viable H. ducreyi, one of the MAbs partially inhibited binding of fibronectin, fibrinogen, and vitronectin to class I H. ducreyi strain 35000HP, suggesting a role for anti-DsrA antibodies in preventing binding of H. ducreyi to extracellular matrix proteins. Standard ELISA and surface plasmon resonance using a peptide library representing full-length, mature DsrAI revealed the smallest nominal epitope bound by one of the MAbs to be MEQNTHNINKLS. Taken together, our findings suggest that this epitope is a potential target for an H. ducreyi vaccine.

  4. Specificity of antibodies directed against the cytolethal distending toxin of Haemophilus ducreyi in patients with chancroid.

    Science.gov (United States)

    Mbwana, Judica; Ahmed, Hinda J; Ahlman, Karin; Sundaeus, Vivian; Dahlén, Gunnar; Lyamuya, Eligius; Lagergård, Teresa

    2003-09-01

    Antibodies specific for the cytolethal-distending toxin of Haemophilus ducreyi (HdCDT) complex and for the CdtA, CdtB, and CdtC components were measured by ELISA in the sera of 50 patients with culture and/or PCR proven chancroid, 42 patients with periodontitis, 50 blood donors from Tanzania, 50 blood donors from Sweden. In addition, the biological activity e.g. neutralization capacity of the sera were tested. Our results demonstrate that majority of chancroid patients and healthy individuals had detectable levels of serum antibodies to HdCDT complex and to separate toxin components. However, high levels (> or =100 units) of antibodies to HdCDT complex were significantly more prevalent in the sera of patients with both chancroid and periodontitis than in the sera of the corresponding controls (P=0.001 and P=0.04, respectively). In the sera of the 50 patients with chancroid, antibodies to CdtA, CdtB, and CdtC were detected in 50, 35, and 34 individuals, respectively. Antibodies to CdtC, being less frequently detected than the antibodies to other components, show a good correlation with the neutralizing capacity of sera. High levels of neutralizing antibodies (> or =160) were detected in only 22 and 2% of the patients with chancroid and periodontitis, respectively. The data suggest that the low levels of anti-HdCDT antibodies, which include neutralizing antibodies, may contribute to limited protection in chancroid and since anti-HdCDT antibodies, may be detected in healthy individuals and in patients with certain disease conditions (e.g. periodontitis), they may not be specific markers for chancroid infection.

  5. Trimeric autotransporter DsrA is a major mediator of fibrinogen binding in Haemophilus ducreyi.

    Science.gov (United States)

    Fusco, William G; Elkins, Christopher; Leduc, Isabelle

    2013-12-01

    Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. In both natural and experimental chancroid, H. ducreyi colocalizes with fibrin at the base of the ulcer. Fibrin is obtained by cleavage of the serum glycoprotein fibrinogen (Fg) by thrombin to initiate formation of the blood clot. Fg binding proteins are critical virulence factors in medically important Gram-positive bacteria. H. ducreyi has previously been shown to bind Fg in an agglutination assay, and the H. ducreyi Fg binding protein FgbA was identified in ligand blotting with denatured proteins. To better characterize the interaction of H. ducreyi with Fg, we examined Fg binding to intact, viable H. ducreyi bacteria and identified a novel Fg binding protein. H. ducreyi bound unlabeled Fg in a dose-dependent manner, as measured by two different methods. In ligand blotting with total denatured cellular proteins, digoxigenin (DIG)-Fg bound only two H. ducreyi proteins, the trimeric autotransporter DsrA and the lectin DltA; however, only the isogenic dsrA mutant had significantly less cell-associated Fg than parental strains in Fg binding assays with intact bacteria. Furthermore, expression of DsrA, but not DltA or an empty vector, rendered the non-Fg-binding H. influenzae strain Rd capable of binding Fg. A 13-amino-acid sequence in the C-terminal section of the passenger domain of DsrA appears to be involved in Fg binding by H. ducreyi. Taken together, these data suggest that the trimeric autotransporter DsrA is a major determinant of Fg binding at the surface of H. ducreyi.

  6. The Seroepidemiology of Haemophilus Influenzae Type b in Children Under 6 Years Old in Khorramabad, Iran

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    N. Ansari

    2015-05-01

    Full Text Available Background and Objective: Haemophilus influenzae type b (Hib is now recognized as an agent of bacterial meningitis in Asia. Polyribosil Ribitol Phosphate (PRP capsule are in all of the strains and IgG antibodies can be target. Due to limited studies on H. influenzae type b in the country and the lack of sufficient information on the status of carriers, achieve a comprehensive model of the spread of the bacteria in the carrier's most vulnerable children is the aim of this study. Material and Methods: In this study, 194 (49% female and 51% male serum samples were collected from children under 6 years old referred to medical centers in Korram Abad. The serological study of H. influenzae type b was carried using anti-H. influenzae PRP IgG antibodies kit by indirect enzyme linked immunosorbent assay (ELISA. Results: Of 194 children under 6 years-old screened for H. influenzae-specific IgG antibodies with ELISA, 6 (3 percent were IgG seropositive. Prevalence of bacteria in children under 2 years was 67% that demonstrated no significant association between seropositivity in subjects and place of residence of children with the prevalence of bacteria. Conclusion: The prevalence of H. influenzae type b seropositivity in our study was 3% of the total sample. In current study, a higher percentage of males than females were seropositive and frequency of predominant bacteria in patients younger than 2 years. The age factor is a variable that positively affects serum. Regarding to non-Hib vaccination in children, the presence of the bacteria in children can be important.

  7. Economic evaluation of delivering Haemophilus influenzae type b vaccine in routine immunization services in Kenya.

    Science.gov (United States)

    Akumu, Angela Oloo; English, Mike; Scott, J Anthony G; Griffiths, Ulla K

    2007-07-01

    Haemophilus influenzae type b (Hib) vaccine was introduced into routine immunization services in Kenya in 2001. We aimed to estimate the cost-effectiveness of Hib vaccine delivery. A model was developed to follow the Kenyan 2004 birth cohort until death, with and without Hib vaccine. Incidence of invasive Hib disease was estimated at Kilifi District Hospital and in the surrounding demographic surveillance system in coastal Kenya. National Hib disease incidence was estimated by adjusting incidence observed by passive hospital surveillance using assumptions about access to care. Case fatality rates were also assumed dependent on access to care. A price of US$ 3.65 per dose of pentavalent diphtheria-tetanus-pertussis-hep B-Hib vaccine was used. Multivariate Monte Carlo simulations were performed in order to assess the impact on the cost-effectiveness ratios of uncertainty in parameter values. The introduction of Hib vaccine reduced the estimated incidence of Hib meningitis per 100,000 children aged disability adjusted life year (DALY) and per discounted death averted were US$ 38 (95% confidence interval, CI: 26-63) and US$ 1197 (95% CI: 814-2021) respectively. Most of the uncertainty in the results was due to uncertain access to care parameters. The break-even pentavalent vaccine price--where incremental Hib vaccination costs equal treatment costs averted from Hib disease--was US$ 1.82 per dose. Hib vaccine is a highly cost-effective intervention in Kenya. It would be cost-saving if the vaccine price was below half of its present level.

  8. Accelerating policy decisions to adopt haemophilus influenzae type B vaccine: a global, multivariable analysis.

    Science.gov (United States)

    Shearer, Jessica C; Stack, Meghan L; Richmond, Marcie R; Bear, Allyson P; Hajjeh, Rana A; Bishai, David M

    2010-03-16

    Adoption of new and underutilized vaccines by national immunization programs is an essential step towards reducing child mortality. Policy decisions to adopt new vaccines in high mortality countries often lag behind decisions in high-income countries. Using the case of Haemophilus influenzae type b (Hib) vaccine, this paper endeavors to explain these delays through the analysis of country-level economic, epidemiological, programmatic and policy-related factors, as well as the role of the Global Alliance for Vaccines and Immunisation (GAVI Alliance). Data for 147 countries from 1990 to 2007 were analyzed in accelerated failure time models to identify factors that are associated with the time to decision to adopt Hib vaccine. In multivariable models that control for Gross National Income, region, and burden of Hib disease, the receipt of GAVI support speeded the time to decision by a factor of 0.37 (95% CI 0.18-0.76), or 63%. The presence of two or more neighboring country adopters accelerated decisions to adopt by a factor of 0.50 (95% CI 0.33-0.75). For each 1% increase in vaccine price, decisions to adopt are delayed by a factor of 1.02 (95% CI 1.00-1.04). Global recommendations and local studies were not associated with time to decision. This study substantiates previous findings related to vaccine price and presents new evidence to suggest that GAVI eligibility is associated with accelerated decisions to adopt Hib vaccine. The influence of neighboring country decisions was also highly significant, suggesting that approaches to support the adoption of new vaccines should consider supply- and demand-side factors.

  9. Economic Evaluation and Budget Impact Analysis of Vaccination against Haemophilus influenzae Type b Infection in Thailand

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    Surachai Kotirum

    2017-11-01

    Full Text Available Current study aimed to estimate clinical and economic outcomes of providing the Haemophilus influenzae type b (Hib vaccination as a national vaccine immunization program in Thailand. A decision tree combined with Markov model was developed to simulate relevant costs and health outcomes covering lifetime horizon in societal and health care payer perspectives. This analysis considered children aged under 5 years old whom preventive vaccine of Hib infection are indicated. Two combined Hib vaccination schedules were considered: three-dose series (3 + 0 and three-dose series plus a booster does (3 + 1 compared with no vaccination. Budget impact analysis was also performed under Thai government perspective. The outcomes were reported as Hib-infected cases averted and incremental cost-effectiveness ratios (ICERs in 2014 Thai baht (THB ($ per quality-adjusted life year (QALY gained. In base-case scenario, the model estimates that 3,960 infected cases, 59 disability cases, and 97 deaths can be prevented by national Hib vaccination program. The ICER for 3 + 0 schedule was THB 1,099 ($34 per QALY gained under societal perspective. The model was sensitive to pneumonia incidence among aged under 5 years old and direct non-medical care cost per episode of Hib pneumonia. Hib vaccination is very cost-effective in the Thai context. The budget impact analysis showed that Thai government needed to invest an additional budget of 110 ($3.4 million to implement Hib vaccination program. Policy makers should consider our findings for adopting this vaccine into national immunization program.

  10. Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis.

    Science.gov (United States)

    Abdeldaim, Guma M K; Strålin, Kristoffer; Korsgaard, Jens; Blomberg, Jonas; Welinder-Olsson, Christina; Herrmann, Björn

    2010-12-03

    Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients. The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis) in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 10⁵ genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively.In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both bacteria. The PCR provides increased

  11. Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis

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    Welinder-Olsson Christina

    2010-12-01

    Full Text Available Abstract Background Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR for detection of S. pneumoniae (9802 gene fragment, H. influenzae (omp P6 gene and N. meningitidis (ctrA gene. The method was evaluated on bronchoalveolar lavage (BAL samples from 156 adults with lower respiratory tract infection (LRTI and 31 controls, and on 87 cerebrospinal fluid (CSF samples from meningitis patients. Results The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 105 genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively. In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both

  12. Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication

    International Nuclear Information System (INIS)

    Kim, Seong K.; Kim, Seongman; Dai Gan; Zhang Yunfei; Ahn, Byung C.; O'Callaghan, Dennis J.

    2011-01-01

    The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% of control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain. - Highlights: → We examine the functional domains of IR2P that mediates negative regulation. → IR2P inhibits at the transcriptional level. → DNA-binding mutant or TFIIB-binding mutant fails to inhibit. → C-terminal aa 707 to 1116 are required for full inhibition. → Inhibition requires the DNA-binding domain, TFIIB-binding domain, and C-terminus.

  13. The histone demethylase LSD1 is required for estrogen-dependent S100A7 gene expression in human breast cancer cells

    International Nuclear Information System (INIS)

    Yu, Seung Eun; Jang, Yeun Kyu

    2012-01-01

    Highlights: ► S100A7 gene is up-regulated in response to estrogen in breast cancer cells. ► Histone demethylase LSD1 can associate physically with S100A7 gene promoters. ► E2-induced S100A7 expression requires the enzymatic activity of LSD1. ► S100A7 inhibits cell proliferation, implying its tumor suppressor-like function. -- Abstract: S100A7, a member of S100 calcium binding protein family, is highly associated with breast cancer. However, the molecular mechanism of S100A7 regulation remains unclear. Here we show that long-term treatment with estradiol stimulated S100A7 expression in MCF7 breast cancer cells at both the transcriptional and translational levels. Both treatment with a histone demethylase LSD1 inhibitor and shRNA-based knockdown of LSD1 expression significantly decreased 17β-estradiol (E2)-induced S100A7 expression. These reduced E2-mediated S100A7 expression are rescued by the overexpressed wild-type LSD1 but not by its catalytically inactive mutant. Our data showed in vivo association of LSD1 with S100A7 promoters, confirming the potential role of LSD1 in regulating S100A7 expression. S100A7 knockdown increased both normal cell growth and estrogen-induced cell proliferation, suggesting a negative influence by S100A7 on the growth of cancer cells. Together, our data suggest that estrogen-induced S100A7 expression mediated by the histone demethylase LSD1 may downregulate breast cancer cell proliferation, implying a potential tumor suppressor-like function for S100A7.

  14. Arabidopsis Raf-Like Mitogen-Activated Protein Kinase Kinase Kinase Gene Raf43 Is Required for Tolerance to Multiple Abiotic Stresses.

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    Nasar Virk

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are critical signaling modules that mediate the transduction of extracellular stimuli into intracellular response. A relatively large number of MAPKKKs have been identified in a variety of plant genomes but only a few of them have been studied for their biological function. In the present study, we identified an Arabidopsis Raf-like MAPKKK gene Raf43 and studied its function in biotic and abiotic stress response using a T-DNA insertion mutant raf43-1 and two Raf43-overexpressing lines Raf43-OE#1 and Raf43-OE#13. Expression of Raf43 was induced by multiple abiotic and biotic stresses including treatments with drought, mannitol and oxidative stress or defense signaling molecule salicylic acid and infection with necrotrophic fungal pathogen Botrytis cinerea. Seed germination and seedling root growth of raf43-1 were significantly inhibited on MS medium containing mannitol, NaCl, H2O2 or methyl viologen (MV while seed germination and seedling root growth of the Raf43-OE#1 and Raf43-OE#13 lines was similar to wild type Col-0 under the above stress conditions. Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress. Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines. Expression of stress-responsive RD17 and DREB2A genes was significantly down-regulated in raf43-1 plants. However, the raf43-1 and Raf43-overexpressing plants showed similar disease phenotype to the wild type plants after infection with B. cinerea or Pseudomonas syringae pv. tomato DC3000. Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis.

  15. The Muscular Dystrophy Gene TMEM5 Encodes a Ribitol β1,4-Xylosyltransferase Required for the Functional Glycosylation of Dystroglycan.

    Science.gov (United States)

    Manya, Hiroshi; Yamaguchi, Yoshiki; Kanagawa, Motoi; Kobayashi, Kazuhiro; Tajiri, Michiko; Akasaka-Manya, Keiko; Kawakami, Hiroko; Mizuno, Mamoru; Wada, Yoshinao; Toda, Tatsushi; Endo, Tamao

    2016-11-18

    A defect in O-mannosyl glycan is the cause of α-dystroglycanopathy, a group of congenital muscular dystrophies caused by aberrant α-dystroglycan (α-DG) glycosylation. Recently, the entire structure of O-mannosyl glycan, [3GlcAβ1-3Xylα1] n -3GlcAβ1-4Xyl-Rbo5P-1Rbo5P-3GalNAcβ1-3GlcNAcβ1-4 (phospho-6)Manα1-, which is required for the binding of α-DG to extracellular matrix ligands, has been proposed. However, the linkage of the first Xyl residue to ribitol 5-phosphate (Rbo5P) is not clear. TMEM5 is a gene product responsible for α-dystroglycanopathy and was reported as a potential enzyme involved in this linkage formation, although the experimental evidence is still incomplete. Here, we report that TMEM5 is a xylosyltransferase that forms the Xylβ1-4Rbo5P linkage on O-mannosyl glycan. The anomeric configuration and linkage position of the product (β1,4 linkage) was determined by NMR analysis. The introduction of two missense mutations in TMEM5 found in α-dystroglycanopathy patients impaired xylosyltransferase activity. Furthermore, the disruption of the TMEM5 gene by CRISPR/Cas9 abrogated the elongation of the (-3GlcAβ1-3Xylα1-) unit on O-mannosyl glycan. Based on these results, we concluded that TMEM5 acts as a UDP-d-xylose:ribitol-5-phosphate β1,4-xylosyltransferase in the biosynthetic pathway of O-mannosyl glycan. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. The pmr gene, encoding a Ca2+-ATPase, is required for calcium and manganese homeostasis and normal development of hyphae and conidia in Neurospora crassa.

    Science.gov (United States)

    Bowman, Barry J; Abreu, Stephen; Johl, Jessica K; Bowman, Emma Jean

    2012-11-01

    The pmr gene is predicted to encode a Ca(2+)-ATPase in the secretory pathway. We examined two strains of Neurospora crassa that lacked PMR: the Δpmr strain, in which pmr was completely deleted, and pmr(RIP), in which the gene was extensively mutated. Both strains had identical, complex phenotypes. Compared to the wild type, these strains required high concentrations of calcium or manganese for optimal growth and had highly branched, slow-growing hyphae. They conidiated poorly, and the shape and size of the conidia were abnormal. Calcium accumulated in the Δpmr strains to only 20% of the wild-type level. High concentrations of MnCl(2) (1 to 5 mM) in growth medium partially suppressed the morphological defects but did not alter the defect in calcium accumulation. The Δpmr Δnca-2 double mutant (nca-2 encodes a Ca(2+)-ATPase in the plasma membrane) accumulated 8-fold more calcium than the wild type, and the morphology of the hyphae was more similar to that of wild-type hyphae. Previous experiments failed to show a function for nca-1, which encodes a SERCA-type Ca(2+)-ATPase in the endoplasmic reticulum (B. J. Bowman, S. Abreu, E. Margolles-Clark, M. Draskovic, and E. J. Bowman, Eukaryot. Cell 10:654-661, 2011). The pmr(RIP) Δnca-1 double mutant accumulated small amounts of calcium, like the Δpmr strain, but exhibited even more extreme morphological defects. Thus, PMR can apparently replace NCA-1 in the endoplasmic reticulum, but NCA-1 cannot replace PMR. The morphological defects in the Δpmr strain are likely caused, in part, by insufficient concentrations of calcium and manganese in the Golgi compartment; however, PMR is also needed to accumulate normal levels of calcium in the whole cell.

  17. A Tourist-like MITE insertion in the upstream region of the BnFLC.A10 gene is associated with vernalization requirement in rapeseed (Brassica napus L.

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    Hou Jinna

    2012-12-01

    Full Text Available Abstract Background Rapeseed (Brassica napus L. has spring and winter genotypes adapted to different growing seasons. Winter genotypes do not flower before the onset of winter, thus leading to a longer vegetative growth period that promotes the accumulation and allocation of more resources to seed production. The development of winter genotypes enabled the rapeseed to spread rapidly from southern to northern Europe and other temperate regions of the world. The molecular basis underlying the evolutionary transition from spring- to winter- type rapeseed is not known, however, and needs to be elucidated. Results We fine-mapped the spring environment specific quantitative trait locus (QTL for flowering time, qFT10-4,in a doubled haploid (DH mapping population of rapeseed derived from a cross between Tapidor (winter-type and Ningyou7 (semi-winter and delimited the qFT10-4 to an 80-kb region on chromosome A10 of B. napus. The BnFLC.A10 gene, an ortholog of FLOWERING LOCUS C (FLC in Arabidopsis, was cloned from the QTL. We identified 12 polymorphic sites between BnFLC.A10 parental alleles of the TN-DH population in the upstream region and in intron 1. Expression of both BnFLC.A10 alleles decreased during vernalization, but decreased more slowly in the winter parent Tapidor. Haplotyping and association analysis showed that one of the polymorphic sites upstream of BnFLC.A10 is strongly associated with the vernalization requirement of rapeseed (r2 = 0.93, χ2 = 0.50. This polymorphic site is derived from a Tourist-like miniature inverted-repeat transposable element (MITE insertion/deletion in the upstream region of BnFLC.A10. The MITE sequence was not present in the BnFLC.A10 gene in spring-type rapeseed, nor in ancestral ‘A’ genome species B. rapa genotypes. Our results suggest that the insertion may have occurred in winter rapeseed after B. napus speciation. Conclusions Our findings strongly suggest that (i BnFLC.A10 is the gene underlying qFT10

  18. A Tourist-like MITE insertion in the upstream region of the BnFLC.A10 gene is associated with vernalization requirement in rapeseed (Brassica napus L.).

    Science.gov (United States)

    Hou, Jinna; Long, Yan; Raman, Harsh; Zou, Xiaoxiao; Wang, Jing; Dai, Shutao; Xiao, Qinqin; Li, Cong; Fan, Longjiang; Liu, Bin; Meng, Jinling

    2012-12-15

    Rapeseed (Brassica napus L.) has spring and winter genotypes adapted to different growing seasons. Winter genotypes do not flower before the onset of winter, thus leading to a longer vegetative growth period that promotes the accumulation and allocation of more resources to seed production. The development of winter genotypes enabled the rapeseed to spread rapidly from southern to northern Europe and other temperate regions of the world. The molecular basis underlying the evolutionary transition from spring- to winter- type rapeseed is not known, however, and needs to be elucidated. We fine-mapped the spring environment specific quantitative trait locus (QTL) for flowering time, qFT10-4,in a doubled haploid (DH) mapping population of rapeseed derived from a cross between Tapidor (winter-type) and Ningyou7 (semi-winter) and delimited the qFT10-4 to an 80-kb region on chromosome A10 of B. napus. The BnFLC.A10 gene, an ortholog of FLOWERING LOCUS C (FLC) in Arabidopsis, was cloned from the QTL. We identified 12 polymorphic sites between BnFLC.A10 parental alleles of the TN-DH population in the upstream region and in intron 1. Expression of both BnFLC.A10 alleles decreased during vernalization, but decreased more slowly in the winter parent Tapidor. Haplotyping and association analysis showed that one of the polymorphic sites upstream of BnFLC.A10 is strongly associated with the vernalization requirement of rapeseed (r2 = 0.93, χ2 = 0.50). This polymorphic site is derived from a Tourist-like miniature inverted-repeat transposable element (MITE) insertion/deletion in the upstream region of BnFLC.A10. The MITE sequence was not present in the BnFLC.A10 gene in spring-type rapeseed, nor in ancestral 'A' genome species B. rapa genotypes. Our results suggest that the insertion may have occurred in winter rapeseed after B. napus speciation. Our findings strongly suggest that (i) BnFLC.A10 is the gene underlying qFT10-4, the QTL for phenotypic diversity of flowering time in

  19. Genomic DNA fingerprinting of clinical Haemophilus influenzae isolates by polymerase chain reaction amplification: comparison with major outer-membrane protein and restriction fragment length polymorphism analysis

    NARCIS (Netherlands)

    van Belkum, A.; Duim, B.; Regelink, A.; Möller, L.; Quint, W.; van Alphen, L.

    1994-01-01

    Non-capsulate strains of Haemophilus influenzae were genotyped by analysis of variable DNA segments obtained by amplification of genomic DNA with the polymerase chain reaction (PCR fingerprinting). Discrete fragments of 100-2000 bp were obtained. The reproducibility of the procedure was assessed by

  20. Costo-efectividad de la vacunación contra Haemophilus influenzae tipo b : un análisis de decisión para Cuba

    NARCIS (Netherlands)

    Carcía, A; Postma, Maarten; Gálvez, AM; Sierra, G

    2002-01-01

    The objective of the present study was to estimate the cost-effectiveness ratio of the therapeutic and prophylactic alternatives for treatment of meningitis caused by Haemophilus influenzae type b in less than one year old infants applying a mathematical model. A hypothetical setting with two

  1. Contribution of the major and minor subunits to fimbria-mediated adherence of Haemophilus influenzae to human epithelial cells and erythrocytes

    NARCIS (Netherlands)

    van Ham, S. M.; van Alphen, L.; Mooi, F. R.; van Putten, J. P.

    1995-01-01

    Fimbriae are colonization factors of the human pathogen Haemophilus influenzae in that they mediate bacterial adherence to human eukaryotic cells. The contribution of the major (HifA) and putative minor (HifD and HifE) subunits of H. influenzae fimbriae to fimbria-specific adherence was studied by

  2. Frequent carriage of resistance mechanisms to beta-lactams and biofilm formation in Haemophilus influenzae causing treatment failure and recurrent otitis media in young children

    NARCIS (Netherlands)

    Garcia-Cobos, Silvia; Moscoso, Miriam; Pumarola, Felix; Arroyo, Margarita; Lara, Noelia; Perez-Vazquez, Maria; Aracil, Belen; Oteo, Jesus; Garcia, Ernesto; Campos, Jose

    Objectives: Non-typeable Haemophilus influenzae are a major cause of acute otitis media (AOM), including chronic and recurrent otitis in young children. The objective of this study was to determine whether non-typeable H. influenzae isolates causing these infections produce biofilms and carry

  3. Sensibilidad antibiótica de cepas de Haemophilus spp aisladas de pacientes pediátricos en un hospi- tal general entre los años 2003 -2006.

    OpenAIRE

    Alarco Castro, Raúl Rodrigo; Cavero Chávez, Vanessa Yohana; Hernández Díaz, Herminio Renán; Tapia Egoávil, Elena Zoraida

    2012-01-01

    Objetivo: Determinar la sensibilidad antibiótica de cepas de Haemophilus spp aisladas de pacientes pediátricos en un hospital general entre los años 2003 y 2006. Materiales y métodos: Estudio descriptivo retrospectivo en el que se incluyeron cultivos positivos de Haemophilus spp. Se revisaron las historias clínicas de los pacientes de los cuales provenían los cultivos. Resultados: Se encontraron 37 cultivos positivos para Haemophilus spp, entre los cuales se encontró mayor sensibilidad a levo...

  4. Identification of the promoter region required for human adiponectin gene transcription: Association with CCAAT/enhancer binding protein-β and tumor necrosis factor-α

    International Nuclear Information System (INIS)

    Kita, Atsushi; Yamasaki, Hironori; Kuwahara, Hironaga; Moriuchi, Akie; Fukushima, Keiko; Kobayashi, Masakazu; Fukushima, Tetsuya; Takahashi, Ryoko; Abiru, Norio; Uotani, Shigeo; Kawasaki, Eiji; Eguchi, Katsumi

    2005-01-01

    Adiponectin, an adipose tissue-specific plasma protein, is involved in insulin sensitizing and has anti-atherosclerotic properties. Plasma levels of adiponectin are decreased in obese individuals and patients with type 2 diabetes with insulin resistance. Tumor necrosis factor-α (TNF-α) decreases the expression of adiponectin in adipocytes. The aims of the present study were: (1) to identify the promoter region responsible for basal transcription of the human adiponectin gene, and (2) to investigate the mechanism by which adiponectin was regulated by TNF-α. The human adiponectin promoter (2.1 kb) was isolated and used for luciferase reporter analysis by transient transfection into 3T3-L1 adipocytes. Deletion analysis demonstrated that the promoter region from -676 to +41 was sufficient for basal transcriptional activity. Mutation analysis of putative response elements for sterol regulatory element binding protein (SREBP) (-431 to -423) and CCAAT/enhancer binding protein (C/EBP) (-230 to -224) showed that both elements were required for basal promoter activity. Adiponectin transcription was increased 3-fold in cells that over-expressed constitutively active C/EBP-β. Electrophoretic mobility shift assay, using nuclear extract from 3T3-L1 cells and the -258 to -199 region as a probe, demonstrated specific DNA-protein binding, which was abolished by TNF-α treatment. The present data indicate that the putative response elements for SREBP and C/EBP are required for human adiponectin promoter activity, and that suppression by TNF-α may, at least in part, be associated with inactivation of C/EBP-β

  5. The Drosophila Su(var)3-7 gene is required for oogenesis and female fertility, genetically interacts with piwi and aubergine, but impacts only weakly transposon silencing.

    Science.gov (United States)

    Basquin, Denis; Spierer, Anne; Begeot, Flora; Koryakov, Dmitry E; Todeschini, Anne-Laure; Ronsseray, Stéphane; Vieira, Cristina; Spierer, Pierre; Delattre, Marion

    2014-01-01

    Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3-7 protein in Drosophila ovaries. We present evidences that Su(var)3-7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3-7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3-7 is required for genome integrity. Females homozygous for Su(var)3-7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3-7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3-7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3-7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3-7 and piwi or aubergine controls important developmental processes independently of transposon silencing.

  6. The Drosophila Su(var)3–7 Gene Is Required for Oogenesis and Female Fertility, Genetically Interacts with piwi and aubergine, but Impacts Only Weakly Transposon Silencing

    Science.gov (United States)

    Begeot, Flora; Koryakov, Dmitry E.; Todeschini, Anne-Laure; Ronsseray, Stéphane; Vieira, Cristina; Spierer, Pierre; Delattre, Marion

    2014-01-01

    Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3–7 protein in Drosophila ovaries. We present evidences that Su(var)3–7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3–7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3–7 is required for genome integrity. Females homozygous for Su(var)3–7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3–7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3–7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3–7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3–7 and piwi or aubergine controls important developmental processes independently of transposon silencing. PMID:24820312

  7. Final Report Grant No. DE-FG02-98ER20307 Lipopolysaccharide Structures and Genes Required for Root Nodule Development August 1, 2004 to July 31, 2008

    Energy Technology Data Exchange (ETDEWEB)

    Noel, K. Dale [Marquette Univ., Milwaukee,WI (United States)

    2008-12-07

    the roles of other important bacterial factors at multiple stages of nodule development. The project also investigated the biosynthesis of this bacterial factor. It has a complex structure and the first accomplishment was the determination of the sequences of genetic regions known to be important. Next the discovered genes were mutated to identify the 26 that are required for its synthesis. In addition, six others were discovered that are believed to change its structure under various environmental conditions. By studying mutants affected in specific genes, genes were associated with each of the predicted steps in the biosynthesis. Current work is testing the predicted biosynthetic model with studies conducted in vitro with bacterial extracts. Overall, the work funded by this grant establishes this system as a model for host-bacterial interactions based on specific polysaccharide structure. All areas that are needed for a comprehensive model have been significantly advanced: the biological function, the structural features that are crucial, the complete set of bacterial genes involved, and a model for the biosynthesis.

  8. Process development of a New Haemophilus influenzae type b conjugate vaccine and the use of mathematical modeling to identify process optimization possibilities.

    Science.gov (United States)

    Hamidi, Ahd; Kreeftenberg, Hans; V D Pol, Leo; Ghimire, Saroj; V D Wielen, Luuk A M; Ottens, Marcel

    2016-05-01

    Vaccination is one of the most successful public health interventions being a cost-effective tool in preventing deaths among young children. The earliest vaccines were developed following empirical methods, creating vaccines by trial and error. New process development tools, for example mathematical modeling, as well as new regulatory initiatives requiring better understanding of both the product and the process are being applied to well-characterized biopharmaceuticals (for example recombinant proteins). The vaccine industry is still running behind in comparison to these industries. A production process for a new Haemophilus influenzae type b (Hib) conjugate vaccine, including related quality control (QC) tests, was developed and transferred to a number of emerging vaccine manufacturers. This contributed to a sustainable global supply of affordable Hib conjugate vaccines, as illustrated by the market launch of the first Hib vaccine based on this technology in 2007 and concomitant price reduction of Hib vaccines. This paper describes the development approach followed for this Hib conjugate vaccine as well as the mathematical modeling tool applied recently in order to indicate options for further improvements of the initial Hib process. The strategy followed during the process development of this Hib conjugate vaccine was a targeted and integrated approach based on prior knowledge and experience with similar products using multi-disciplinary expertise. Mathematical modeling was used to develop a predictive model for the initial Hib process (the 'baseline' model) as well as an 'optimized' model, by proposing a number of process changes which could lead to further reduction in price. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:568-580, 2016. © 2016 American Institute of Chemical Engineers.

  9. Impacto da vacinação contra o Haemophilus influenzae b na redução de meningites, Goiás

    Directory of Open Access Journals (Sweden)

    Simões Luciana Leite Pineli

    2004-01-01

    Full Text Available OBJETIVO: Avaliar o impacto da vacinação contra o Haemophilus influenzae b na incidência de meningites em crianças menores de cinco anos de idade. MÉTODOS: Utilizou-se o delineamento tipo "antes-depois" para comparar as taxas de incidência de meningites por Haemophilus influenzae b nos períodos pré-vacinação (julho/95-junho/99 e pós-vacinação (julho/99-junho/2001 no Estado de Goiás. A definição de caso de meningite bacteriana seguiu os critérios da Organização Mundial de Saúde. As taxas de meningite por Streptococcus pneumoniae e Neisseria. meningitidis foram utilizadas para efeito de comparação. Para análise estatística foram utilizados o teste de chi2 e o t de Student. Valores de p<0,05 foram considerados estatisticamente significantes. RESULTADOS: Foi detectada meningite bacteriana aguda em 979 crianças no período de estudo. A incidência de meningite por Haemophilus influenzae b diminuiu de 10,8x10(5 no período pré-vacinal para 2,3x10(5 no segundo ano pós-vacina, significando 78% de redução no risco, principalmente na faixa etária de 7-23 meses (p<0,05. Foram prevenidos 65 casos de meningite por Haemophilus influenzae b. Observou-se aumento na incidência de meningite por S. pneumoniae. Foi observada falha vacinal em um caso. CONCLUSÕES: Expressivo declínio da incidência de meningite por Haemophilus influenzae b foi detectado, precocemente, logo após o primeiro ano de introdução da vacina contra o Haemophilus influenzae b. Assim, se faz necessária a vigilância contínua com instrumental de alta acurácia para: (i detectar re-emergência do Haemophilus influenzae b; (ii avaliar possibilidade de falha vacinal; (iii identificar mudanças no padrão dos sorotipos do H. influenzae.

  10. Autophagy genes Smatg8 and Smatg4 are required for fruiting-body development, vegetative growth and ascospore germination in the filamentous ascomycete Sordaria macrospora.

    Science.gov (United States)

    Voigt, Oliver; Pöggeler, Stefanie

    2013-01-01

    Autophagy is a tightly controlled degradation process involved in various developmental aspects of eukaryotes. However, its involvement in developmental processes of multicellular filamentous ascomycetes is largely unknown. Here, we analyzed the impact of the autophagic proteins SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. A Saccharomyces cerevisiae complementation assay demonstrated that the S. macrospora Smatg8 and Smatg4 genes can functionally replace the yeast homologs. By generating homokaryotic deletion mutants, we showed that the S. macrospora SmATG8 and SmATG4 orthologs were associated with autophagy-dependent processes. Smatg8 and Smatg4 deletions abolished fruiting-body formation and impaired vegetative growth and ascospore germination, but not hyphal fusion. We demonstrated that SmATG4 was capable of processing the SmATG8 precursor. SmATG8 was localized to autophagosomes, whereas SmATG4 was distributed throughout the cytoplasm of S. macrospora. Furthermore, we could show that Smatg8 and Smatg4 are not only required for nonselective macroautophagy, but for selective macropexophagy as well. Taken together, our results suggest that in S. macrospora, autophagy seems to be an essential and constitutively active process to sustain high energy levels for filamentous growth and multicellular development even under nonstarvation conditions.

  11. Proposal of Histophilus somni gen. nov., sp nov for the three species incertae sedis 'Haemophilus somnus', 'Haemophilus agni' and 'Histophilus ovis'

    DEFF Research Database (Denmark)

    Angen, Øystein; Ahrens, Peter; Kuhnert, P.

    2003-01-01

    previously by DNA-DNA hybridization. These results clearly support the allocation of this species to a novel genus within the family Pasteurellaceae. The phenotypic separation of Histophilus somni gen. nov., sp. nov. from other members of the family can, for most strains, be based on capnophilia, yellowish...... pigmentation and indole production. However, due to phenotypic variation, the use of a species-specific PCR test based on the 16S rRNA gene is included in the species description. This is justified by the high sequence similarity of the 16S rRNA gene within the species and the fact that the highest sequence...

  12. Biased distribution of DNA uptake sequences towards genome maintenance genes

    DEFF Research Database (Denmark)

    Davidsen, T.; Rodland, E.A.; Lagesen, K.

    2004-01-01

    Repeated sequence signatures are characteristic features of all genomic DNA. We have made a rigorous search for repeat genomic sequences in the human pathogens Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae and found that by far the most frequent 9-10mers residing within...... in these organisms. Pasteurella multocida also displayed high frequencies of a putative DUS identical to that previously identified in H. influenzae and with a skewed distribution towards genome maintenance genes, indicating that this bacterium might be transformation competent under certain conditions....

  13. GyrA and/or ParC alterations of Haemophilus influenzae strains isolated from the urethra of men with acute urethritis.

    Science.gov (United States)

    Kondo, Hiromi; Ito, Shin; Hatazaki, Kyoko; Horie, Kengo; Nakane, Keita; Mizutani, Kosuke; Tsuchiya, Tomohiro; Yasuda, Mitsuru; Yokoi, Shigeaki; Nakano, Masahiro; Deguchi, Takashi

    2018-03-01

    Of 73 clinical strains of Haemophilus influenzae isolated from the urethra of men with urogenital infections, we enrolled 6 strains (8.2%) with levofloxacin (LVFX) minimum inhibitory concentrations (MICs) of ≥0.03 μg/ml in this study. All the strains were isolated from non-gonococcal urethritis (NGU). We amplified the quinolone resistance-determining region of the gyrA gene and the analogous region of the parC gene from bacterial DNAs by PCR and sequenced the PCR products. Two strains with a LVFX MIC of 0.03 μg/ml had an amino acid change of Asp88 to Gly in GyrA. One with a LVFX MIC of 0.06 μg/ml had a change of Asp88 to Tyr in GyrA. Two with respective LVFX MICs of 0.12 and 0.25 μg/ml had a change of Ser84 to Leu in GyrA. One with a LVFX MIC of 1 μg/ml had changes of Ser84 to Leu in GyrA and of Ser84 to Ile in ParC. Multilocus sequence typing showed two strains with a change of Asp88 to Gly in GyrA had the same sequence type, but the others had sequence types different from each other. Single amino acid changes in GyrA alone or single changes in both GyrA and ParC could contribute to decreased susceptibility to fluoroquinolones in H. influenzae isolates from NGU. Most of the isolates with GyrA and/or ParC alterations would be multiclonal. The prevalence of such isolates would be relatively low, and they would still be susceptible to fluoroquinolones commonly prescribed for treatment of NGU. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  14. Diesel exhaust alters the response of cultured primary bronchial epithelial cells from patients with chronic obstructive pulmonary disease (COPD) to non-typeable Haemophilus influenzae.

    Science.gov (United States)

    Zarcone, Maria C; van Schadewijk, Annemarie; Duistermaat, Evert; Hiemstra, Pieter S; Kooter, Ingeborg M

    2017-01-28

    Exacerbations constitute a major cause of morbidity and mortality in patients suffering from chronic obstructive pulmonary disease (COPD). Both bacterial infections, such as those with non-typeable Haemophilus influenzae (NTHi), and exposures to diesel engine emissions are known to contribute to exacerbations in COPD patients. However, the effect of diesel exhaust (DE) exposure on the epithelial response to microbial stimulation is incompletely understood, and possible differences in the response to DE of epithelial cells from COPD patients and controls have not been studied. Primary bronchial epithelial cells (PBEC) were obtained from age-matched COPD patients (n = 7) and controls (n = 5). PBEC were cultured at the air-liquid interface (ALI) to achieve mucociliary differentiation. ALI-PBECs were apically exposed for 1 h to a stream of freshly generated whole DE or air. Exposure was followed by 3 h incubation in presence or absence of UV-inactivated NTHi before analysis of epithelial gene expression. DE alone induced an increase in markers of oxidative stress (HMOX1, 50-100-fold) and of the integrated stress response (CHOP, 1.5-2-fold and GADD34, 1.5-fold) in cells from both COPD patients and controls. Exposure of COPD cultures to DE followed by NTHi caused an additive increase in GADD34 expression (up to 3-fold). Importantly, DE caused an inhibition of the NTHi-induced expression of the antimicrobial peptide S100A7, and of the chaperone protein HSP5A/BiP. Our findings show that DE exposure of differentiated primary airway epithelial cells causes activation of the gene expression of HMOX1 and markers of integrated stress response to a similar extent in cells from COPD donors and controls. Furthermore, DE further increased the NTHi-induced expression of GADD34, indicating a possible enhancement of the integrated stress response. DE reduced the NTHi-induced expression of S100A7. These data suggest that DE exposure may cause adverse health effects in part by

  15. The structure of Haemophilus influenzae prephenate dehydrogenase suggests unique features of bifunctional TyrA enzymes

    International Nuclear Information System (INIS)

    Chiu, Hsiu-Ju; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Carlton, Dennis; Clayton, Thomas; Das, Debanu; Deller, Marc C.; Duan, Lian; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Reyes, Ron; Tien, Henry J.; Trame, Christine B.; Bedem, Henry van den; Weekes, Dana; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    The crystal structure of the prephenate dehydrogenase component of the bifunctional H. influenzae TyrA reveals unique structural differences between bifunctional and monofunctional TyrA enzymes. Chorismate mutase/prephenate dehydrogenase from Haemophilus influenzae Rd KW20 is a bifunctional enzyme that catalyzes the rearrangement of chorismate to prephenate and the NAD(P) + -dependent oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate in tyrosine biosynthesis. The crystal structure of the prephenate dehydrogenase component (HinfPDH) of the TyrA protein from H. influenzae Rd KW20 in complex with the inhibitor tyrosine and cofactor NAD + has been determined to 2.0 Å resolution. HinfPDH is a dimeric enzyme, with each monomer consisting of an N-terminal α/β dinucleotide-binding domain and a C-terminal α-helical dimerization domain. The structure reveals key active-site residues at the domain interface, including His200, Arg297 and Ser179 that are involved in catalysis and/or ligand binding and are highly conserved in TyrA proteins from all three kingdoms of life. Tyrosine is bound directly at the catalytic site, suggesting that it is a competitive inhibitor of HinfPDH. Comparisons with its structural homologues reveal important differences around the active site, including the absence of an α–β motif in HinfPDH that is present in other TyrA proteins, such as Synechocystis sp. arogenate dehydrogenase. Residues from this motif are involved in discrimination between NADP + and NAD + . The loop between β5 and β6 in the N-terminal domain is much shorter in HinfPDH and an extra helix is present at the C-terminus. Furthermore, HinfPDH adopts a more closed conformation compared with TyrA proteins that do not have tyrosine bound. This conformational change brings the substrate, cofactor and active-site residues into close proximity for catalysis. An ionic network consisting of Arg297 (a key residue for tyrosine binding), a water molecule, Asp206 (from

  16. Single-Dose Azithromycin for the Treatment of Haemophilus ducreyi Skin Ulcers in Papua New Guinea.

    Science.gov (United States)

    González-Beiras, Camila; Kapa, August; Vall-Mayans, Marti; Paru, Raymond; Gavilán, Sergi; Houinei, Wendy; Bieb, Sibauk; Sanz, Sergi; Martins, Rosario; Mitjà, Oriol

    2017-11-29

    Haemophilus ducreyi (HD) and Treponema pallidum subspecies pertenue (TP) are major causative agents of cutaneous ulcer (CU) in the tropics. Azithromycin is recommended to treat sexually transmitted HD infections and has good in vitro activity against HD strains from both genital and skin ulcers. We investigated the efficacy of oral single-dose azithromycin on HD-CU. We conducted a community-based cohort study in Lihir Island, Papua New Guinea, from October 2014 through May 2016. Consenting patients with skin ulcers >1 cm in diameter were eligible for this study and had collected a lesional swab for polymerase chain reaction (PCR). All participants were treated with single-dose azithromycin (30 mg/kg) and were followed up for assessment of clinical resolution. We retrospectively classified patients according to PCR results into HD, TP, and PCR-negative groups. The primary endpoint was healing rates of HD-CU at 14 days after treatment. We obtained full outcome data from 246 patients; 131 (53.3%) were HD PCR positive, 37 (15.0%) were TP positive, and 78 (31.7%) were negative for all tests. Healing rates were 88.5% (95% confidence interval [CI], .82-.93) in the HD group, 78.4% [95% CI, .63-.89] in the TP group, and 74.4% (95% CI, .64-.83) in the PCR-negative group. If we included the participants with improved ulcers, the healing rates increased to 94.7%, 97.3%, and 89.7% respectively. HD cases classified as not healed all converted to HD-negative PCR. Based upon clinical resolution and PCR conversion to HD negative, a single oral dose of azithromycin is efficacious for the treatment of HD-CU. These results have implications for the treatment of individual patients and for the use of antibiotics in public health strategies to control CU in the tropics. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  17. Expression of the Flp proteins by Haemophilus ducreyi is necessary for virulence in human volunteers

    Directory of Open Access Journals (Sweden)

    Zwickl Beth W

    2011-09-01

    Full Text Available Abstract Background Haemophilus ducreyi, the causative agent of the sexually transmitted disease chancroid, contains a flp (fimbria like protein operon that encodes proteins predicted to contribute to adherence and pathogenesis. H. ducreyi mutants that lack expression of Flp1 and Flp2 or TadA, which has homology to NTPases of type IV secretion systems, have decreased abilities to attach to and form microcolonies on human foreskin fibroblasts (HFF. A tadA mutant is attenuated in its ability to cause disease in human volunteers and in the temperature dependent rabbit model, but a flp1flp2 mutant is virulent in rabbits. Whether a flp deletion mutant would cause disease in humans is not clear. Results We constructed 35000HPΔflp1-3, a deletion mutant that lacks expression of all three Flp proteins but has an intact tad secretion system. 35000HPΔflp1-3 was impaired in its ability to form microcolonies and to attach to HFF in vitro when compared to its parent (35000HP. Complementation of the mutant with flp1-3 in trans restored the parental phenotype. To test whether expression of Flp1-3 was necessary for virulence in humans, ten healthy adult volunteers were experimentally infected with a fixed dose of 35000HP (ranging from 54 to 67 CFU on one arm and three doses of 35000HPΔflp1-3 (ranging from 63 to 961 CFU on the other arm. The overall papule formation rate for the parent was 80% (95% confidence interval, CI, 55.2%-99.9% and for the mutant was 70.0% (95% CI, 50.5%-89.5% (P = 0.52. Mutant papules were significantly smaller (mean, 11.2 mm2 than were parent papules (21.8 mm2 24 h after inoculation (P = 0.018. The overall pustule formation rates were 46.7% (95% CI 23.7-69.7% at 30 parent sites and 6.7% (95% CI, 0.1-19.1% at 30 mutant sites (P = 0.001. Conclusion These data suggest that production and secretion of the Flp proteins contributes to microcolony formation and attachment to HFF cells in vitro. Expression of flp1-3 is also necessary for H

  18. Expression of the Flp proteins by Haemophilus ducreyi is necessary for virulence in human volunteers.

    Science.gov (United States)

    Janowicz, Diane M; Cooney, Sean A; Walsh, Jessica; Baker, Beth; Katz, Barry P; Fortney, Kate R; Zwickl, Beth W; Ellinger, Sheila; Munson, Robert S

    2011-09-22

    Haemophilus ducreyi, the causative agent of the sexually transmitted disease chancroid, contains a flp (fimbria like protein) operon that encodes proteins predicted to contribute to adherence and pathogenesis. H. ducreyi mutants that lack expression of Flp1 and Flp2 or TadA, which has homology to NTPases of type IV secretion systems, have decreased abilities to attach to and form microcolonies on human foreskin fibroblasts (HFF). A tadA mutant is attenuated in its ability to cause disease in human volunteers and in the temperature dependent rabbit model, but a flp1flp2 mutant is virulent in rabbits. Whether a flp deletion mutant would cause disease in humans is not clear. We constructed 35000HPΔflp1-3, a deletion mutant that lacks expression of all three Flp proteins but has an intact tad secretion system. 35000HPΔflp1-3 was impaired in its ability to form microcolonies and to attach to HFF in vitro when compared to its parent (35000HP). Complementation of the mutant with flp1-3 in trans restored the parental phenotype. To test whether expression of Flp1-3 was necessary for virulence in humans, ten healthy adult volunteers were experimentally infected with a fixed dose of 35000HP (ranging from 54 to 67 CFU) on one arm and three doses of 35000HPΔflp1-3 (ranging from 63 to 961 CFU) on the other arm. The overall papule formation rate for the parent was 80% (95% confidence interval, CI, 55.2%-99.9%) and for the mutant was 70.0% (95% CI, 50.5%-89.5%) (P = 0.52). Mutant papules were significantly smaller (mean, 11.2 mm2) than were parent papules (21.8 mm2) 24 h after inoculation (P = 0.018). The overall pustule formation rates were 46.7% (95% CI 23.7-69.7%) at 30 parent sites and 6.7% (95% CI, 0.1-19.1%) at 30 mutant sites (P = 0.001). These data suggest that production and secretion of the Flp proteins contributes to microcolony formation and attachment to HFF cells in vitro. Expression of flp1-3 is also necessary for H. ducreyi to initiate disease and progress to

  19. Ampicillin resistance in Haemophilus influenzae from COPD patients in the UK

    Directory of Open Access Journals (Sweden)

    Maddi S

    2017-05-01

    Full Text Available Satyanarayana Maddi,1 Umme Kolsum,1 Sarah Jackson,1 Richard Barraclough,2 Barbara Maschera,3 Karen D Simpson,3 Thierry G Pascal,4 Serge Durviaux,4 Edith M Hessel,3 Dave Singh1 1Division of Infection, Immunity and Respiratory Medicine, Medicines Evaluation Unit, University Hospital of South Manchester Foundation Trust, University of Manchester, 2Department of Respiratory Medicine, University Hospital of South Manchester Foundation Trust, Manchester, 3Refractory Respiratory Inflammation DPU, GlaxoSmithKline Medicines Research Centre, Stevenage, Hertfordshire, UK; 4Clinical Laboratory Sciences, GlaxoSmithKline Vaccines, Wavre, Belgium Background: Haemophilus influenzae is commonly isolated from the airways of COPD patients. Antibiotic treatment may cause the emergence of resistant H. influenzae strains, particularly ampicillin-resistant strains, including β-lactamase-negative ampicillin resistance (BLNAR strains. Genetic identification using ftsI sequencing is the optimum method for identifying mutations within BLNAR strains. The prevalence of BLNAR in COPD patients during the stable state has not been reported. We investigated the antibiotic resistance patterns of H. influenzae present in the sputum of stable COPD patients, focusing on ampicillin resistance; the prevalence of enzyme and non-enzyme-mediated ampicillin resistance was determined. A subset of patients was followed up longitudinally to study H. influenzae strain switching and antibiotic sensitivity changes.Patients and methods: Sputum sampling was performed in 61 COPD patients, with 42 samples obtained at baseline; H. influenzae was detected by polymerase chain reaction in 28 samples. In all, 45 patients completed the follow-up for 2 years; 24 H. influenzae isolates were obtained.Results: Disk diffusion showed the highest antibiotic resistance in the penicillin antibiotic group (eg, 67% for ampicillin and macrolides (eg, 46% for erythromycin, whereas all isolates were susceptible to

  20. The Drosophila Translational Control Element (TCE) is required for high-level transcription of many genes that are specifically expressed in testes.

    Science.gov (United States)

    Katzenberger, Rebeccah J; Rach, Elizabeth A; Anderson, Ashley K; Ohler, Uwe; Wassarman, David A

    2012-01-01

    To investigate the importance of core promoter elements for tissue-specific transcription of RNA polymerase II genes, we examined testis-specific transcription in Drosophila melanogaster. Bioinformatic analyses of core promoter sequences from 190 genes that are specifically expressed in testes identified a 10 bp A/T-rich motif that is identical to the translational control element (TCE). The TCE functions in the 5' untranslated region of Mst(3)CGP mRNAs to repress translation, and it also functions in a heterologous gene to regulate transcription. We found that among genes with focused initiation patterns, the TCE is significantly enriched in core promoters of genes that are specifically expressed in testes but not in core promoters of genes that are specifically expressed in other tissues. The TCE is variably located in core promoters and is conserved in melanogaster subgroup species, but conservation dramatically drops in more distant species. In transgenic flies, short (300-400 bp) genomic regions containing a TCE directed testis-specific transcription of a reporter gene. Mutation of the TCE significantly reduced but did not abolish reporter gene transcription indicating that the TCE is important but not essential for transcription activation. Finally, mutation of testis-specific TFIID (tTFIID) subunits significantly reduced the transcription of a subset of endogenous TCE-containing but not TCE-lacking genes, suggesting that tTFIID activity is limited to TCE-containing genes but that tTFIID is not an obligatory regulator of TCE-containing genes. Thus, the TCE is a core promoter element in a subset of genes that are specifically expressed in testes. Furthermore, the TCE regulates transcription in the context of short genomic regions, from variable locations in the core promoter, and both dependently and independently of tTFIID. These findings set the stage for determining the mechanism by which the TCE regulates testis-specific transcription and understanding the

  1. The Haemophilus ducreyi LspA1 protein inhibits phagocytosis by using a new mechanism involving activation of C-terminal Src kinase.

    Science.gov (United States)

    Dodd, Dana A; Worth, Randall G; Rosen, Michael K; Grinstein, Sergio; van Oers, Nicolai S C; Hansen, Eric J

    2014-05-20

    Haemophilus ducreyi causes chancroid, a sexually transmitted infection. A primary means by which this pathogen causes disease involves eluding phagocytosis; however, the molecular basis for this escape mechanism has been poorly understood. Here, we report that the LspA virulence factors of H. ducreyi inhibit phagocytosis by stimulating the catalytic activity of C-terminal Src kinase (Csk), which itself inhibits Src family protein tyrosine kinases (SFKs) that promote phagocytosis. Inhibitory activity could be localized to a 37-kDa domain (designated YL2) of the 456-kDa LspA1 protein. The YL2 domain impaired ingestion of IgG-opsonized targets and decreased levels of active SFKs when expressed in mammalian cells. YL2 contains tyrosine residues in two EPIYG motifs that are phosphorylated in mammalian cells. These tyrosine residues were essential for YL2-based inhibition of phagocytosis. Csk was identified as the predominant mammalian protein interacting with YL2, and a dominant-negative Csk rescued phagocytosis in the presence of YL2. Purified Csk phosphorylated the tyrosines in the YL2 EPIYG motifs. Phosphorylated YL2 increased Csk catalytic activity, resulting in positive feedback, such that YL2 can be phosphorylated by the same kinase that it activates. Finally, we found that the Helicobacter pylori CagA protein also inhibited phagocytosis in a Csk-dependent manner, raising the possibility that this may be a general mechanism among diverse bacteria. Harnessing Csk to subvert the Fcγ receptor (FcγR)-mediated phagocytic pathway represents a new bacterial mechanism for circumventing a crucial component of the innate immune response and may potentially affect other SFK-involved cellular pathways. Phagocytosis is a critical component of the immune system that enables pathogens to be contained and cleared. A number of bacterial pathogens have developed specific strategies to either physically evade phagocytosis or block the intracellular signaling required for

  2. A Zinc-Finger-Family Transcription Factor, AbVf19, Is Required for the Induction of a Gene Subset Important for Virulence in Alternaria brassicicola

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, Akhil [Univ. of Hawaii, Manoa, HI (United States); Ohm, Robin A. [USDOE Joint Genome Inst., Walnut Creek, CA (United States); Oxiles, Lindsay [Univ. of Hawaii, Manoa, HI (United States); Brooks, Fred [Univ. of Hawaii, Manoa, HI (United States); Lawrence, Christopher B. [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Grigoriev, Igor V. [USDOE Joint Genome Inst., Walnut Creek, CA (United States); Cho, Yangrae [Univ. of Hawaii, Manoa, HI (United States)

    2011-10-26

    Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen with a broad host range within the family Brassicaceae. It produces secondary metabolites that marginally affect virulence. Cell wall degrading enzymes (CDWE) have been considered important for pathogenesis but none of them individually have been identified as significant virulence factors in A. brassicicola. In this study, knockout mutants of a gene, AbVf19, were created and produced considerably smaller lesions than the wild type on inoculated host plants. The presence of tandem zinc-finger domains in the predicted amino acid sequence and nuclear localization of AbVf19- reporter protein suggested that it was a transcription factor. Gene expression comparisons using RNA-seq identified 74 genes being downregulated in the mutant during a late stage of infection. Among the 74 downregulated genes, 28 were putative CWDE genes. These were hydrolytic enzyme genes that composed a small fraction of genes within each family of cellulases, pectinases, cutinases, and proteinases. The mutants grew slower than the wild type on an axenic medium with pectin as a major carbon source. This study demonstrated the existence and the importance of a transcription factor that regulates a suite of genes that are important for decomposing and utilizing plant material during the late stage of plant infection.

  3. Impact of the Haemophilus influenzae type b vaccination program on HIB meningitis in Brazil Impacto do programa de vacinação contra meningites causadas por Haemophilus influenzae tipo b no Brasil

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    Sybelle de Souza Castro Miranzi

    2007-07-01

    Full Text Available This study aimed to evaluate the impact of vaccination against Haemophilus influenzae type b (HIB in Brazil on the morbidity, mortality, and case fatality of HIB meningitis, using the Ministry of Health database and population data from the Brazilian Institute of Geography and Statistics (Instituto Brasileiro de Geografia e Estatística - IBGE. Impact was evaluated through a time series analysis (1983-2002, using regression forecasting (RF by dividing the time series into two periods: (a historical (1983-1998 and (b validation (1999-2002. Impact of the vaccination was positive, although more significant for incidence and mortality than for case fatality rates.A proposta deste trabalho foi avaliar o impacto da vacinação contra Haemophilus influenzae tipo b (HIB no Brasil sobre a morbi-mortalidade e a letalidade das meningites por HIB, a partir de base de dados fornecida pelo Ministério da Saúde e as estimativas populacionais provenientes do Instituto Brasileiro de Geografia e Estatística (IBGE. Para a avaliação do impacto utilizou-se análise de tendência temporal (1983-2002, aplicando-se a técnica RF (regression forecasting, dividindo-se a série em dois períodos: (a período histórico (1983-1998 e (b período de estimação (1999-2002. O impacto da vacinação foi positivo, embora tenha se revelado mais expressivo sobre a morbi-mortalidade que sobre a letalidade.

  4. Haemophilus influenzae tipo b: situação epidemiológica no Estado de Minas Gerais, Brasil, 1993 a 1997 Haemophilus influenzae type b: epidemiological situation in the State of Minas Gerais, Brazil, 1993-1997

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    Sybelle de Souza Castro Miranzi

    2003-10-01

    Full Text Available Entre as doenças invasivas causadas pelo Haemophilus influenzae tipo b (Hib, destacam-se, pela freqüência e gravidade, as pneumonias e as meningites. No período de 1993 a 1997, foram notificados, em Minas Gerais, 720 casos de meningites por Hib, sendo a causa mais freqüente de meningite bacteriana em menores de um ano e a segunda causa no total de meningites. Entretanto, estimou-se uma ocorrência total de 1.160 casos considerando as meningites bacterianas não especificadas. O total de casos estimados de doença invasiva por Hib parece justificar a recente inclusão da vacina no esquema básico de imunizações. O alto custo da vacina reforça a necessidade de melhorar a vigilância epidemiológica da meningite, que constitui uma das fragilidades das ações de controle desta doença.Among Haemophilus influenzae type b (Hib invasive diseases, pneumonia and meningitis are the most relevant in public health due to their frequency and severity. From 1993 to 1997, there were 720 cases of Hib meningitis in Minas Gerais State, Brazil, representing the most frequent cause of bacterial meningitis in infants (< 1 year and the second most frequent among all causes of meningitis. The total estimated cases of invasive Hib diseases thus appear to justify the recent inclusion of the vaccine in the basic immunization protocol. The vaccine's high cost reinforces the need for more precise monitoring of the etiological diagnosis of meningitis cases, representing one of the weaknesses in the prevailing epidemiological surveillance system.

  5. A Pectate Lyase-Coding Gene Abundantly Expressed during Early Stages of Infection Is Required for Full Virulence in Alternaria brassicicola.

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    Yangrae Cho

    Full Text Available Alternaria brassicicola causes black spot disease of Brassica species. The functional importance of pectin digestion enzymes and unidentified phytotoxins in fungal pathogenesis has been suspected but not verified in A. brassicicola. The fungal transcription factor AbPf2 is essential for pathogenicity and induces 106 genes during early pathogenesis, including the pectate lyase-coding gene, PL1332. The aim of this study was to test the importance and roles of PL1332 in pathogenesis. We generated deletion strains of the PL1332 gene, produced heterologous PL1332 proteins, and evaluated their association with virulence. Deletion strains of the PL1332 gene were approximately 30% less virulent than wild-type A. brassicicola, without showing differences in colony expansion on solid media and mycelial growth in nutrient-rich liquid media or minimal media with pectins as a major carbon source. Heterologous PL1332 expressed as fusion proteins digested polygalacturons in vitro. When the fusion proteins were injected into the apoplast between leaf veins of host plants the tissues turned dark brown and soft, resembling necrotic leaf tissue. The PL1332 gene was the first example identified as a general toxin-coding gene and virulence factor among the 106 genes regulated by the transcription factor, AbPf2. It was also the first gene to have its functions investigated among the 19 pectate lyase genes and several hundred putative cell-wall degrading enzymes in A. brassicicola. These results further support the importance of the AbPf2 gene as a key pathogenesis regulator and possible target for agrochemical development.

  6. Limited agreement of independent RNAi screens for virus-required host genes owes more to false-negative than false-positive factors.

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    Linhui Hao

    Full Text Available Systematic, genome-wide RNA interference (RNAi analysis is a powerful approach to identify gene functions that support or modulate selected biological processes. An emerging challenge shared with some other genome-wide approaches is that independent RNAi studies often show limited agreement in their lists of implicated genes. To better understand this, we analyzed four genome-wide RNAi studies that identified host genes involved in influenza virus replication. These studies collectively identified and validated the roles of 614 cell genes, but pair-wise overlap among the four gene lists was only 3% to 15% (average 6.7%. However, a number of functional categories were overrepresented in multiple studies. The pair-wise overlap of these enriched-category lists was high, ∼19%, implying more agreement among studies than apparent at the gene level. Probing this further, we found that the gene lists implicated by independent studies were highly connected in interacting networks by independent functional measures such as protein-protein interactions, at rates significantly higher than predicted by chance. We also developed a general, model-based approach to gauge the effects of false-positive and false-negative factors and to estimate, from a limited number of studies, the total number of genes involved in a process. For influenza virus replication, this novel statistical approach estimates the total number of cell genes involved to be ∼2,800. This and multiple other aspects of our experimental and computational results imply that, when following good quality control practices, the low overlap between studies is primarily due to false negatives rather than false-positive gene identifications. These results and methods have implications for and applications to multiple forms of genome-wide analysis.

  7. Non-Type b Haemophilus influenzae Invasive Infections in North Dakota and South Dakota, 2013-2015.

    Science.gov (United States)

    Antony, Stephanie; Kaushik, Ashlesha; Mauriello, Clifford; Chatterjee, Archana

    2017-09-01

    Reports of children with non-type b Haemophilus influenzae infection in the United States in recent years have been limited. Here, we report the spectrum and severity of disease associated with invasive non-type b H influenzae infection in 17 patients at 2 tertiary-care children's hospitals over a 2-year period. Meningitis was the most common diagnosis. The majority of the patients had neurologic sequelae, and 1 patient died. The high proportions of hospitalization, intensive care utilization, and neurologic complications reveal that non-type b H influenzae infection was associated with significant morbidity in this pediatric population. © The Author 2016. Published by Oxford University Press on behalf of the Pediatric Infectious Diseases Society. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. The determination of minimum inhibitory concentrations of selected antimicrobials for porcine Haemophilus parasuis isolates from the Czech Republic

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    Kateřina Nedbalcová

    2017-01-01

    Full Text Available Haemophilus parasuis isolates obtained from pigs in the Czech Republic were tested for their susceptibility to amoxicillin, penicillin, ceftiofur, enrofloxacin, tetracycline, and tulathromycin by determination of minimum inhibitory concentrations using the broth microdilution method. The H. parasuis isolates were mostly susceptible to majority of tested antimicrobials (amoxicillin 90%, penicillin 73.3%, enrofloxacin 83.3%, and tulathromycin 83.3%. All isolates were susceptible to ceftiofur. On the other hand, no isolate was susceptible to tetracycline, 30% of tested isolates were intermediately susceptible, and 70% were resistant. These findings indicate that tested antimicrobials with the exception of tetracycline should be the preferred option used for the treatment of infection caused by H. parasuis but due to the potential transmission of resistance from animals to humans, the use of ceftiofur is considered as a last resort option in antimicrobial treatment of animals.

  9. Chancroid in Sheffield. A report of 22 cases diagnosed by isolating Haemophilus ducreyi in a modified medium.

    Science.gov (United States)

    Hafiz, S; Kinghorn, G R; McEntegart, M G

    1981-01-01

    The causative organism of chancroid, Haemophilus ducreyi, is generally considered to be very fastidious and its isolation, maintenance, and detailed study very demanding. In this study a modified medium was developed, which allowed the organism to be isolated more frequently than previously would have been expected. Twenty-two cases of chancroid were confirmed by the isolation of H ducreyi in 160 patients with genital ulceration examined over a one-year period. The cases were apparently unrelated, and in only five was there a history of recent sexual contact abroad. Concurrent infection with other sexually transmitted diseases was present in 18 (81.8%) patients, and in 14 (63.6%) both H ducreyi and herpes simplex virus were isolated form the same genital ulcers. Thus, these findings indicate that chancroid is underdiagnosed in England and that H ducreyi may frequently occur as a secondary invader of damaged genital skin and mucosa. PMID:6976815

  10. In vitro stimulation of peripheral blood mononuclear cells (PBMC) from HIV- and HIV+ chancroid patients by Haemophilus ducreyi antigens.

    Science.gov (United States)

    Van Laer, L; Vingerhoets, J; Vanham, G; Kestens, L; Bwayo, J; Otido, J; Piot, P; Roggen, E

    1995-11-01

    The cellular immune responses to fractionated Haemophilus ducreyi antigens, coated on latex beads, were assessed in patients with chancroid and in controls, using an in vitro lymphocyte proliferation assay. Several fractions of H. ducreyi antigen revealed stimulating activity. However, only the molecular size ranges 91-78 kD, 59-29 kD, and 25-21 kD induced proliferation that may be specifically related to H. ducreyi infection. Lymphocytes from four HIV- patients, successfully treated for chancroid, were not stimulated by H. ducreyi antigen. In general, lymphocytes from HIV+ chancroid patients were less responsive to H. ducreyi antigen compared with those from HIV- chancroid patients. However, two HIV-infected patients showed exceptionally strong responses to high molecular weight fractions. To our knowledge this is the first report demonstrating that H. ducreyi contains specific T cell-stimulating antigens. Based on this work, further identification and purification of the T cell antigens is feasible.

  11. Recurrent meningitis due to pneumococci and non-typable Haemophilus influenzae in a child with a Mondini malformation.

    Science.gov (United States)

    Valmari, P; Palva, A

    1986-01-01

    An eight-year-old boy with a congenital inner ear malformation and recurrent otitis media had three episodes of bacteriologically confirmed meningitis within seven months. The first episode was caused by pneumococci, the other two by non-typable Haemophilus influenzae. All episodes were characterized by an abrupt onset. The CSF cultures were positive within 0.5 to 12 hours after the onset of symptoms. Despite misleading laboratory studies, surgical exploration revealed a CSF fistula associated to the inner ear anomaly. No further episodes occurred after the fistula was closed. Careful roentgenographic evaluation, including recently introduced special computed tomography (CT) methods, is indicated in recurrent meningitis. In addition, such evaluations should be considered even after the first episode, when special clinical features suggest a CSF fistula. Such features include an extremely rapid onset and detection of common non-invasive bacteria as causative agents, as illustrated by the present case.

  12. Erythromycin and azithromycin transport into Haemophilus influenzae ATCC 19418 under conditions of depressed proton motive force (delta mu H)

    Energy Technology Data Exchange (ETDEWEB)

    Capobianco, J.O.; Goldman, R.C. (Abbott Laboratories, IL (USA))

    1990-09-01

    The effect of collapsing the electrochemical proton gradient (delta mu H) on ({sup 3}H)erythromycin and ({sup 14}C)azithromycin transport in Haemophilus influenzae ATCC 19418 was studied. The proton gradient and membrane potential were determined from the distribution of (2-{sup 14}C)dimethadione and rubidium-86, respectively. delta mu H was reduced from 124 to 3 mV in EDTA-valinomycin-treated cells at 22{degrees}C with 150 mM KCl and 0.1 mM carbonyl cyanide m-chlorophenylhydrazone. During the collapse of delta mu H, macrolide uptake increased. Erythromycin efflux studies strongly suggested that this increase was not due to an energy-dependent efflux pump but was likely due to increased outer membrane permeability. These data indicated that macrolide entry was not a delta mu H-driven active transport process but rather a passive diffusion process.

  13. Molecular study on the carAB operon reveals that carB gene is required for swimming and biofilm formation in Xanthomonas citri subsp. citri.

    Science.gov (United States)

    Zhuo, Tao; Rou, Wei; Song, Xue; Guo, Jing; Fan, Xiaojing; Kamau, Gicharu Gibson; Zou, Huasong

    2015-10-23

    The carA and carB genes code the small and large subunits of carbamoyl-phosphate synthase (CPS) that responsible for arginine and pyrimidine production. The purpose of this work was to study the gene organization and expression pattern of carAB operon, and the biological functions of carA and carB genes in Xanthomonas citri subsp. citri. RT-PCR method was employed to identify the full length of carAB operon transcript in X. citri subsp. citri. The promoter of carAB operon was predicted and analyzed its activity by fusing a GUS reporter gene. The swimming motility was tested on 0.25% agar NY plates with 1% glucose. Biofilm was measured by cell adhesion to polyvinyl chloride 96-well plate. The results indicated that carAB operon was composed of five gene members carA-orf-carB-greA-rpfE. A single promoter was predicted from the nucleotide sequence upstream of carAB operon, and its sensitivity to glutamic acid, uracil and arginine was confirmed by fusing a GUS reporter gene. Deletion mutagenesis of carB gene resulted in reduced abilities in swimming on soft solid media and in forming biofilm on polystyrene microtiter plates. From these results, we concluded that carAB operon was involved in multiple biological processes in X. citri subsp. citri.

  14. Characterization of RAD4 gene required for ultraviolet-induced excision repair of Saccharomyces cerevisiae propagated in Escherichia coli without inactivation

    International Nuclear Information System (INIS)

    Choi, I.S.; Kim, J.B.; Lee, K.N.; Park, S.D.

    1990-01-01

    The previously isolated RAD4 gene designated as pPC1 from the genomic library of Saccharomyces cerevisiae appeared to propagate in Escherichia coli and yet retained its complementing activity of rad4 mutants without inactivation. The subcloned RAD4 gene was found to be localized within a 2.5 kb DNA fragment flanking Bg/II and BamHI sites in the insert DNA, and was shown to have the same restriction map as a yeast chromosomal DNA, as determined by Southern hybridization. Tetrad analysis and pulse-field chromosome mapping have revealed that the cloned RAD4 gene can be mapped and integrated into the yeast chromosome V, the actual site of this gene. DNA-tRNA hybridization has shown that the isolated RAD4 gene did not contain a suppressor tRNA gene. These results have indicated that the pPC1 is a functional RAD4 gene playing a unique role involved in the nucleotide excision repair of yeast without any genetic change during amplification in E. coli. (author)

  15. Comparative transcript profiling of Candida albicans and Candida dubliniensis identifies SFL2, a C. albicans gene required for virulence in a reconstituted epithelial infection model.

    LENUS (Irish Health Repository)

    Spiering, Martin J

    2010-02-01

    Candida albicans and Candida dubliniensis are closely related species displaying differences in virulence and genome content, therefore providing potential opportunities to identify novel C. albicans virulence genes. C. albicans gene arrays were used for comparative analysis of global gene expression in the two species in reconstituted human oral epithelium (RHE). C. albicans (SC5314) showed upregulation of hypha-specific and virulence genes within 30 min postinoculation, coinciding with rapid induction of filamentation and increased RHE damage. C. dubliniensis (CD36) showed no detectable upregulation of hypha-specific genes, grew as yeast, and caused limited RHE damage. Several genes absent or highly divergent in C. dubliniensis were upregulated in C. albicans. One such gene, SFL2 (orf19.3969), encoding a putative heat shock factor, was deleted in C. albicans. DeltaDeltasfl2 cells failed to filament under a range of hypha-inducing conditions and exhibited greatly reduced RHE damage, reversed by reintroduction of SFL2 into the DeltaDeltasfl2 strain. Moreover, SFL2 overexpression in C. albicans triggered hyphal morphogenesis. Although SFL2 deletion had no apparent effect on host survival in the murine model of systemic infection, DeltaDeltasfl2 strain-infected kidney tissues contained only yeast cells. These results suggest a role for SFL2 in morphogenesis and an indirect role in C. albicans pathogenesis in epithelial tissues.

  16. Genes and Gene Therapy

    Science.gov (United States)

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  17. The Drosophila Translational Control Element (TCE is required for high-level transcription of many genes that are specifically expressed in testes.

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    Rebeccah J Katzenberger

    Full Text Available To investigate the importance of core promoter elements for tissue-specific transcription of RNA polymerase II genes, we examined testis-specific transcription in Drosophila melanogaster. Bioinformatic analyses of core promoter sequences from 190 genes that are specifically expressed in testes identified a 10 bp A/T-rich motif that is identical to the translational control element (TCE. The TCE functions in the 5' untranslated region of Mst(3CGP mRNAs to repress translation, and it also functions in a heterologous gene to regulate transcription. We found that among genes with focused initiation patterns, the TCE is significantly enriched in core promoters of genes that are specifically expressed in testes but not in core promoters of genes that are specifically expressed in other tissues. The TCE is variably located in core promoters and is conserved in melanogaster subgroup species, but conservation dramatically drops in more distant species. In transgenic flies, short (300-400 bp genomic regions containing a TCE directed testis-specific transcription of a reporter gene. Mutation of the TCE significantly reduced but did not abolish reporter gene transcription indicating that the TCE is important but not essential for transcription activation. Finally, mutation of testis-specific TFIID (tTFIID subunits significantly reduced the transcription of a subset of endogenous TCE-containing but not TCE-lacking genes, suggesting that tTFIID activity is limited to TCE-containing genes but that tTFIID is not an obligatory regulator of TCE-containing genes. Thus, the TCE is a core promoter element in a subset of genes that are specifically expressed in testes. Furthermore, the TCE regulates transcription in the context of short genomic regions, from variable locations in the core promoter, and both dependently and independently of tTFIID. These findings set the stage for determining the mechanism by which the TCE regulates testis-specific transcription and

  18. Susceptibilities of Haemophilus influenzae and Moraxella catarrhalis to ABT-773 compared to their susceptibilities to 11 other agents.

    Science.gov (United States)

    Credito, K L; Lin, G; Pankuch, G A; Bajaksouzian, S; Jacobs, M R; Appelbaum, P C

    2001-01-01

    The activity of the ketolide ABT-773 against Haemophilus and Moraxella was compared to those of 11 other agents. Against 210 Haemophilus influenzae strains (39.0% beta-lactamase positive), microbroth dilution tests showed that azithromycin and ABT-773 had the lowest MICs (0.5 to 4.0 and 1.0 to 8.0 microg/ml, respectively), followed by clarithromycin and roxithromycin (4.0 to >32.0 microg/ml). Of the beta-lactams, ceftriaxone had the lowest MICs (32.0 microg/ml). Against 50 Moraxella catarrhalis strains, all of the compounds except amoxicillin and cefprozil were active. Time-kill studies against 10 H. influenzae strains showed that ABT-773, at two times the MIC, was bactericidal against 9 of 10 strains, with 99% killing of all strains at the MIC after 24 h; at 12 h, ABT-773 gave 90% killing of all strains at two times the MIC. At 3 and 6 h, killing by ABT-773 was slower, with 99.9% killing of four strains at two times the MIC after 6 h. Similar results were found for azithromycin, with slightly slower killing by erythromycin, clarithromycin, and roxithromycin, especially at earlier times. beta-Lactams were bactericidal against 8 to 10 strains at two times the MIC after 24 h, with slower killing at earlier time periods. Most compounds gave good killing of five M. catarrhalis strains, with beta-lactams killing more rapidly than other drugs. ABT-773 and azithromycin gave the longest postantibiotic effects (PAEs) of the ketolide-macrolide-azalide group tested (4.4 to >8.0 h), followed by clarithromycin, erythromycin, and roxithromycin. beta-Lactam PAEs were similar and shorter than those of the ketolide-macrolide-azalide group for all strains tested.

  19. Impacto de la vacuna conjugada en la incidencia de meningitis por Haemophilus influenzae en el Distrito Federal de Brasil: resultados de tres años de seguimiento Impact of anti-Hib conjugate vaccine on the incidence of Haemophilus influenzae meningitis in Brazil's Federal District: results of a three-year follow-up

    Directory of Open Access Journals (Sweden)

    Helen Selma de Abreu Freitas

    2006-01-01

    Full Text Available INTRODUCCIÓN: Haemophilus influenzae del serotipo b (Hib es todavía un importante agente causal de procesos infecciosos. Su variante encapsulada es la causa de formas invasoras de enfermedad. En algunas poblaciones aborígenes, la incidencia de enfermedades causadas por Hib es mayor de 400 por 100 000 niños menores de 5 años. En los decenios de 1970 y 1980, tras la identificación de anticuerpos protectores contra la cápsula de Hib, se desarrollaron vacunas contra este microorganismo. OBJETIVO: Estimar el impacto que ha tenido desde su introducción, en marzo de 1998, la vacunación contra Hib en el Distrito Federal de Brasil. MÉTODO: Con los datos de base poblacional del Sistema de Vigilancia de la Secretaría de Salud del Distrito Federal de Brasil, se compararon las tasas de incidencia de meningitis correspondientes a los tres años anteriores y posteriores a la introducción de la vacuna. Se compararon también los cambios en su tendencia. RESULTADOS: Al comparar los dos períodos se advierte una reducción de aproximadamente 90% en la incidencia de meningitis por Hib, cambio que no ocurre en el caso de las meningitis ocasionadas por otros agentes bacterianos. Se registró un aumento proporcional de los casos entre los niños de 6 meses de edad y menores, debido a la reducción de la incidencia entre los mayores de esa edad. CONCLUSIÓN: La introducción de la vacuna conjugada en el Distrito Federal de Brasil redujo de 168 por 100 000 (1995-1997 a 15 por 100 000 (1999-2001 la incidencia de meningitis por Hib entre niños de 7 a 35 meses. Esto representa una reducción de 91,1%.INTRODUCTION: Type b Haemophilus influenzae (Hib continues to be an important causative agent of various infectious processes, and its encapsulated strains cause invasive disease. In some aboriginal populations, the incidence of Hib infections in children under five is greater than 400 per 100 000. In the seventies and eighties, vaccines against Hib were

  20. Antimicrobial activity of innate immune molecules against Streptococcus pneumoniae, Moraxella catarrhalis and nontypeable Haemophilus influenzae

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    Teufert Karen

    2004-05-01

    Full Text Available Abstract Background Despite its direct connection to the nasopharynx which harbors otitis media pathogens as part of its normal flora, the middle ear cavity is kept free of these bacteria by as yet unknown mechanisms. Respiratory mucosal epithelia, including those of the middle ear and eustachian tube, secrete antimicrobial effectors including lysozyme, lactoferrin and β defensins-1 and -2. To elucidate the role of these innate immune molecules in the normal defense and maintenance of sterility of respiratory mucosa such as that of the middle ear, we assessed their effect on the respiratory pathogens nontypeable Haemophilus influenzae (NTHi 12, Moraxella catarrhalis 035E, and Streptococcus pneumoniae 3, and 6B. Methods Two assay methods, the radial assay and the liquid broth assay, were employed for testing the antimicrobial activity of the molecules. This was done in order to minimize the possibility that the observed effects were artifacts of any single assay system employed. Also, transmission electron microscopy (TEM was employed to evaluate the effect of antimicrobial innate immune molecules on OM pathogens. For the statistical analysis of the data, Student's t-test was performed. Results Results of the radial diffusion assay showed that β defensin-2 was active against all four OM pathogens tested, while treatment with β defensin-1 appeared to only affect M. catarrhalis. The radial assay results also showed that lysozyme was quite effective against S. pneumoniae 3 and 6B and was partially bacteriostatic/bactericidal against M. catarrhalis. Lysozyme however, appeared not to affect the growth of NTHi. Thus, lysozyme seems to have a more pronounced impact on the growth of the Gram-positive S. pneumoniae as compared to that of Gram-negative pathogens. Lactoferrin on the other hand, enhanced the growth of the bacteria tested. The results of the radial assays were confirmed using liquid broth assays for antimicrobial activity, and showed that

  1. Vigilancia de los serotipos y susceptibilidad antimicrobiana de Haemophilus influenzae en Colombia, 1994-2002.

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    María Victoria Ovalle

    2003-06-01

    Full Text Available La enfermedad invasora causada por Haemophilus influenzae, serotipo b, ha sido una de las mayores causas de morbilidad y mortalidad en la población infantil; afortunadamente, en algunos países con amplia cobertura de la vacuna conjugada esta situación ha cambiado. En 1994 se inició en el Grupo de Microbiología un programa de vigilancia de la susceptibilidad antimicrobiana y de los serotipos de aislamientos invasores de H. influenzae, remitidos por los hospitales y Laboratorios de Salud Pública del país como componente de los programas de vigilancia en red de infección respiratoria aguda y meningitis bacteriana aguda. El objetivo de este trabajo fue determinar la evolución de los serotipos y los patrones de susceptibilidad antimicrobiana de los aislamientos invasores de H. influenzae obtenidos de 1994 al 2002 y realizar un nuevo análisis sobre el impacto de la vacuna conjugada de H. influenzae, serotipo b, en Colombia. De 1994 a 2002 se han estudiado 683 aislamientos; 379 (55,5% de pacientes del género masculino; 370 (54,2% de menores de 1 año; 227 (33,2% de 1 a 5 años; 19 (2,8% de 6 a 14 años; 38 (5,6% de mayores de 14 años, y de 29 (4,2% no se tenía el dato de la edad; 493 (72,2% fueron recuperados de pacientes con meningitis, 181 (26,5% de neumonía y 9 (0,9% de otras enfermedades. El 85,1% de los aislamientos fueron H. influenzae, serotipo b, 12,9% no capsulares y 2,0% de otros serotipos (10 a, 1 d, 1 e y 2 f. Del total de aislamientos, 12% fueron productores de beta-lactamasa; 13,9%, resistentes a ampicilina; 12,7%, a trimetoprim sulfametoxazol (SXT; 5,4%, a cloranfenicol, y 1% a cefuroxima; todos fueron sensibles a ceftriaxona. Durante este período se observó un incremento en la resistencia de los aislamientos a SXT (5% al 13%, pero la diferencia no fue estadísticamente significativa (p=0,1. Con la vigilancia se pudo determinar una disminución significativa de los casos de meningitis en los menores de 1 año y en el

  2. Virulence phenotypes of low-passage clinical isolates of Nontypeable Haemophilus influenzae assessed using the chinchilla laniger model of otitis media

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    Hogg Justin

    2007-06-01

    Full Text Available Abstract Background The nontypeable Haemophilus influenzae (NTHi are associated with a spectrum of respiratory mucosal infections including: acute otitis media (AOM; chronic otitis media with effusion (COME; otorrhea; locally invasive diseases such as mastoiditis; as well as a range of systemic disease states, suggesting a wide range of virulence phenotypes. Genomic studies have demonstrated that each clinical strain contains a unique genic distribution from a population-based supragenome, the distributed genome hypothesis. These diverse clinical and genotypic findings suggest that each NTHi strain possesses a unique set of virulence factors that contributes to the course of the disease. Results The local and systemic virulence patterns of ten genomically characterized low-passage clinical NTHi strains (PittAA – PittJJ obtained from children with COME or otorrhea were stratified using the chinchilla model of otitis media (OM. Each isolate was used to bilaterally inoculate six animals and thereafter clinical assessments were carried out daily for 8 days by blinded observers. There was no statistical difference in the time it took for any of the 10 NTHi strains to induce otologic (local disease with respect to any or all of the other strains, however the differences in time to maximal local disease and the severity of local disease were both significant between the strains. Parameters of systemic disease indicated that the strains were not all equivalent: time to development of the systemic disease, maximal systemic scores and mortality were all statistically different among the strains. PittGG induced 100% mortality while PittBB, PittCC, and PittEE produced no mortality. Overall Pitt GG, PittII, and Pitt FF produced the most rapid and most severe local and systemic disease. A post hoc determination of the clinical origins of the 10 NTHi strains revealed that these three strains were of otorrheic origin, whereas the other 7 were from patients

  3. The Four Arabidopsis Reduced Wall Acetylation Genes are Expressed in Secondary Wall-Containing Cells and Required for the Acetylation of Xylan

    Science.gov (United States)

    Xylan is one of the major polysaccharides in cellulosic biomass, and understanding the mechanisms underlying xylan biosynthesis will potentially help us design strategies to produce cellulosic biomass better suited for biofuel production. Although a number of genes have been show...

  4. Synergism between a half-site and an imperfect estrogen-responsive element, and cooperation with COUP-TFI are required for estrogen receptor (ER) to achieve a maximal estrogen-stimulation of rainbow trout ER gene.

    Science.gov (United States)

    Petit, F G; Métivier, R; Valotaire, Y; Pakdel, F

    1999-01-01

    In all oviparous, liver represents one of the main E2-target tissues where estrogen receptor (ER) constitutes the key mediator of estrogen action. The rainbow trout estrogen receptor (rtER) gene expression is markedly up-regulated by estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region. Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect estrogen-responsive element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation. Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone-responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-elements and/or trans-acting factors, such as ER itself or COUP-TF.

  5. The Fdb3 transcription factor of the Fusarium Detoxification of Benzoxazolinone gene cluster is required for MBOA but not BOA degradation in Fusarium pseudograminearum.

    Science.gov (United States)

    Kettle, Andrew J; Carere, Jason; Batley, Jacqueline; Manners, John M; Kazan, Kemal; Gardiner, Donald M

    2016-03-01

    A number of cereals produce the benzoxazolinone class of phytoalexins. Fusarium species pathogenic towards these hosts can typically degrade these compounds via an aminophenol intermediate, and the ability to do so is encoded by a group of genes found in the Fusarium De