WorldWideScience

Sample records for antitoxins

  1. Production of Clostridium difficile antitoxin.

    OpenAIRE

    Ehrich, M.; Van Tassell, R L; Libby, J M; Wilkins, T D

    1980-01-01

    We have produced antitoxin to the toxin of Clostridium difficile in rabbits and in goats. Antitoxin dilutions of 1/8,000 and 1/5,120 were capable of neutralizing lethal doses of the toxin in mice and in tissue culture, respectively.

  2. Bacterial toxin-antitoxin systems

    OpenAIRE

    Guglielmini, Julien; Van Melderen, Laurence

    2011-01-01

    Toxin-antitoxin (TA) systems are composed of two elements: a toxic protein and an antitoxin which is either an RNA (type I and III) or a protein (type II). Type II systems are abundant in bacterial genomes in which they move via horizontal gene transfer. They are generally composed of two genes organized in an operon, encoding a toxin and a labile antitoxin. When carried by mobile genetic elements, these small modules contribute to their stability by a phenomenon denoted as addiction. Recentl...

  3. Toxin-Antitoxin Battle in Bacteria

    DEFF Research Database (Denmark)

    Cataudella, Ilaria

    This PhD thesis consists of three research projects revolving around the common thread of investigation of the properties and biological functions of Toxin-Antitoxin loci. Toxin-Antitoxin (TA) loci are transcriptionally regulated via an auto-inhibition mechanism called conditional cooperativity, ...

  4. Keeping the Wolves at Bay: Antitoxins of Prokaryotic Type II Toxin-Antitoxin Systems

    Science.gov (United States)

    Chan, Wai Ting; Espinosa, Manuel; Yeo, Chew Chieng

    2016-01-01

    In their initial stages of discovery, prokaryotic toxin-antitoxin (TA) systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I–VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA antitoxins. We shall

  5. Keeping the Wolves at Bay: Antitoxins of Prokaryotic Type II Toxin-Antitoxin Systems.

    Science.gov (United States)

    Chan, Wai Ting; Espinosa, Manuel; Yeo, Chew Chieng

    2016-01-01

    In their initial stages of discovery, prokaryotic toxin-antitoxin (TA) systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I-VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA antitoxins. We shall

  6. Keeping the wolves at bay: antitoxins of prokaryotic type II toxin-antitoxin systems

    Directory of Open Access Journals (Sweden)

    Wai Ting eChan

    2016-03-01

    Full Text Available In their initial stages of discovery, prokaryotic toxin-antitoxin (TA systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I – VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA

  7. Evolution of the SpoIISABC Toxin-Antitoxin-Antitoxin System in Bacilli.

    Science.gov (United States)

    Gabriško, Marek; Barák, Imrich

    2016-01-01

    Programmed cell death in bacteria is generally associated with two-component toxin-antitoxin systems. The SpoIISABC system, originally identified in Bacillus subtilis, consists of three components: a SpoIISA toxin and the SpoIISB and SpoIISC antitoxins. SpoIISA is a membrane-bound protein, while SpoIISB and SpoIISC are small cytosolic antitoxins, which are able to bind SpoIISA and neutralize its toxicity. In the presented bioinformatics analysis, a taxonomic distribution of the genes of the SpoIISABC system is investigated; their conserved regions and residues are identified; and their phylogenetic relationships are inferred. The SpoIISABC system is part of the core genome in members of the Bacillus genus of the Firmicutes phylum. Its presence in some non-bacillus species is likely the result of horizontal gene transfer. The SpoIISB and SpoIISC antitoxins originated by gene duplications, which occurred independently in the B. subtilis and B. cereus lineages. In the B. cereus lineage, the SpoIIS module is present in two different architectures. PMID:27294956

  8. Toxin-Antitoxin Systems in Clinical Pathogens

    Science.gov (United States)

    Fernández-García, Laura; Blasco, Lucia; Lopez, Maria; Bou, German; García-Contreras, Rodolfo; Wood, Thomas; Tomas, María

    2016-01-01

    Toxin-antitoxin (TA) systems are prevalent in bacteria and archaea. Although not essential for normal cell growth, TA systems are implicated in multiple cellular functions associated with survival under stress conditions. Clinical strains of bacteria are currently causing major human health problems as a result of their multidrug resistance, persistence and strong pathogenicity. Here, we present a review of the TA systems described to date and their biological role in human pathogens belonging to the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) and others of clinical relevance (Escherichia coli, Burkholderia spp., Streptococcus spp. and Mycobacterium tuberculosis). Better understanding of the mechanisms of action of TA systems will enable the development of new lines of treatment for infections caused by the above-mentioned pathogens. PMID:27447671

  9. Algal chloroplast produced camelid VHH antitoxins are capable of neutralizing botulinum neurotoxin

    OpenAIRE

    Daniel J Barrera; Rosenberg, Julian N.; Chiu, Joanna G.; Chang, Yung-Nien; Debatis, Michelle; Ngoi, Soo-Mun; Chang, John T.; Shoemaker, Charles B.; George A Oyler; Mayfield, Stephen P

    2014-01-01

    We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VHH) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydom...

  10. Postbooster Antibodies from Humans as Source of Diphtheria Antitoxin

    OpenAIRE

    Bermejo-Martin, Jesús F; Avila-Alonso, Ana; González-Rivera, Milagros; Tamayo, Eduardo; Eiros, Jose María; Almansa, Raquel

    2016-01-01

    Diphtheria antitoxin for therapeutic use is in limited supply. A potential source might be affinity-purified antibodies originally derived from plasma of adults who received a booster dose of a vaccine containing diphtheria toxoid. These antibodies might be useful for treating even severe cases of diphtheria.

  11. Postbooster Antibodies from Humans as Source of Diphtheria Antitoxin.

    Science.gov (United States)

    Bermejo-Martin, Jesús F; Avila-Alonso, Ana; González-Rivera, Milagros; Tamayo, Eduardo; Eiros, Jose María; Almansa, Raquel

    2016-07-01

    Diphtheria antitoxin for therapeutic use is in limited supply. A potential source might be affinity-purified antibodies originally derived from plasma of adults who received a booster dose of a vaccine containing diphtheria toxoid. These antibodies might be useful for treating even severe cases of diphtheria. PMID:27314309

  12. Radiosensitivity of antibody responses and radioresistant secondary tetanus antitoxin responses

    International Nuclear Information System (INIS)

    Primary tetanus antitoxin responses were increasingly repressed in mice when gamma radiation doses of 100 to 400 rads were delivered by whole-body exposure prior to immunization with fluid tetanus toxoid (FTT). Nearly normal secondary antitoxin responses were obtained in mice exposed to 600 rads of gamma radiation 4 days after secondary antigenic stimulation with FTT. A rapid transition from radiosensitivity of the antibody-forming system on days 1 to 3 was followed by relative radioresistance on day 4 after the booster injection of toxoid. Studies on lymphoid cellular kinetics in popliteal lymph nodes after injection of 3H--thymidine (3H--TdR) and incorporation of 3H--L-histidine into circulating antitoxin were carried out. Analysis of tritium radioactivity in antigen--antibody precipitates of serums 2 hr after injection of the labeled amino acid revealed maximum incorporation into antibody around day 7 after the booster in nonirradiated controls and about day 12, i.e., 8 days after irradiation, in experimental mice. The shift from radiosensitivity to relative radioresistance was attributed to a marked peak of plasma-cell proliferation in the medulla of lymph nodes on day 3. Many medullary plasma cells survived and continued to proliferate after exposure to radiation. Germinal centers were destroyed by radiation within 1 day. Since antibody formation continued after exposure to radiation and after the loss of germinal centers, this supports the view that germinal-center cells were involved more in the generation of memory cells than in antibody synthesis

  13. Crystallization of the HigBA2 toxin-antitoxin complex from Vibrio cholerae

    DEFF Research Database (Denmark)

    Hadǽi, San; Garcia-Pino, Abel; Martinez-Rodriguez, Sergio;

    2013-01-01

    The genome of Vibrio cholerae encodes two higBA toxin-antitoxin (TA) modules that are activated by amino-acid starvation. Here, the TA complex of the second module, higBA2, as well as the C-terminal domain of the corresponding HigA2 antitoxin, have been purified and crystallized. The HigBA2 complex...

  14. Doc of prophage P1 is inhibited by its antitoxin partner Phd through fold complementation

    DEFF Research Database (Denmark)

    Garcia-Pino, Abel; Christensen-Dalsgaard, Mikkel; Wyns, Lode;

    2008-01-01

    Prokaryotic toxin-antitoxin modules are involved in major physiological events set in motion under stress conditions. The toxin Doc (death on curing) from the phd/doc module on phage P1 hosts the C-terminal domain of its antitoxin partner Phd (prevents host death) through fold complementation. This...

  15. sRNA Antitoxins: More than One Way to Repress a Toxin

    Directory of Open Access Journals (Sweden)

    Jia Wen

    2014-08-01

    Full Text Available Bacterial toxin-antitoxin loci consist of two genes: one encodes a potentially toxic protein, and the second, an antitoxin to repress its function or expression. The antitoxin can either be an RNA or a protein. For type I and type III loci, the antitoxins are RNAs; however, they have very different modes of action. Type I antitoxins repress toxin protein expression through interacting with the toxin mRNA, thereby targeting the mRNA for degradation or preventing its translation or both; type III antitoxins directly bind to the toxin protein, sequestering it. Along with these two very different modes of action for the antitoxin, there are differences in the functions of the toxin proteins and the mobility of these loci between species. Within this review, we discuss the major differences as to how the RNAs repress toxin activity, the potential consequences for utilizing different regulatory strategies, as well as the confirmed and potential biological roles for these loci across bacterial species.

  16. A common origin for the bacterial toxin-antitoxin systems parD and ccd, suggested by analyses of toxin/target and toxin/antitoxin interactions.

    Directory of Open Access Journals (Sweden)

    Andrew B Smith

    Full Text Available Bacterial toxin-antitoxin (TA systems encode two proteins, a potent inhibitor of cell proliferation (toxin and its specific antidote (antitoxin. Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets.

  17. Repurposing a Prokaryotic Toxin-Antitoxin System for the Selective Killing of Oncogenically Stressed Human Cells.

    Science.gov (United States)

    Preston, Mark A; Pimentel, Belén; Bermejo-Rodríguez, Camino; Dionne, Isabelle; Turnbull, Alice; de la Cueva-Méndez, Guillermo

    2016-07-15

    Prokaryotes express intracellular toxins that pass unnoticed to carrying cells until coexpressed antitoxin partners are degraded in response to stress. Although not evolved to function in eukaryotes, one of these toxins, Kid, induces apoptosis in mammalian cells, an effect that is neutralized by its cognate antitoxin, Kis. Here we engineered this toxin-antitoxin pair to create a synthetic system that becomes active in human cells suffering a specific oncogenic stress. Inspired by the way Kid becomes active in bacterial cells, we produced a Kis variant that is selectively degraded in human cells expressing oncoprotein E6. The resulting toxin-antitoxin system functions autonomously in human cells, distinguishing those that suffer the oncogenic insult, which are killed by Kid, from those that do not, which remain protected by Kis. Our results provide a framework for developing personalized anticancer strategies avoiding off-target effects, a challenge that has been hardly tractable by other means thus far. PMID:26230535

  18. Conditional cooperativity of toxin - antitoxin regulation can mediate bistability between growth and dormancy.

    Directory of Open Access Journals (Sweden)

    Ilaria Cataudella

    Full Text Available Many toxin-antitoxin operons are regulated by the toxin/antitoxin ratio by mechanisms collectively coined "conditional cooperativity". Toxin and antitoxin form heteromers with different stoichiometric ratios, and the complex with the intermediate ratio works best as a transcription repressor. This allows transcription at low toxin level, strong repression at intermediate toxin level, and then again transcription at high toxin level. Such regulation has two interesting features; firstly, it provides a non-monotonous response to the concentration of one of the proteins, and secondly, it opens for ultra-sensitivity mediated by the sequestration of the functioning heteromers. We explore possible functions of conditional regulation in simple feedback motifs, and show that it can provide bistability for a wide range of parameters. We then demonstrate that the conditional cooperativity in toxin-antitoxin systems combined with the growth-inhibition activity of free toxin can mediate bistability between a growing state and a dormant state.

  19. Conditional cooperativity of toxin - antitoxin regulation can mediate bistability between growth and dormancy.

    Science.gov (United States)

    Cataudella, Ilaria; Sneppen, Kim; Gerdes, Kenn; Mitarai, Namiko

    2013-01-01

    Many toxin-antitoxin operons are regulated by the toxin/antitoxin ratio by mechanisms collectively coined "conditional cooperativity". Toxin and antitoxin form heteromers with different stoichiometric ratios, and the complex with the intermediate ratio works best as a transcription repressor. This allows transcription at low toxin level, strong repression at intermediate toxin level, and then again transcription at high toxin level. Such regulation has two interesting features; firstly, it provides a non-monotonous response to the concentration of one of the proteins, and secondly, it opens for ultra-sensitivity mediated by the sequestration of the functioning heteromers. We explore possible functions of conditional regulation in simple feedback motifs, and show that it can provide bistability for a wide range of parameters. We then demonstrate that the conditional cooperativity in toxin-antitoxin systems combined with the growth-inhibition activity of free toxin can mediate bistability between a growing state and a dormant state. PMID:24009488

  20. FDA Approves First Botulism Antitoxin for Use in Neutralizing All Seven Known Botulinum Nerve Toxin Serotypes

    Science.gov (United States)

    ... Antitoxin for use in neutralizing all seven known botulinum nerve toxin serotypes Product to be stored in Strategic National ... antibody fragments that neutralize all of the seven botulinum nerve toxin serotypes known to cause botulism. Botulism is a ...

  1. Discovery of functional toxin/antitoxin systems in bacteria by shotgun cloning

    Energy Technology Data Exchange (ETDEWEB)

    Sberro, Hila; Leavitt, Azita; Kiro, Ruth; Koh, Eugene; Peleg, Yoav; Qimron, Udi; Sorek, Rotem

    2013-04-01

    Toxin-antitoxin (TA) modules, composed of a toxic protein and a counteracting antitoxin, play important roles in bacterial physiology. We examined the experimental insertion of 1.5 million genes from 388 microbial genomes into an Escherichia coli host using over 8.5 million random clones. This revealed hundreds of genes (toxins) that could only be cloned when the neighboring gene (antitoxin) was present on the same clone. Clustering of these genes revealed TA families widespread in bacterial genomes, some of which deviate from the classical characteristics previously described for such modules. Introduction of these genes into E. coli validated that the toxin toxicity is mitigated by the antitoxin. Infection experiments with T7 phage showed that two of the new modules can provide resistance against phage. Moreover, our experiments revealed an 'anti-defense' protein in phage T7 that neutralizes phage resistance. Our results expose active fronts in the arms race between bacteria and phage.

  2. Identification and classification of bacterial Type III toxin–antitoxin systems encoded in chromosomal and plasmid genomes

    OpenAIRE

    Blower, Tim R.; Short, Francesca L.; Rao, Feng; Mizuguchi, Kenji; Pei, Xue Y.; Fineran, Peter C.; Luisi, Ben F.; Salmond, George P. C.

    2012-01-01

    Toxin–antitoxin systems are widespread in bacteria and archaea. They perform diverse functional roles, including the generation of persistence, maintenance of genetic loci and resistance to bacteriophages through abortive infection. Toxin–antitoxin systems have been divided into three types, depending on the nature of the interacting macromolecules. The recently discovered Type III toxin–antitoxin systems encode protein toxins that are inhibited by pseudoknots of antitoxic RNA, encoded by sho...

  3. Effect of biotherapeutics on antitoxin IgG in experimentally induced Clostridium difficile infection

    Directory of Open Access Journals (Sweden)

    S Kaur

    2012-01-01

    Full Text Available Purpose: Recurrent diarrhoea after successful treatment of primary Clostridium difficile associated disease (CDAD occurs due to bowel flora alterations and failure to mount an effective antibody response. Apart from antibiotics, risk factors include immunosuppressive and acid-suppressive drug administration. Biotherapeutics such as probiotic and epidermal growth factor (EGF may offer potential effective therapy for CDAD. Materials and Methods: The effect of biotherapeutics in mounting an antibody response against C. difficile toxins was studied in BALB/c mice challenged with C. difficile after pre-treatment with ampicillin, lansoprazole or cyclosporin. Sera from sacrificed animals were estimated for antitoxin IgG by enzyme linked immunosorbent assay. Results: Antitoxin IgG was significantly higher (P0.05 in animals in which C. difficile was given after pre-treatment with cyclosporin compared to those without any pre-treatment, or pre-treatment with antibiotic or lansoprazole. In inter-subgroup comparisons also significant anomaly in production of antitoxin IgG was found. The antitoxin IgG levels were raised in animals administered C. difficile after pre-treatment with ampicillin, but lower in animals administered cyclosporin. High levels of antitoxin IgG were also found in the serum samples of animals receiving lansoprazole and C. difficile. Conclusions: Probiotics showed their beneficial effect by boosting the immune response as seen by production of antitoxin IgG. Oral administration of EGF did not affect the immune response to C. difficile toxins as significant increase was not observed in the serum antitoxin IgG levels in any of the groups investigated.

  4. Interactions of Kid-Kis toxin-antitoxin complexes with the parD operator-promotor region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid-Kis oligomers

    NARCIS (Netherlands)

    Monti, M.C.; Hernandez-Arriaga, A.M.; Kamphuis, M.B.; Lopez-Villarejo, J.; Heck, A.J.R.; Boelens, R.; Diaz-Orejas, R.; van den Heuvel, R.H.H.

    2007-01-01

    The parD operon of Escherichia coli plasmid R1 encodes a toxin–antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly unders

  5. Regulating Toxin-Antitoxin Expression: Controlled Detonation of Intracellular Molecular Timebombs

    Directory of Open Access Journals (Sweden)

    Finbarr Hayes

    2014-01-01

    Full Text Available Genes for toxin-antitoxin (TA complexes are widely disseminated in bacteria, including in pathogenic and antibiotic resistant species. The toxins are liberated from association with the cognate antitoxins by certain physiological triggers to impair vital cellular functions. TAs also are implicated in antibiotic persistence, biofilm formation, and bacteriophage resistance. Among the ever increasing number of TA modules that have been identified, the most numerous are complexes in which both toxin and antitoxin are proteins. Transcriptional autoregulation of the operons encoding these complexes is key to ensuring balanced TA production and to prevent inadvertent toxin release. Control typically is exerted by binding of the antitoxin to regulatory sequences upstream of the operons. The toxin protein commonly works as a transcriptional corepressor that remodels and stabilizes the antitoxin. However, there are notable exceptions to this paradigm. Moreover, it is becoming clear that TA complexes often form one strand in an interconnected web of stress responses suggesting that their transcriptional regulation may prove to be more intricate than currently understood. Furthermore, interference with TA gene transcriptional autoregulation holds considerable promise as a novel antibacterial strategy: artificial release of the toxin factor using designer drugs is a potential approach to induce bacterial suicide from within.

  6. Plasma as alternatively sample to quantify tetanus antitoxin

    Directory of Open Access Journals (Sweden)

    Ariel Menéndez-Barrios

    2015-08-01

    Full Text Available Tetanus antitoxin is quantified in Cuba at blood banks, from the serum of immunized donors, to produce aspecific human gamma globulin. A heterogeneous indirect immunoenzymatic assay is used, using the serum as analytical sample. The possible use of plasma obtained from plasmapheresis as alternative sample was evaluated in this research, to minimize the volume of total blood extracted to the donors. One hundred plasma donors who came to donate between October and November 2013 were selected by simple random sampling. Serum sample was obtained for extraction of 5 mL of blood, deposited in dry glass tube. While the other sample took 1.5 mL of plasma in a plastic tube with cover, at the end of the donation directly of the unit of plasma collected. Comparison of the difference between the means of both groups was done using SPSS for Windows. It was found that the values obtained in serum were bigger than those obtained in plasma. Difference between the means of both groups was statistically significant (p 0.00. It is not advisable to use the obtained plasma of the plasmapheresis as analytic sample in this assay.

  7. Interactions of Kid–Kis toxin–antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid–Kis oligomers

    OpenAIRE

    Monti, M. C.; Hernandez-Arriaga, A.M.; Kamphuis, M.B.; Lopez-Villarejo, J.; Heck, A.J.R.; Boelens, R.; Diaz-Orejas, R.; van den Heuvel, R.H.H.

    2007-01-01

    The parD operon of Escherichia coli plasmid R1 encodes a toxin–antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid–kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the...

  8. Diphtheria antitoxin levels in the Netherlands: a population-based study.

    OpenAIRE

    de Melker, H. E.; Berbers, G A; Nagelkerke, N. J.; Conyn-van Spaendonck, M. A.

    1999-01-01

    In a population-based study in the Netherlands, diphtheria antitoxin antibodies were measured with a toxin-binding inhibition assay in 9, 134 sera from the general population and religious communities refusing vaccination. The Dutch immunization program appears to induce long-term protection against diphtheria. However, a substantial number of adults born before the program was introduced had no protective diphtheria antibody levels. Although herd immunity seems adequate, long-term population...

  9. Identification of a Human Monoclonal Antibody To Replace Equine Diphtheria Antitoxin for Treatment of Diphtheria Intoxication

    OpenAIRE

    Sevigny, Leila M; Booth, Brian J.; Rowley, Kirk J.; Leav, Brett A.; Cheslock, Peter S.; Kerry A Garrity; Sloan, Susan E.; Thomas, William; Babcock, Gregory J.; Wang, Yang

    2013-01-01

    Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available ...

  10. Type II Toxin-antitoxin distribution and adaptive aspects on Xanthomonas genomes: focus on Xanthomonas citri

    Directory of Open Access Journals (Sweden)

    Paula Maria Moreira Martins

    2016-05-01

    Full Text Available Prokaryotic toxin-antitoxin (TA systems were first described as being designed to prevent plasmid loss in bacteria. However, with the increase in prokaryotic genome sequencing, recently many TAs have been found in bacterial chromosomes, having other biological functions, such as environmental stress response. To date, only few studies have focused on TA systems in phytopathogens, and their possible impact on the bacterial fitness. This may be especially important for pathogens like Xanthomonas spp., which live epiphytically before entering the host. In this study, we looked for TA systems in the genomes of ten Xanthomonas strains. We verified that citrus-infecting pathovars have, on average, 50% more TAs than other Xanthomonas spp. and no genome harbors classical toxins such as MqsR, RelB and HicA. Only one TA system (PIN_VapC-FitB-like/SpoVT_AbrB was conserved among the Xanthomonas genomes, suggesting adaptive aspects concerning its broad occurrence. We also detected a trend of toxin gene loss in this genus, while the antitoxin gene was preferably maintained. This study discovers the quantitative and qualitative differences among the type II TA systems present in Xanthomonas spp., especially concerning the citrus-infecting strains. In addition, the antitoxin retention in the genomes is possibly related with the resistance mechanism of further TA infections as an anti-addiction system or might also be involved in regulation of certain specific genes.

  11. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery.

    Science.gov (United States)

    Cataudella, Ilaria; Trusina, Ala; Sneppen, Kim; Gerdes, Kenn; Mitarai, Namiko

    2012-08-01

    Many toxin-antitoxin (TA) loci are known to strongly repress their own transcription. This auto-inhibition is often called 'conditional cooperativity' as it relies on cooperative binding of TA complexes to operator DNA that occurs only when toxins are in a proper stoichiometric relationship with antitoxins. There has recently been an explosion of interest in TA systems due to their role in bacterial persistence, however the role of conditional cooperativity is still unclear. We reveal the biological function of conditional cooperativity by constructing a mathematical model of the well studied TA system, relBE of Escherichia coli. We show that the model with the in vivo and in vitro established parameters reproduces experimentally observed response to nutritional stress. We further demonstrate that conditional cooperativity stabilizes the level of antitoxin in rapidly growing cells such that random induction of relBE is minimized. At the same time it enables quick removal of free toxin when the starvation is terminated. PMID:22495927

  12. Characterization of a novel toxin-antitoxin module, VapBC, encoded by Leptospira interrogans chromosome

    Institute of Scientific and Technical Information of China (English)

    Yi Xuan ZHANG; Xiao Kui GUO; Chuan WU; Bo BI; Shuang Xi REN; Chun Fu WU; Guo Ping ZHAO

    2004-01-01

    Comparative genomic analysis of the coding sequences (CDSs) of Leptospira interrogans revealed a pair of closely linked genes homologous to the vapBC loci of many other bacteria with respect to both deduced amino acid sequences and operon organizations. Expression of single vapC gene in Escherichia coli resulted in inhibition of bacterial growth,whereas co-expression of vapBC restored the growth effectively. This phenotype is typical for three other characterized toxin-antitoxin systems of bacteria, i.e., mazEF[1], relBE[2] and chpIK[3]. The VapC proteins of bacteria and a thermophilic archeae, Solfolobus tokodaii, form a structurally distinguished group of toxin different from the other known toxins of bacteria. Phylogenetic analysis of both toxins and antitoxins of all categories indicated that although toxins were evolved from divergent sources and may or may not follow their speciation paths (as indicated by their 16s RNA sequences), co-evolution with their antitoxins was obvious.

  13. Structural characterizations of phage antitoxin Dmd and its interactions with bacterial toxin RnlA.

    Science.gov (United States)

    Wei, Yong; Gao, Zengqiang; Zhang, Heng; Dong, Yuhui

    2016-04-15

    Toxin-antitoxin (TA) loci are widespread in bacteria plasmids and chromosomes, and target various cellular functions to regulate cell growth and death. A type II TA system RnlA-RnlB from Escherichia coli is associated with phage-resistance. After the infection of bacteriophage T4 with Dmd defection, RnlA is activated by the disappearance of RnlB, resulting in the rapid degradation of T4 mRNAs. Dmd can bind to RnlA directly and neutralize RnlA toxicity to allow phage reproduction. Dmd represent a heterogenous antitoxin of RnlA replacing antitoxin RnlB. Here, we reported two structures of Dmd from T4 phage and RB69 phage. Both Dmd structures are high similar with a compacted domain composed of a four-stranded anti-parallel β-sheet and an α-helix. Chromatography and SAXS suggest Dmd forms a dimer in solution consistent with that in crystal. Structure-based mutagenesis of Dmd reveals key residues involved in RnlA-binding. Possibility cavities in Dmd used for compounds design were modeled. Our structural study revealed the recognition and inhibition mechanism of RnlA by Dmd and providing a potential laboratory phage prevention target for drug design. PMID:26972252

  14. Characterization of a novel toxin-antitoxin module, VapBC, encoded by Leptospira interrogans chromosome.

    Science.gov (United States)

    Zhang, Yi Xuan; Li, Juan; Guo, Xiao Kui; Wu, Chuan; Bi, Bo; Ren, Shuang Xi; Wu, Chun Fu; Zhao, Guo Ping

    2004-06-01

    Comparative genomic analysis of the coding sequences (CDSs) of Leptospira interrogans revealed a pair of closely linked genes homologous to the vapBC loci of many other bacteria with respect to both deduced amino acid sequences and operon organizations. Expression of single vapC gene in Escherichia coli resulted in inhibition of bacterial growth, whereas co-expression of vapBC restored the growth effectively. This phenotype is typical for three other characterized toxin-antitoxin systems of bacteria, i.e., mazEF, relBE and chpIK. The VapC proteins of bacteria and a thermophilic archeae, Solfolobus tokodaii, form a structurally distinguished group of toxin different from the other known toxins of bacteria. Phylogenetic analysis of both toxins and antitoxins of all categories indicated that although toxins were evolved from divergent sources and may or may not follow their speciation paths (as indicated by their 16s RNA sequences), co-evolution with their antitoxins was obvious. PMID:15225414

  15. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications.

    Science.gov (United States)

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-02-01

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies. PMID:26907343

  16. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    Directory of Open Access Journals (Sweden)

    Chew Chieng Yeo

    2016-02-01

    Full Text Available Toxin-antitoxin (TA systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  17. Type II Toxin-Antitoxin Distribution and Adaptive Aspects on Xanthomonas Genomes: Focus on Xanthomonas citri.

    Science.gov (United States)

    Martins, Paula M M; Machado, Marcos A; Silva, Nicholas V; Takita, Marco A; de Souza, Alessandra A

    2016-01-01

    Prokaryotic toxin-antitoxin (TA) systems were first described as being designed to prevent plasmid loss in bacteria. However, with the increase in prokaryotic genome sequencing, recently many TAs have been found in bacterial chromosomes, having other biological functions, such as environmental stress response. To date, only few studies have focused on TA systems in phytopathogens, and their possible impact on the bacterial fitness. This may be especially important for pathogens like Xanthomonas spp., which live epiphytically before entering the host. In this study, we looked for TA systems in the genomes of 10 Xanthomonas strains. We verified that citrus-infecting pathovars have, on average, 50% more TAs than other Xanthomonas spp. and no genome harbors classical toxins such as MqsR, RelB, and HicA. Only one TA system (PIN_VapC-FitB-like/SpoVT_AbrB) was conserved among the Xanthomonas genomes, suggesting adaptive aspects concerning its broad occurrence. We also detected a trend of toxin gene loss in this genus, while the antitoxin gene was preferably maintained. This study discovers the quantitative and qualitative differences among the type II TA systems present in Xanthomonas spp., especially concerning the citrus-infecting strains. In addition, the antitoxin retention in the genomes is possibly related with the resistance mechanism of further TA infections as an anti-addiction system or might also be involved in regulation of certain specific genes. PMID:27242687

  18. Growth control switch by a DNA-damage-inducible toxin-antitoxin system in Caulobacter crescentus.

    Science.gov (United States)

    Kirkpatrick, Clare L; Martins, Daniel; Redder, Peter; Frandi, Antonio; Mignolet, Johann; Chapalay, Julien Bortoli; Chambon, Marc; Turcatti, Gerardo; Viollier, Patrick H

    2016-01-01

    Bacterial toxin-antitoxin systems (TASs) are thought to respond to various stresses, often inducing growth-arrested (persistent) sub-populations of cells whose housekeeping functions are inhibited. Many such TASs induce this effect through the translation-dependent RNA cleavage (RNase) activity of their toxins, which are held in check by their cognate antitoxins in the absence of stress. However, it is not always clear whether specific mRNA targets of orthologous RNase toxins are responsible for their phenotypic effect, which has made it difficult to accurately place the multitude of TASs within cellular and adaptive regulatory networks. Here, we show that the TAS HigBA of Caulobacter crescentus can promote and inhibit bacterial growth dependent on the dosage of HigB, a toxin regulated by the DNA damage (SOS) repressor LexA in addition to its antitoxin HigA, and the target selectivity of HigB's mRNA cleavage activity. HigB reduced the expression of an efflux pump that is toxic to a polarity control mutant, cripples the growth of cells lacking LexA, and targets the cell cycle circuitry. Thus, TASs can have outcome switching activity in bacterial adaptive (stress) and systemic (cell cycle) networks. PMID:27572440

  19. Interactions between the toxin kid of the bacterial parD system and the antitoxins Kis and MazE

    OpenAIRE

    Kamphuis, M.B.; Monti, M. C.; van den Heuvel, R.H.H.; Santos-Sierra, S.; Folkers, G E; Lemonnier, M.; Diaz-Orejas, R.; Heck, A.J.R.; Boelens, R.

    2007-01-01

    The proteins Kid and Kis are the toxin and antitoxin, respectively, encoded by the parD operon of Escherichia coli plasmid R1. Kis prevents the inhibition of E. coli cell growth caused by the RNA cleavage activity of Kid. Overproduction of MazE, the chromosome-encoded homologue of Kis, has been demonstrated to neutralize Kid toxicity to a certain extent in the absence of native Kis. Here,we show that a high structural similarity exists between these antitoxins, using NMR spectroscopy. We repo...

  20. Killing Effect and Antitoxic Activity of the Leptospira interrogans Toxin-Antitoxin System in Escherichia coli

    OpenAIRE

    Picardeau, Mathieu; Ren, Shuangxi; Saint Girons, Isabelle

    2001-01-01

    We report the first evidence of a chromosome-encoded toxin-antitoxin locus in spirochetes. This locus has been found in the pathogenic spirochete Leptospira interrogans and exhibits homologies with the pem/chp loci. The L. interrogans chp locus consists of two genes: chpK (for “killer protein”) and its upstream partner chpI (for “inhibitory protein”). Expression of ChpK in Escherichia coli results in the inhibition of bacterial growth. The coexpression of ChpI neutralizes ChpK toxicity. By So...

  1. A viral nanoparticle with dual function as an anthrax antitoxin and vaccine.

    Directory of Open Access Journals (Sweden)

    Darly J Manayani

    2007-10-01

    Full Text Available The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel antitoxins and vaccines that act rapidly and with minimal adverse effects. B. anthracis produces an AB-type toxin composed of the receptor-binding moiety protective antigen (PA and the enzymatic moieties edema factor and lethal factor. PA is a key target for both antitoxin and vaccine development. We used the icosahedral insect virus Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor. The chimeric virus-like particles (VLPs correctly displayed the receptor von Willebrand A domain on their surface and inhibited lethal toxin action in in vitro and in vivo models of anthrax intoxication. Moreover, VLPs complexed with PA elicited a potent toxin-neutralizing antibody response that protected rats from anthrax lethal toxin challenge after a single immunization without adjuvant. This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax.

  2. Diverse distribution of Toxin-Antitoxin II systems in Salmonella enterica serovars

    Science.gov (United States)

    Di Cesare, Andrea; Losasso, Carmen; Barco, Lisa; Eckert, Ester M.; Conficoni, Daniele; Sarasini, Giulia; Corno, Gianluca; Ricci, Antonia

    2016-01-01

    Type II Toxin-Antitoxin systems (TAs), known for their presence in virulent and antibiotic resistant bacterial strains, were recently identified in Salmonella enterica isolates. However, the relationships between the presence of TAs (ccdAB and vapBC) and the epidemiological and genetic features of different non-typhoidal Salmonella serovars are largely unknown, reducing our understanding of the ecological success of different serovars. Salmonella enterica isolates from different sources, belonging to different serovars and epidemiologically unrelated according to ERIC profiles, were investigated for the presence of type II TAs, plasmid content, and antibiotic resistance. The results showed the ubiquitous presence of the vapBC gene in all the investigated Salmonella isolates, but a diverse distribution of ccdAB, which was detected in the most widespread Salmonella serovars, only. Analysis of the plasmid toxin ccdB translated sequence of four selected Salmonella isolates showed the presence of the amino acid substitution R99W, known to impede in vitro the lethal effect of CcdB toxin in the absence of its cognate antitoxin CcdA. These findings suggest a direct role of the TAs in promoting adaptability and persistence of the most prevalent Salmonella serovars, thus implying a wider eco-physiological role for these type II TAs. PMID:27357537

  3. Interactions between the toxin kid of the bacterial parD system and the antitoxins Kis and MazE

    NARCIS (Netherlands)

    Kamphuis, M.B.; Monti, M.C.; van den Heuvel, R.H.H.; Santos-Sierra, S.; Folkers, G.E.; Lemonnier, M.; Diaz-Orejas, R.; Heck, A.J.R.; Boelens, R.

    2007-01-01

    The proteins Kid and Kis are the toxin and antitoxin, respectively, encoded by the parD operon of Escherichia coli plasmid R1. Kis prevents the inhibition of E. coli cell growth caused by the RNA cleavage activity of Kid. Overproduction of MazE, the chromosome-encoded homologue of Kis, has been demo

  4. Early and enhanced antitoxin responses elicited with complexes of tetanus toxoid and specific mouse and human antibodies

    International Nuclear Information System (INIS)

    Primary tetanus antitoxin responses were early and enhanced in mice when tetanus toxoid was administered in complex with specific isologous antitoxin or specific mouse γ-globulin. Antitoxin responses were enhanced when fluid tetanus toxoid was complexed in vitro in antigen-to-antibody ratios of equivalence or antigen excess; responses to complexed toxoid in antibody excess were comparatively repressed. Primary responses were greatly inhibited in mice immunized with the same amount of toxoid complexed at equivalence or in antibody excess with specific human γ-globulin. Although primary responses were totally repressed, a primed state developed; a second injection of fluid toxoid within a few days produced excellent antitoxin responses. Separate injections of antigen and antibody at different sites produced an excellent in vivo primed state for early and high responses. Antibody production after stimulation with complexed toxoid was also enhanced in mice irradiated with 400 rads, a dose that ordinarily completely suppresses primary responses with fluid toxoid alone. These data provide evidence for the efficacy of antigen-antibody complexes in early and active immunization. (U.S.)

  5. Crystallization of two operator complexes from the Vibrio cholerae HigBA2 toxin-antitoxin module

    DEFF Research Database (Denmark)

    Hadzi, San; Garcia-Pino, Abel; Gerdes, Kenn;

    2015-01-01

    The HigA2 antitoxin and the HigBA2 toxin-antitoxin complex from Vibrio cholerae were crystallized in complex with their operator box. Screening of 22 different DNA duplexes led to two crystal forms of HigA2 complexes and one crystal form of a HigBA2 complex. Crystals of HigA2 in complex with a 17...

  6. Kinetics of epsilon antitoxin antibodies in different strategies for active immunization of lambs against enterotoxaemia

    Directory of Open Access Journals (Sweden)

    Heni F. Costa

    2013-08-01

    Full Text Available Enterotoxaemia, a common disease that affects domestic small ruminants, is mainly caused by the epsilon toxin of Clostridium perfringens type D. The present study tested four distinct immunization protocols to evaluate humoral response in lambs, a progeny of non-vaccinated sheep during gestation. Twenty-four lambs were randomly allocated into four groups according to age (7, 15, 30 and 45 days, receiving the first dose of epsilon toxoid commercial vaccine against clostridiosis with booster after 30 days post vaccination. Indirect ELISA was performed after the first vaccine dose and booster to evaluate the immune response of the lambs. Results showed that for the four protocols tested all lambs presented serum title considered protective (≥0.2UI/ml epsilon antitoxin antibodies and also showed that the anticipation of primovaccination of lambs against enterotoxaemia conferred serum title considered protective allowing the optimization of mass vaccination of lambs.

  7. Preliminary crystallographic analysis of the Escherichia coli antitoxin MqsA (YgiT/b3021) in complex with mqsRA promoter DNA

    International Nuclear Information System (INIS)

    The E. coli antitoxin MqsA (YgiT/b3021) has been cocrystallized with mqsRA promoter DNA. A native data set has been collected to a resolution of 2.1 Å. The Escherichia coli proteins MqsR and MqsA comprise a novel toxin–antitoxin (TA) system. MqsA, the antitoxin, defines a new family of antitoxins because unlike other antitoxins MqsA is structured throughout its entire sequence, binds zinc and coordinates DNA via its C-terminal and not its N-terminal domain. In order to understand how bacterial antitoxins, and MqsA in particular, regulate transcription, the MqsA protein was cocrystallized with a 26-mer duplex DNA corresponding to the palindromic region of the mqsRA promoter. The merohedrally twinned crystal belonged to space group P41, with unit-cell parameters a = 60.99, b = 60.99, c = 148.60 Å. A complete data set was collected to a resolution of 2.1 Å. The solvent content of the crystal was consistent with the presence of two MqsA molecules bound to the duplex DNA in the asymmetric unit

  8. A Chimeric Protein That Functions as both an Anthrax Dual-Target Antitoxin and a Trivalent Vaccine▿

    OpenAIRE

    Wu, Gaobing; Hong, Yuzhi; Guo, Aizhen; Feng, Chunfang; Cao, Sha; Zhang, Cheng-Cai; Shi, Ruiping; Tan, Yadi; Liu, Ziduo

    2010-01-01

    Effective measures for the prophylaxis and treatment of anthrax are still required for counteracting the threat posed by inhalation anthrax. In this study, we first demonstrated that the chimeric protein LFn-PA, created by fusing the protective antigen (PA)-binding domain of lethal factor (LFn) to PA, retained the functions of the respective molecules. On the basis of this observation, we attempted to develop an antitoxin that targets the binding of lethal factor (LF) and/or edema factor (EF)...

  9. A toxin-antitoxin module in Bacillus subtilis can both mitigate and amplify effects of lethal stress.

    Directory of Open Access Journals (Sweden)

    Xiangli Wu

    Full Text Available BACKGROUND: Bacterial type-2 (protein-protein toxin-antitoxin (TA modules are two-gene operons that are thought to participate in the response to stress. Previous work with Escherichia coli has led to a debate in which some investigators conclude that the modules protect from stress, while others argue that they amplify lethal stress and lead to programmed cell death. To avoid ambiguity arising from the presence of multiple TA modules in E. coli, the effect of the sole type-2 toxin-antitoxin module of Bacillus subtilis was examined for several types of lethal stress. METHODOLOGY/PRINCIPAL FINDINGS: Genetic knockout of the toxin gene, ndoA (ydcE, conferred protection to lethal stressors that included kanamycin, moxifloxacin, hydrogen peroxide, and UV irradiation. However, at low doses of UV irradiation the ndoA deficiency increased lethality. Indeed, gradually increasing UV dose with the ndoA mutant revealed a crossover response--from the mutant being more sensitive than wild-type cells to being less sensitive. For high temperature and nutrient starvation, the toxin deficiency rendered cells hypersensitive. The ndoA deficiency also reduced sporulation frequency, indicating a role for toxin-antitoxin modules in this developmental process. In the case of lethal antimicrobial treatment, deletion of the toxin eliminated a surge in hydrogen peroxide accumulation observed in wild-type cells. CONCLUSIONS: A single toxin-antitoxin module can mediate two opposing effects of stress, one that lowers lethality and another that raises it. Protective effects are thought to arise from toxin-mediated inhibition of translation based on published work. The enhanced, stress-mediated killing probably involves toxin-dependent accumulation of reactive oxygen species, since a deficiency in the NdoA toxin suppressed peroxide accumulation following antimicrobial treatment. The type and perhaps the level of stress appear to be important for determining whether this toxin

  10. In Vitro Characterization of the Type I Toxin-Antitoxin System bsrE/SR5 from Bacillus subtilis.

    Science.gov (United States)

    Meißner, Christin; Jahn, Natalie; Brantl, Sabine

    2016-01-01

    BsrE/SR5 is a new type I toxin/antitoxin system located on the prophage-like region P6 of the Bacillus subtilis chromosome. The bsrE gene encoding a 30-amino acid hydrophobic toxin and the antitoxin gene sr5 overlap at their 3' ends by 112 bp. Overexpression of bsrE causes cell lysis on agar plates. Here, we present a detailed in vitro analysis of bsrE/SR5. The secondary structures of SR5, bsrE mRNA, and the SR5/bsrE RNA complex were determined. Apparent binding rate constants (kapp) of wild-type and mutated SR5 species with wild-type bsrE mRNA were calculated, and SR5 regions required for efficient inhibition of bsrE mRNA narrowed down. In vivo studies confirmed the in vitro data but indicated that a so far unknown RNA binding protein might exist in B. subtilis that can promote antitoxin/toxin RNA interaction. Using time course experiments, the binding pathway of SR5 and bsrE RNA was elucidated. A comparison with the previously well characterized type I TA system from the B. subtilis chromosome, bsrG/SR4, reveals similarities but also significant differences. PMID:26565032

  11. The axe-txe complex of Enterococcus faecium presents a multilayered mode of toxin-antitoxin gene expression regulation.

    Directory of Open Access Journals (Sweden)

    Lidia Boss

    Full Text Available Multidrug-resistant variants of human pathogens from the genus Enterococcus represent a significant health threat as leading agents of nosocomial infections. The easy acquisition of plasmid-borne genes is intimately involved in the spread of antibiotic resistance in enterococci. Toxin-antitoxin (TA systems play a major role in both maintenance of mobile genetic elements that specify antibiotic resistance, and in bacterial persistence and virulence. Expression of toxin and antitoxin genes must be in balance as inappropriate levels of toxin can be dangerous to the host. The controlled production of toxin and antitoxin is usually achieved by transcriptional autoregulation of TA operons. One of the most prevalent TA modules in enterococcal species is axe-txe which is detected in a majority of clinical isolates. Here, we demonstrate that the axe-txe cassette presents a complex pattern of gene expression regulation. Axe-Txe cooperatively autorepress expression from a major promoter upstream of the cassette. However, an internal promoter that drives the production of a newly discovered transcript from within axe gene combined with a possible modulation in mRNA stability play important roles in the modulation of Axe:Txe ratio to ensure controlled release of the toxin.

  12. Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes

    Science.gov (United States)

    Abu Bakar, Fauziah; Yeo, Chew Chieng; Harikrishna, Jennifer Ann

    2016-01-01

    Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells. PMID:27104531

  13. Crystal Structures of Phd-Doc, HigA, and YeeU Establish Multiple Evolutionary Links between Microbial Growth-Regulating Toxin-Antitoxin Systems

    Energy Technology Data Exchange (ETDEWEB)

    Arbing, Mark A.; Handelman, Samuel K.; Kuzin, Alexandre P.; Verdon, Grégory; Wang, Chi; Su, Min; Rothenbacher, Francesca P.; Abashidze, Mariam; Liu, Mohan; Hurley, Jennifer M.; Xiao, Rong; Acton, Thomas; Inouye, Masayori; Montelione, Gaetano T.; Woychik, Nancy A.; Hunt, John F. (Rutgers); (Columbia); (RWJ-Med)

    2010-09-27

    Bacterial toxin-antitoxin (TA) systems serve a variety of physiological functions including regulation of cell growth and maintenance of foreign genetic elements. Sequence analyses suggest that TA families are linked by complex evolutionary relationships reflecting likely swapping of functional domains between different TA families. Our crystal structures of Phd-Doc from bacteriophage P1, the HigA antitoxin from Escherichia coli CFT073, and YeeU of the YeeUWV systems from E. coli K12 and Shigella flexneri confirm this inference and reveal additional, unanticipated structural relationships. The growth-regulating Doc toxin exhibits structural similarity to secreted virulence factors that are toxic for eukaryotic target cells. The Phd antitoxin possesses the same fold as both the YefM and NE2111 antitoxins that inhibit structurally unrelated toxins. YeeU, which has an antitoxin-like activity that represses toxin expression, is structurally similar to the ribosome-interacting toxins YoeB and RelE. These observations suggest extensive functional exchanges have occurred between TA systems during bacterial evolution.

  14. Studies of the distribution of intrathecally injected 125I-tetanus antitoxin-F(ab')2

    International Nuclear Information System (INIS)

    Overall F(ab')2 and antitetanus-f(ab')2 - fragments were labelled with 125I and injected i.th. into normal juvenile cats and adult rats. One group of rats was normal; in the other, unilateral local tetanus had been induced by injection of tetanus toxin into a M. gastrocnemius. The animals were sacrificed 24 h after the i.th. injection, and tissue samples were taken for histoautoradiography. 125I-antitetanus-F(ab')2 permeated into the extracellular space of the spinal cord, roots, and ganglia but not into the neuronal intracellular space. 125I-overall-F(ab') showed identical permeation behaviour. 125I-antitetanus-F(ab')2 reacted with tetanus toxin issuing from the motoneurons after i.th. injection, forming an immunocomplex around the motorneurons. The immunocomplex was not formed around pseudo-unipolar ganglian cells in the spinal ganglia even though some of the ganglian cells contained tetanus toxin, and 125I-antitetanus-F(ab')2 was present in the extracellular space. As an explanation, it was suggested that tetanus toxin does not permeate into the extracellular space through the membrane of the pseudo-unipolar ganglian cells so that immune reactions will not occur. These findings help to explain the widely divergent results of tetanus therapy by means of i.th. injection of tetanus antitoxin. Recommendations for future therapy measures are derived from the findings. (orig./MG)

  15. Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

    Directory of Open Access Journals (Sweden)

    M.H. Sonobe

    2007-01-01

    Full Text Available A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test. In this study, the serum titers (values between 1.0 and 19.5 IU measured by a modified TOBI test (Modi-TOBI test and toxin neutralization assays were correlated (P < 0.0001. Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001 and for diphtheria (r² = 0.93, P < 0.0001 between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

  16. A VapBC Toxin-Antitoxin Module Is a Posttranscriptional Regulator of Metabolic Flux in Mycobacteria

    OpenAIRE

    McKenzie, Joanna L.; Robson, Jennifer; Berney, Michael; Smith, Tony C.; Ruthe, Alaine; Gardner, Paul P.; Vickery L Arcus; Cook, Gregory M.

    2012-01-01

    The largest family of toxin-antitoxin (TA) modules are encoded by the vapBC operons, but their roles in bacterial physiology remain enigmatic. Microarray analysis in Mycobacterium smegmatis overexpressing VapC/VapBC revealed a high percentage of downregulated genes with annotated roles in carbon transport and metabolism, suggesting that VapC was targeting specific metabolic mRNA transcripts. To validate this hypothesis, purified VapC was used to identify the RNA cleavage site in vitro. VapC h...

  17. The interaction of the antitoxin DM43 with a snake venom metalloproteinase analyzed by mass spectrometry and surface plasmon resonance

    DEFF Research Database (Denmark)

    Brand, Guilherme D; Salbo, Rune; Jørgensen, Thomas J D;

    2012-01-01

    that, in contrast to the toxin-antitoxin complex, hydrophobic interactions are the primary driving force for the inhibitor dimerization. For the real-time interaction analysis, the toxin was captured on a sensor chip derivatized with the anti-jararhagin monoclonal antibody MAJar 2. The sensorgrams...... equilibrium dissociation constant (K(D) ) of 0.33 ± 0.06 nM. Taken together, MS and SPR results show that DM43 binds to its target toxin with high affinity and constitute the first accurate quantitative study on the extent of the interaction between a natural inhibitor and a metalloproteinase toxin, with...

  18. Structural and functional studies of the Mycobacterium tuberculosis VapBC30 toxin-antitoxin system: implications for the design of novel antimicrobial peptides.

    Science.gov (United States)

    Lee, In-Gyun; Lee, Sang Jae; Chae, Susanna; Lee, Ki-Young; Kim, Ji-Hun; Lee, Bong-Jin

    2015-09-01

    Toxin-antitoxin (TA) systems play important roles in bacterial physiology, such as multidrug tolerance, biofilm formation, and arrest of cellular growth under stress conditions. To develop novel antimicrobial agents against tuberculosis, we focused on VapBC systems, which encompass more than half of TA systems in Mycobacterium tuberculosis. Here, we report that theMycobacterium tuberculosis VapC30 toxin regulates cellular growth through both magnesium and manganese ion-dependent ribonuclease activity and is inhibited by the cognate VapB30 antitoxin. We also determined the 2.7-Å resolution crystal structure of the M. tuberculosis VapBC30 complex, which revealed a novel process of inactivation of the VapC30 toxin via swapped blocking by the VapB30 antitoxin. Our study on M. tuberculosis VapBC30 leads us to design two kinds of VapB30 and VapC30-based novel peptides which successfully disrupt the toxin-antitoxin complex and thus activate the ribonuclease activity of the VapC30 toxin. Our discovery herein possibly paves the way to treat tuberculosis for next generation. PMID:26150422

  19. Characterisation of the stbD/E toxin-antitoxin system of pEP36, a plasmid of the plant pathogen Erwinia pyrifoliae.

    Science.gov (United States)

    Unterholzner, Simon J; Hailer, Barbara; Poppenberger, Brigitte; Rozhon, Wilfried

    2013-09-01

    pEP36 is a plasmid ubiquitously present in Erwinia pyrifoliae, a pathogen which causes black stem blight of Asian pear. pEP36 is highly stable in its host, even in the absence of selective pressure. The plasmid is closely related to pEA29, which is widespread in E. amylovora, the causative agent of fire blight of apple and pear trees. Here we report that pEP36 possesses a functional hybrid toxin-antitoxin module, stbD/E(pEP36), with the toxin showing homology to the RelE/ParE proteins and the antidote belonging to the Phd/YefM antitoxin family. Bacteria expressing the StbE(pEP36) toxin arrest cell growth and enter a viable but non-culturable stage. However, they maintain their typical cell length and do not show filamentation. Pulse-chase experiments revealed that StbE(pEP36) acts as a global inhibitor of protein synthesis while it does not interfere with DNA and RNA synthesis. The StbD(pEP36) antitoxin is capable of neutralising StbE(pEP36) toxicity. Additional experiments show that the stbD/E(pEP36) module can stabilise plasmids at least 20-fold. Thus the toxin-antitoxin system may contribute to the remarkable stability of pEP36. PMID:23632277

  20. Is the mazEF toxin-antitoxin system responsible for vancomycin resistance in clinical isolates of Enterococcus faecalis?

    Directory of Open Access Journals (Sweden)

    Sadeghifard, Nourkhoda

    2014-03-01

    Full Text Available [english] The current study was conducted to investigate the relationship between vancomycin-resistant (VRE and the presence of toxin-antitoxin (TA system, which may be useful as target for novel antimicrobial therapy concepts. The susceptibility of was determined by MIC, and the presence of the TA system was evaluated by PCR. Among 200 isolates 39.5% showed resistance to vancomycin (VRE, while 60.5% were susceptible strains (VSE. The TA system was positive in all VRE isolates (100%, but less prevalent (38/121, 31.4% among the 121 VSE strains. In conclusion, our study demonstrated a positive relationship between the presence of vancomycin resistance and TA system. This observation may introduce therapeutic options against a novel antimicrobial target in enterococci.

  1. Wound Botulism in Injection Drug Users: Time to Antitoxin Correlates with Intensive Care Unit Length of Stay

    Directory of Open Access Journals (Sweden)

    Offerman, Steven R

    2009-11-01

    Full Text Available Objectives: We sought to identify factors associated with need for mechanical ventilation (MV, length of intensive care unit (ICU stay, length of hospital stay, and poor outcome in injection drug users (IDUs with wound botulism (WB.Methods: This is a retrospective review of WB patients admitted between 1991-2005. IDUs were included if they had symptoms of WB and diagnostic confirmation. Primary outcome variables were the need for MV, length of ICU stay, length of hospital stay, hospital-related complications, and death.Results: Twenty-nine patients met inclusion criteria. Twenty-two (76% admitted to heroin use only and seven (24% admitted to heroin and methamphetamine use. Chief complaints on initial presentation included visual changes, 13 (45%; weakness, nine (31%; and difficulty swallowing, seven (24%. Skin wounds were documented in 22 (76%. Twenty-one (72% patients underwent mechanical ventilation (MV. Antitoxin (AT was administered to 26 (90% patients but only two received antitoxin in the emergency department (ED. The time from ED presentation to AT administration was associated with increased length of ICU stay (Regression coefficient = 2.5; 95% CI 0.45, 4.5. The time from ED presentation to wound drainage was also associated with increased length of ICU stay (Regression coefficient = 13.7; 95% CI = 2.3, 25.2. There was no relationship between time to antibiotic administration and length of ICU stay.Conclusion: MV and prolonged ICU stays are common in patients identified with WB. Early AT administration and wound drainage are recommended as these measures may decrease ICU length of stay.[West J Emerg Med. 2009;10(4:251-256.

  2. Antiradiation Antitoxin IgG : Immunological neutralization of Radiation Toxins at Acute Radiation Syndromes.

    Science.gov (United States)

    Popov, Dmitri; Maliev, Slava

    radiation toxins to induce hyperimmune serum: Group A -Toxoid form of CV ARS toxins ( SRD-1); Group B-Toxoid form of CR ARS (SRD-2)toxins ; Group C -Toxoid form of GI ARS (SRD-3); Group D -Toxoid form of HP ARS (SRD-4). After the hyperimmune serum was pooled from several animals, purified, and concentrated, the IgG fraction was separated. Enzyme-linked immunosorbent assays of the hyper-immune serum had revealed high titers of IgG with specific binding to radi-ation toxins. The antiradiation IgG preparation was injected into laboratory animals one hour before and three hours after irradiation, and was evaluated for its ability to protect inoculated animals against the development of acute radiation syndromes. Results: Animals that were inoculated with specific antiradiation antibodies before and after receiving lethal irradiation at LD 100/30 exhibited 60-75% survival rate within 30 days. Also, these animals inoculated with the Antiradiation Antitoxin had exhibited markedly reduced clinical symptoms of the ARS, even those ones that did not survive irradiation. Discussion: The results of our experiments have demonstrated that the rabbit hyperimmune IgG preparations directed against SRD toxins provide a significant protection against high doses of radiation. In comparison, the mortality rate of irradiated control animals was 100% in the same time period. The mortality rates of animals treated by the hyperimmune IgG antidote have varied in the different groups of ani-mals and different forms of the ARS. However, significant radioprotection was observed in each group treated with the IgGs. The specific antiradiation antidote IGg isolated from hyperim-mune serum of immunized horses is under study. The specific antiradiation antidote contains antibodies to neurotoxins -SAAN IgG includes 50% IgG to Cv ARS, 25% IgG to Cr ARS and 25 % IgG to Gi ARS. The other type of the Specific antiradiation antidote containes antibodies to hematotoxins -SAAH IgG -100%. A combined variant is under

  3. Systemically Administered IgG Anti-Toxin Antibodies Protect the Colonic Mucosa during Infection with Clostridium difficile in the Piglet Model

    OpenAIRE

    Cohen, Ocean R.; Steele, Jennifer A.; Zhang, Quanshun; Schmidt, Diane J.; Wang, Yuankai; Hamel, Philip E. S.; Beamer, Gillian; Xu, Bingling; Tzipori, Saul

    2014-01-01

    The use of anti-toxin human monoclonal antibodies (HMab) as treatment for C. difficile infection has been investigated in animal models and human clinical trials as an alternative to or in combination with traditional antibiotic therapy. While HMab therapy appears to be a promising option, how systemically administered IgG antibodies protect the colonic mucosa during Clostridium difficile infection is unknown. Using the gnotobiotic piglet model of Clostridium difficile infection, we administe...

  4. Coupling between the Basic Replicon and the Kis-Kid Maintenance System of Plasmid R1: Modulation by Kis Antitoxin Levels and Involvement in Control of Plasmid Replication

    Directory of Open Access Journals (Sweden)

    Juan López-Villarejo

    2015-02-01

    Full Text Available kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now report that: (i the Kis antitoxin levels do decrease in cells containing a mini-R1 plasmid carrying a repA mutation that reduces plasmid copy number; (ii kid-dependent replication rescue is abolished in cells in which the Kis antitoxin levels or the CopB levels are increased. Unexpectedly we found that this coordination significantly increases both the copy number of the repA mutant and of the wt mini-R1 plasmid. This indicates that the coordination between plasmid replication functions and kis-kid system contributes significantly to control plasmid R1 replication.

  5. Divergently overlapping cis-encoded antisense RNA regulating toxin-antitoxin systems from E. coli: hok/sok, ldr/rdl, symE/symR.

    Science.gov (United States)

    Kawano, Mitsuoki

    2012-12-01

    Toxin-antitoxin (TA) systems are categorized into three classes based on the type of antitoxin. In type I TA systems, the antitoxin is a small antisense RNA that inhibits translation of small toxic proteins by binding to the corresponding mRNAs. Those type I TA systems were originally identified as plasmid stabilization modules rendering a post-segregational killing (PSK) effect on the host cells. The type I TA loci also exist on the Escherichia coli chromosome but their biological functions are less clear. Genetic organization and regulatory elements of hok/sok and ldr/rdl families are very similar and the toxins are predicted to contain a transmembrane domain, but otherwise share no detectable sequence similarity. This review will give an overview of the type I TA modules of E. coli K-12, especially hok/sok, ldr/rdl and SOS-inducible symE/symR systems, which are regulated by divergently overlapping cis-encoded antisense RNAs. PMID:23131729

  6. Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.

    Science.gov (United States)

    Burbank, Lindsey P; Stenger, Drake C

    2016-08-01

    The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention. PMID:27088393

  7. Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.

    Directory of Open Access Journals (Sweden)

    Alene Kast

    2015-05-01

    Full Text Available Cytoplasmic virus like elements (VLEs from Kluyveromyces lactis (Kl, Pichia acaciae (Pa and Debaryomyces robertsiae (Dr are extremely A/T-rich (>75% and encode toxic anticodon nucleases (ACNases along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5 results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle.

  8. Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.

    Science.gov (United States)

    Kast, Alene; Voges, Raphael; Schroth, Michael; Schaffrath, Raffael; Klassen, Roland; Meinhardt, Friedhelm

    2015-05-01

    Cytoplasmic virus like elements (VLEs) from Kluyveromyces lactis (Kl), Pichia acaciae (Pa) and Debaryomyces robertsiae (Dr) are extremely A/T-rich (>75%) and encode toxic anticodon nucleases (ACNases) along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5) results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE) as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A) site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle. PMID:25973601

  9. Improving the anti-toxin abilities of the CMG2-Fc fusion protein with the aid of computational design.

    Directory of Open Access Journals (Sweden)

    Yongyi Xi

    Full Text Available CMG2-Fc is a fusion protein composed of the extracellular domain of capillary morphogenesis protein 2 (CMG2 and the Fc region of human immunoglobulin G; CMG2-Fc neutralizes anthrax toxin and offers protection against Bacillus anthracis challenge. To enhance the efficacy of CMG2-Fc against anthrax toxin, we attempted to engineer a CMG2-Fc with an improved affinity for PA. Using the automatic design algorithm FoldX and visual inspection, we devised two CMG2-Fc variants that introduce mutations in the CMG2 binding interface and improve the computationally assessed binding affinity for PA. An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections. This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable. Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.

  10. VapC from the Leptospiral VapBC Toxin-Antitoxin Module Displays Ribonuclease Activity on the Initiator tRNA

    Science.gov (United States)

    Lopes, Alexandre P. Y.; Lopes, Luana M.; Fraga, Tatiana R.; Chura-Chambi, Rosa M.; Sanson, André L.; Cheng, Elisabeth; Nakajima, Erika; Morganti, Ligia; Martins, Elizabeth A. L.

    2014-01-01

    The prokaryotic ubiquitous Toxin-Antitoxin (TA) operons encode a stable toxin and an unstable antitoxin. The most accepted hypothesis of the physiological function of the TA system is the reversible cessation of cellular growth under stress conditions. The major TA family, VapBC is present in the spirochaete Leptospira interrogans. VapBC modules are classified based on the presence of a predicted ribonucleasic PIN domain in the VapC toxin. The expression of the leptospiral VapC in E. coli promotes a strong bacterial growth arrestment, making it difficult to express the recombinant protein. Nevertheless, we showed that long term induction of expression in E. coli enabled the recovery of VapC in inclusion bodies. The recombinant protein was successfully refolded by high hydrostatic pressure, providing a new method to obtain the toxin in a soluble and active form. The structural integrity of the recombinant VapB and VapC proteins was assessed by circular dichroism spectroscopy. Physical interaction between the VapC toxin and the VapB antitoxin was demonstrated in vivo and in vitro by pull down and ligand affinity blotting assays, respectively, thereby indicating the ultimate mechanism by which the activity of the toxin is regulated in bacteria. The predicted model of the leptospiral VapC structure closely matches the Shigella's VapC X-ray structure. In agreement, the ribonuclease activity of the leptospiral VapC was similar to the activity described for Shigella's VapC, as demonstrated by the cleavage of tRNAfMet and by the absence of unspecific activity towards E. coli rRNA. This finding suggests that the cleavage of the initiator transfer RNA may represent a common mechanism to a larger group of bacteria and potentially configures a mechanism of post-transcriptional regulation leading to the inhibition of global translation. PMID:25047537

  11. Potency of a human monoclonal antibody to diphtheria toxin relative to equine diphtheria anti-toxin in a guinea pig intoxication model

    Science.gov (United States)

    Smith, Heidi L.; Cheslock, Peter; Leney, Mark; Barton, Bruce; Molrine, Deborah C.

    2016-01-01

    ABSTRACT Prompt administration of anti-toxin reduces mortality following Corynebacterium diphtheriae infection. Current treatment relies upon equine diphtheria anti-toxin (DAT), with a 10% risk of serum sickness and rarely anaphylaxis. The global DAT supply is extremely limited; most manufacturers have ceased production. S315 is a neutralizing human IgG1 monoclonal antibody to diphtheria toxin that may provide a safe and effective alternative to equine DAT and address critical supply issues. To guide dose selection for IND-enabling pharmacology and toxicology studies, we dose-ranged S315 and DAT in a guinea pig model of diphtheria intoxication based on the NIH Minimum Requirements potency assay. Animals received a single injection of antibody premixed with toxin, were monitored for 30 days, and assigned a numeric score for clinical signs of disease. Animals receiving ≥ 27.5 µg of S315 or ≥ 1.75 IU of DAT survived whereas animals receiving ≤ 22.5 µg of S315 or ≤ 1.25 IU of DAT died, yielding a potency estimate of 17 µg S315/IU DAT (95% CI 16–21) for an endpoint of survival. Because some surviving animals exhibited transient limb weakness, likely a systemic sign of toxicity, DAT and S315 doses required to prevent hind limb paralysis were also determined, yielding a relative potency of 48 µg/IU (95% CI 38–59) for this alternate endpoint. To support advancement of S315 into clinical trials, potency estimates will be used to evaluate the efficacy of S315 versus DAT in an animal model with antibody administration after toxin exposure, more closely modeling anti-toxin therapy in humans. PMID:27070129

  12. Potency of a human monoclonal antibody to diphtheria toxin relative to equine diphtheria anti-toxin in a guinea pig intoxication model.

    Science.gov (United States)

    Smith, Heidi L; Cheslock, Peter; Leney, Mark; Barton, Bruce; Molrine, Deborah C

    2016-08-17

    Prompt administration of anti-toxin reduces mortality following Corynebacterium diphtheriae infection. Current treatment relies upon equine diphtheria anti-toxin (DAT), with a 10% risk of serum sickness and rarely anaphylaxis. The global DAT supply is extremely limited; most manufacturers have ceased production. S315 is a neutralizing human IgG1 monoclonal antibody to diphtheria toxin that may provide a safe and effective alternative to equine DAT and address critical supply issues. To guide dose selection for IND-enabling pharmacology and toxicology studies, we dose-ranged S315 and DAT in a guinea pig model of diphtheria intoxication based on the NIH Minimum Requirements potency assay. Animals received a single injection of antibody premixed with toxin, were monitored for 30 days, and assigned a numeric score for clinical signs of disease. Animals receiving ≥ 27.5 µg of S315 or ≥ 1.75 IU of DAT survived whereas animals receiving ≤ 22.5 µg of S315 or ≤ 1.25 IU of DAT died, yielding a potency estimate of 17 µg S315/IU DAT (95% CI 16-21) for an endpoint of survival. Because some surviving animals exhibited transient limb weakness, likely a systemic sign of toxicity, DAT and S315 doses required to prevent hind limb paralysis were also determined, yielding a relative potency of 48 µg/IU (95% CI 38-59) for this alternate endpoint. To support advancement of S315 into clinical trials, potency estimates will be used to evaluate the efficacy of S315 versus DAT in an animal model with antibody administration after toxin exposure, more closely modeling anti-toxin therapy in humans. PMID:27070129

  13. Coupling between the Basic Replicon and the Kis-Kid Maintenance System of Plasmid R1: Modulation by Kis Antitoxin Levels and Involvement in Control of Plasmid Replication

    OpenAIRE

    Juan López-Villarejo; Damián Lobato-Márquez; Ramón Díaz-Orejas

    2015-01-01

    kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now repo...

  14. Structural and thermodynamic characterization of the Escherichia coli RelBE toxin-antitoxin system: indication for a functional role of differential stability

    DEFF Research Database (Denmark)

    Cherny, Izhack; Overgaard, Martin; Borch, Jonas;

    2007-01-01

    on the folding and stability of the protein pair in solution. Here we structurally and thermodynamically characterize the RelBE system components from E. coli in solution, both separately and in their complexed state. The RelB antitoxin, an alpha-helical protein according to circular dichroism and...... of 52.5 degrees C, and exhibits exceptional sensitivity to heat. Complex formation, accompanied by a structural transition, leads to a 12 degrees C increase in the TM and substantial heat resistance. Moreover, in vivo interaction and protein footprint experiments indicate that the C-terminal part of...

  15. A VapBC toxin-antitoxin module is a posttranscriptional regulator of metabolic flux in mycobacteria.

    Science.gov (United States)

    McKenzie, Joanna L; Robson, Jennifer; Berney, Michael; Smith, Tony C; Ruthe, Alaine; Gardner, Paul P; Arcus, Vickery L; Cook, Gregory M

    2012-05-01

    The largest family of toxin-antitoxin (TA) modules are encoded by the vapBC operons, but their roles in bacterial physiology remain enigmatic. Microarray analysis in Mycobacterium smegmatis overexpressing VapC/VapBC revealed a high percentage of downregulated genes with annotated roles in carbon transport and metabolism, suggesting that VapC was targeting specific metabolic mRNA transcripts. To validate this hypothesis, purified VapC was used to identify the RNA cleavage site in vitro. VapC had RNase activity that was sequence specific, cleaving single-stranded RNA substrates at AUAU and AUAA in vitro and in vivo (viz., MSMEG_2121 to MSMEG_2124). A bioinformatic analysis of these regions suggested that an RNA hairpin 3' of the AUA(U/A) motif is also required for efficient cleavage. VapC-mediated regulation in vivo was demonstrated by showing that MSMEG_2124 (dhaF) and MSMEG_2121 (dhaM) were upregulated in a ΔvapBC mutant growing on glycerol. The ΔvapBC mutant had a specific rate of glycerol consumption that was 2.4-fold higher than that of the wild type during exponential growth. This increased rate of glycerol consumption was not used for generating bacterial biomass, suggesting that metabolism by the ΔvapBC mutant was uncoupled from growth. These data suggest a model in which VapC regulates the rate of glycerol utilization to match the anabolic demands of the cell, allowing for fine-tuning of the catabolic rate at a posttranscriptional level. PMID:22366418

  16. Three dimensional structure of the MqsR:MqsA complex: a novel TA pair comprised of a toxin homologous to RelE and an antitoxin with unique properties.

    Directory of Open Access Journals (Sweden)

    Breann L Brown

    2009-12-01

    Full Text Available One mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the Escherichia coli gene mqsR (b3022 was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021, which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA pair. MqsR adopts an alpha/beta fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology (e.g., mcbR, spy. Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state.

  17. Three dimensional structure of the MqsR:MqsA complex: a novel TA pair comprised of a toxin homologous to RelE and an antitoxin with unique properties.

    Science.gov (United States)

    Brown, Breann L; Grigoriu, Simina; Kim, Younghoon; Arruda, Jennifer M; Davenport, Andrew; Wood, Thomas K; Peti, Wolfgang; Page, Rebecca

    2009-12-01

    One mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the Escherichia coli gene mqsR (b3022) was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021), which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA) pair. MqsR adopts an alpha/beta fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology (e.g., mcbR, spy). Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state. PMID:20041169

  18. Three Dimensional Structure of the MqsR:MqsA Complex: a Novel TA Pair Comprised of a Toxin Homologous to RelE and an Antitoxin with Unique Properties

    Energy Technology Data Exchange (ETDEWEB)

    Brown, B.; Grigoriu, S; Kim, Y; Arruda, J; Davenport, A; wood, T; Peti, W; Page, R

    2009-01-01

    One mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the Escherichia coli gene mqsR (b3022) was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021), which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA) pair. MqsR adopts an alpha/beta fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology (e.g., mcbR, spy). Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state.

  19. Toxin-antitoxin loci as stress-response-elements: ChpAK/MazF and ChpBK cleave translated RNAs and are counteracted by tmRNA

    DEFF Research Database (Denmark)

    Christensen, S.K.; Pedersen, K.; Hansen, Flemming G.;

    2003-01-01

    Prokaryotic chromosomes encode toxin-antitoxin loci, often in multiple copies. In most cases, the function of these genes is not known. The chpA (mazEF) locus of Escherichia coli has been described as a cell killing module that induces bacterial apoptosis during nutritional stress. However, we...... found recently that ChpAK (MazF) does not confer cell killing but rather, induces a bacteriostatic condition from which the cells could be resuscitated. Results presented here yield a mechanistic explanation for the detrimental effect on cell growth exerted by ChpAK and the homologous ChpBK protein of E....... coli. We show that both proteins inhibit translation by inducing cleavage of translated mRNAs. Consistently, the inhibitory effect of the proteins was counteracted by tmRNA. Amino acid starvation induced strong transcription of chpA that depended on Lon protease but not on ppGpp. Simultaneously, Chp...

  20. VapC toxins from Mycobacterium tuberculosis are ribonucleases that differentially inhibit growth and are neutralized by cognate VapB antitoxins.

    Directory of Open Access Journals (Sweden)

    Bintou Ahmadou Ahidjo

    Full Text Available The chromosome of Mycobacterium tuberculosis (Mtb encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as 'non-toxic'. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of 'non-toxic' VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase activity demonstrated for Rv0065 and Rv0617--VapC proteins with similarity to Rv0549c and Rv3320c, respectively--these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism.

  1. Systemically administered IgG anti-toxin antibodies protect the colonic mucosa during infection with Clostridium difficile in the piglet model.

    Science.gov (United States)

    Cohen, Ocean R; Steele, Jennifer A; Zhang, Quanshun; Schmidt, Diane J; Wang, Yuankai; Hamel, Philip E S; Beamer, Gillian; Xu, Bingling; Tzipori, Saul

    2014-01-01

    The use of anti-toxin human monoclonal antibodies (HMab) as treatment for C. difficile infection has been investigated in animal models and human clinical trials as an alternative to or in combination with traditional antibiotic therapy. While HMab therapy appears to be a promising option, how systemically administered IgG antibodies protect the colonic mucosa during Clostridium difficile infection is unknown. Using the gnotobiotic piglet model of Clostridium difficile infection, we administered a mixture of anti-TcdA and anti-TcdB HMabs systemically to piglets infected with either pathogenic or non-pathogenic C. difficile strains. The HMabs were present throughout the small and large intestinal tissue of both groups, but significant HMabs were present in the lumen of the large intestines only in the pathogenic strain-infected group. Similarly, HMabs measured in the large intestine over a period of 2-4 days following antibody administration were not significantly different over time in the gut mucosa among the groups, but concentrations in the lumen of the large intestine were again consistently higher in the pathogenic strain-infected group. These results indicate that systemically administered HMab IgG reaches the gut mucosa during the course of CDI, protecting the host against systemic intoxication, and that leakage through the damaged colon likely protects the mucosa from further damage, allowing initiation of repair and recovery. PMID:25347821

  2. A homogeneous fluorescence polarization assay for detection of Clostridium perfringenstype D epsilon antitoxin in serum of goats Un ensayo de fluorescencia polarizada homogéneo para detectar anticuerpos contra toxina epsilon de Clostridium perfringenstipo D en suero de cabras

    Directory of Open Access Journals (Sweden)

    F.A. UZAL

    1999-01-01

    Full Text Available A fluorescence polarization assay (FPA was developed to detect Clostridium perfringens type D epsilon antitoxin in serum of goats. Purified epsilon toxin was labelled with fluorescein isothiocyanate and used as a tracer in the test. Seven goat sera with different known concentrations of epsilon antitoxin (as measured by a mouse neutralization test-MNT were used as positive controls. Sera from eleven colostrum-deprived kids were used as negative controls. The inter-assay variation of ten measurements of the seven positive sera varied between 1.56% and 4.85%. The correlation between the FPA values and concentration of epsilon antitoxin (as measured by the MNT was 0.90. This test appears to be a good alternative to the MNT without most of the inconveniences of the laterSe desarrolló un test de fluorescencia polarizada (TFP para detectar antitoxina epsilon de Clostridium perfringens tipo D en suero de cabras. Se marcó toxina epsilon purificada con isothiocianato de fluoresceína y se la usó como tracer en el test. Siete sueros caprinos con diferentes concentraciones conocidas de antitoxina epsilon (titulados por una prueba de seroneutralización en ratones-PSR se utilizaron como controles positivos. Sueros de once chivitos deprivados de calostro se utilizaron como controles negativos. La variación entre ensayos de diez mediciones de los siete sueros positivos control varió entre 1.56% y 4.85%. La correlación entre los valores de TFP y la concentración de antitoxina epsilon (medida por PSR fue de 0.90. Este test parece ser una buena alternativa para la PSR sin los inconvenientes de esta última

  3. AvrRxo1 Is a Bifunctional Type III Secreted Effector and Toxin-Antitoxin System Component with Homologs in Diverse Environmental Contexts.

    Science.gov (United States)

    Triplett, Lindsay R; Shidore, Teja; Long, John; Miao, Jiamin; Wu, Shuchi; Han, Qian; Zhou, Changhe; Ishihara, Hiromichi; Li, Jianyong; Zhao, Bingyu; Leach, Jan E

    2016-01-01

    Toxin-antitoxin (TA) systems are ubiquitous bacterial systems that may function in genome maintenance and metabolic stress management, but are also thought to play a role in virulence by helping pathogens survive stress. We previously demonstrated that the Xanthomonas oryzae pv. oryzicola protein AvrRxo1 is a type III-secreted virulence factor that has structural similarities to the zeta family of TA toxins, and is toxic to plants and bacteria in the absence of its predicted chaperone Arc1. In this work, we confirm that AvrRxo1 and its binding partner Arc1 function as a TA system when expressed in Escherichia coli. Sequences of avrRxo1 homologs were culled from published and newly generated phytopathogen genomes, revealing that avrRxo1:arc1 modules are rare or frequently inactivated in some species and highly conserved in others. Cloning and functional analysis of avrRxo1 from Acidovorax avenae, A. citrulli, Burkholderia andropogonis, Xanthomonas translucens, and Xanthomonas euvesicatoria showed that some AvrRxo1 homologs share the bacteriostatic and Rxo1-mediated cell death triggering activities of AvrRxo1 from X. oryzae. Additional distant putative homologs of avrRxo1 and arc1 were identified in genomic or metagenomic sequence of environmental bacteria with no known pathogenic role. One of these distant homologs was cloned from the filamentous soil bacterium Cystobacter fuscus. avrRxo1 from C. fuscus caused watersoaking and triggered Rxo1-dependent cell collapse in Nicotiana benthamiana, but no growth suppression in E. coli was observed. This work confirms that a type III effector can function as a TA system toxin, and illustrates the potential of microbiome data to reveal new environmental origins or reservoirs of pathogen virulence factors. PMID:27391081

  4. The relBE2Spn toxin-antitoxin system of Streptococcus pneumoniae: role in antibiotic tolerance and functional conservation in clinical isolates.

    Directory of Open Access Journals (Sweden)

    Concha Nieto

    Full Text Available Type II (proteic chromosomal toxin-antitoxin systems (TAS are widespread in Bacteria and Archaea but their precise function is known only for a limited number of them. Out of the many TAS described, the relBE family is one of the most abundant, being present in the three first sequenced strains of Streptococcus pneumoniae (D39, TIGR4 and R6. To address the function of the pneumococcal relBE2Spn TAS in the bacterial physiology, we have compared the response of the R6-relBE2Spn wild type strain with that of an isogenic derivative, Delta relB2Spn under different stress conditions such as carbon and amino acid starvation and antibiotic exposure. Differences on viability between the wild type and mutant strains were found only when treatment directly impaired protein synthesis. As a criterion for the permanence of this locus in a variety of clinical strains, we checked whether the relBE2Spn locus was conserved in around 100 pneumococcal strains, including clinical isolates and strains with known genomes. All strains, although having various types of polymorphisms at the vicinity of the TA region, contained a functional relBE2Spn locus and the type of its structure correlated with the multilocus sequence type. Functionality of this TAS was maintained even in cases where severe rearrangements around the relBE2Spn region were found. We conclude that even though the relBE2Spn TAS is not essential for pneumococcus, it may provide additional advantages to the bacteria for colonization and/or infection.

  5. AvrRxo1 Is a Bifunctional Type III Secreted Effector and Toxin-Antitoxin System Component with Homologs in Diverse Environmental Contexts

    Science.gov (United States)

    Triplett, Lindsay R.; Shidore, Teja; Long, John; Miao, Jiamin; Wu, Shuchi; Han, Qian; Zhou, Changhe; Ishihara, Hiromichi; Li, Jianyong; Zhao, Bingyu; Leach, Jan E.

    2016-01-01

    Toxin-antitoxin (TA) systems are ubiquitous bacterial systems that may function in genome maintenance and metabolic stress management, but are also thought to play a role in virulence by helping pathogens survive stress. We previously demonstrated that the Xanthomonas oryzae pv. oryzicola protein AvrRxo1 is a type III-secreted virulence factor that has structural similarities to the zeta family of TA toxins, and is toxic to plants and bacteria in the absence of its predicted chaperone Arc1. In this work, we confirm that AvrRxo1 and its binding partner Arc1 function as a TA system when expressed in Escherichia coli. Sequences of avrRxo1 homologs were culled from published and newly generated phytopathogen genomes, revealing that avrRxo1:arc1 modules are rare or frequently inactivated in some species and highly conserved in others. Cloning and functional analysis of avrRxo1 from Acidovorax avenae, A. citrulli, Burkholderia andropogonis, Xanthomonas translucens, and Xanthomonas euvesicatoria showed that some AvrRxo1 homologs share the bacteriostatic and Rxo1-mediated cell death triggering activities of AvrRxo1 from X. oryzae. Additional distant putative homologs of avrRxo1 and arc1 were identified in genomic or metagenomic sequence of environmental bacteria with no known pathogenic role. One of these distant homologs was cloned from the filamentous soil bacterium Cystobacter fuscus. avrRxo1 from C. fuscus caused watersoaking and triggered Rxo1-dependent cell collapse in Nicotiana benthamiana, but no growth suppression in E. coli was observed. This work confirms that a type III effector can function as a TA system toxin, and illustrates the potential of microbiome data to reveal new environmental origins or reservoirs of pathogen virulence factors. PMID:27391081

  6. Novel Clostridium difficile Anti-Toxin (TcdA and TcdB) Humanized Monoclonal Antibodies Demonstrate In Vitro Neutralization across a Broad Spectrum of Clinical Strains and In Vivo Potency in a Hamster Spore Challenge Model

    Science.gov (United States)

    Qiu, Hongyu; Cassan, Robyn; Johnstone, Darrell; Han, Xiaobing; Joyee, Antony George; McQuoid, Monica; Masi, Andrea; Merluza, John; Hrehorak, Bryce; Reid, Ross; Kennedy, Kieron; Tighe, Bonnie; Rak, Carla; Leonhardt, Melanie; Dupas, Brian; Saward, Laura; Berry, Jody D.; Nykiforuk, Cory L.

    2016-01-01

    Clostridium difficile (C. difficile) infection (CDI) is the main cause of nosocomial antibiotic-associated colitis and increased incidence of community-associated diarrhea in industrialized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA), and cytotoxin, toxin B (TcdB), which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin antibodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti-TcdB monoclonal antibodies were generated and their protective potency was characterized in a hamster infection model. The humanized anti-TcdA (CANmAbA4) and anti-TcdB (CANmAbB4 and CANmAbB1) antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail) provided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively) in a hamster gastrointestinal infection (GI) model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonal antibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI. PMID:27336843

  7. Novel Clostridium difficile Anti-Toxin (TcdA and TcdB Humanized Monoclonal Antibodies Demonstrate In Vitro Neutralization across a Broad Spectrum of Clinical Strains and In Vivo Potency in a Hamster Spore Challenge Model.

    Directory of Open Access Journals (Sweden)

    Hongyu Qiu

    Full Text Available Clostridium difficile (C. difficile infection (CDI is the main cause of nosocomial antibiotic-associated colitis and increased incidence of community-associated diarrhea in industrialized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA, and cytotoxin, toxin B (TcdB, which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin antibodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti-TcdB monoclonal antibodies were generated and their protective potency was characterized in a hamster infection model. The humanized anti-TcdA (CANmAbA4 and anti-TcdB (CANmAbB4 and CANmAbB1 antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail provided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively in a hamster gastrointestinal infection (GI model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonal antibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI.

  8. Síntese, caracterização e estudos de interação de um análogo da antitoxina CcdA empregando fluorescência no estado estacionário Synthesis, characterization and interaction studies of an analog of CcdA antitoxin by steady state fluorescence

    Directory of Open Access Journals (Sweden)

    Camila Aparecida Cotrim

    2010-01-01

    Full Text Available Toxin-antitoxin (TA systems contribute to plasmid stability by a mechanism called post-segregational killing. The ccd was the first TA system to be discovered with CcdB being the toxin and CcdA the antitoxin. CcdA, an 8.3 kDa protein, interacts with CcdB (11.7 kDa, preventing the cytotoxic activity of CcdB on the DNA gyrase. As an approach to understanding this interaction, CcdA41, a polypeptide derived from CcdA, was synthesized by solid-phase methodology and its interaction with CcdB was analyzed by steady state fluorescence. CcdA41 formed a stable complex with CcdBET2, a peptide based on CcdB, the more recently described bacterial topoisomerase inhibitor.

  9. 高致病性2型猪链球菌毒素-抗毒素系统SezAT的鉴定与活性研究%Identification and activity assay of the SezAT toxin-antitoxin system of highly pathogenic Streptococcus suis serotype 2

    Institute of Scientific and Technical Information of China (English)

    王敏; 李明; 钟秋; 赵岩; 饶贤才; 黎庶; 谭银玲; 胡福泉

    2012-01-01

    [目的]对我国高致病性2型猪链球菌05ZYH33基因组的89K毒力岛序列进行生物信息学分析,发现存在一对与化脓链球菌Epsilon-zeta(ε-ζ)同源的Ⅱ型毒素-抗毒素系统(Toxin-antitoxin system,TA)-SezAT,推测该系统具有稳定89K毒力岛使其不易丢失的作用.验证SezAT为有活性的TA系统.[方法]对SezAT进行了生物信息学分析;RT-PCR验证SezAT共转录特性;在大肠杆菌中选择性地诱导表达毒素蛋白SezT和抗毒素蛋白SezA;最后通过同源重组技术敲除SezAT系统.[结果]sezAT由同一操纵子控制,SezT可抑制细菌生长,SezA可中和SezT的毒性作用,同源重组成功获得sezT敲除突变株.[结论]证实SezAT为一对有活性的毒素-抗毒素(TA)系统,为进一步研究SezAT可能发挥稳定89K毒力岛的功能,同时获得89K毒力岛缺失突变株并深入认识89K在我国高致病性SS2中的作用奠定了基础.%[Objective] Bioinformatics analysis revealed that the 89K pathogenicity island(PAI) of highly pathogenic Streptococcus suis serotype 2 (SS2) in China encodes a putative type II toxin-antitoxin (TA) system named SezAT, which is homologous to the epsilon-zeta system from S. Pyogenes. SezAT is presumed to be requisite for the stability of the 89K PAI in SS2. To confirm the SezAT system is a functional TA system. [Methods] The sequence characteristics of SezAT were subjected to further bioinformatics analysis. RT-PCR was performed to analyze the transcriptions of the sezAT locus and the flanking genes. The SezT toxin and SezA antitoxin proteins were selectively overexpressed in Escherichia coli. Deletion mutagenesis was carried out to obtain a SezAT-deficient mutant. [Results] Bioinformatics analysis and RT-PCR results suggest that sezAT are in the same operon. Overexpression of SezT led to severe growth inhibition of the host bacteria, while this toxicity was counteracted by the expression of SezA. Finally, the toxin-encoding gene sezT was successfully

  10. 9 CFR 113.451 - Tetanus Antitoxin.

    Science.gov (United States)

    2010-01-01

    ... injected subcutaneously into a 340 to 380 gram guinea pig, results in death of that guinea pig within 60 to... shall be held at 20° to 25 ° C for 1 hour before injections of guinea pigs are made. (4) A sample from... dating and 20 percent overage for 3 year dating. (5) Normal guinea pigs weighing within a range of 340...

  11. 结核分枝杆菌毒素-抗毒素系统 maz EF6缺失突变株的构建及其表型的初步探讨%Construction and characterization of toxin-antitoxin system mazEF6 deletion in Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    申爱平; 曹帅丽; 邢建新; 袁俐

    2015-01-01

    To study the toxin‐antitoxin system mazEF6 of Mycobacterium tuberculosis (M . tuberculosis) , deletion mutants were constructed and subjected to phenotype analysis .First ,the flanking (homology arms) of mazEF6 gene from H37Rv and kanamycin resistance gene ( kan gene ) from plasmid PUC‐19K were amplified by polymerase chain reaction (PCR) respectively .Second ,fusion PCR was used for the hybrid splicing of mazEF6 homology arms and kan gene ,and the desired fusion fragment was obtained .Then the fragment was cloned into pMD‐19T (simple) vector to form a suicide plasmid (pMD‐19T‐ΔmazEF6‐kan) , and the suicide plasmid was transformed into Escherichia coli (E . coli) DH5α.At last ,the constructed plasmid was transformed into H37Rv by electroporation .Single colonies of M . tuberculosis were screened on L‐J medium with kanamycin , the genomic DNA of positive strains were extracted , and the targeted fragments were amplified by PCR and sequenced .The genetic stability and other phenotypes of the H 37RvΔmazEF6 deletion mutant were studied .The results showed that the deletion mutant strains did not present reverse mutant within 15 generations . Compared with the wild‐type strains , H37Rv ΔmazEF6 deletion mutant strains grew more slowly and the bacterial cell was relatively shorter .This study demonstrated that it is practical to obtain M . tuberculosis deletion mutant by fusion PCR technology ,and the survival ability of M . tuberculosis without toxin‐antitoxin mazEF6 gene is decreased .%为构建结核分枝杆菌毒素‐抗毒素系统 m azEF6缺失突变株,并对其表型进行初步探讨,首先用聚合酶链反应(PCR)分别从H37Rv标准株和PUC‐19K质粒扩增出 mazEF6基因的同源臂及卡那霉素抗性基因kan ;然后应用融合PCR技术将 mazEF6基因的同源臂与 kan基因进行杂交拼接,获得目的融合片段,将该融合片段克隆于pMD‐19T(simple)载体形成自杀质粒pMD‐19T‐ΔmazEF6

  12. Proteção do recém-nascido contra o tétano pela imunização ativa da gestante com antitoxina tetânica: estudo original de 1953 Protection of newborn infants against tetanus by active immunization of the pregnant women with tetanus antitoxin: the 1953 original study

    Directory of Open Access Journals (Sweden)

    Augusto Gomes Mattos

    2008-12-01

    Full Text Available OBJETIVO: Determinar, em cobaias prenhes e em gestantes, a produção de antitoxina tetânica induzida pela aplicação da anatoxina tetânica e estudar a sua passagem para o recém-nascido. MÉTODOS: Na primeira fase, em estudo experimental, cobaias prenhes foram vacinadas com duas doses de toxóide tetânico em um intervalo de 15 dias, seguida da dosagem de anticorpos na cobaia imunizada, na prole ao nascer e 15 dias após o nascimento. Outro grupo de animais previamente vacinado recebeu uma dose de reforço 30 dias antes do parto, medindo-se o nível de anticorpos na cobaia e na prole. Na segunda fase, em ensaio clínico, as gestantes humanas foram vacinadas com três injeções de anatoxina tetânica, com um intervalo de 30 dias, em qualquer período da gravidez, medindo-se, a seguir, a antitoxina tetânica. Nos recém-nascidos, os anticorpos foram medidos ao nascer e aos 15 dias de vida. RESULTADOS: O título de antitoxina no sangue da prole de cobaias vacinadas com anatoxina tetânica foi elevado ao nascimento e aos 15 dias de vida. A dose de reforço provocou elevação do título basal. Nas gestantes, a aplicação de três doses de toxóide antitetânico conferiu imunidade a 95% dos recém-nascidos estudados. Os recém-nascidos de mães vacinadas apresentaram títulos elevados de antitoxina que persistiram por mais de 15 dias de vida. CONCLUSÕES: A vacinação durante a gestação foi acompanhada de títulos protetores de antitoxina contra o tétano tanto nos filhotes de cobaias quanto nos recém-nascidos humanos.OBJECTIVE: To measure, in pregnant guinea pigs and women, the production of tetanus antitoxin, induced by vaccination with tetanus toxin, and to study the transmission of these antibodies to the offspring. METHODS: In an experimental design, pregnant guinea pigs were vaccinated with two doses of tetanus toxoid with a 15-day interval followed by determination of antibodies in the immunized guinea pig, in the offspring at birth

  13. Circulating antitoxin in rabbits after ingestion of diphtheria toxoid.

    OpenAIRE

    Peri, B A; Rothberg, R M

    1981-01-01

    Immune responses following antigen ingestion vary from stimulation to suppression depending on animal species, antigen, and experimental protocol. Young adult rabbits were given either 0.02% diphtheria toxoid or 0.1% bovine serum albumin in drinking water for 10-day periods, a protocol previously found to immunize human infants fed bovine serum albumin. Specific serum antibody was detected by radioimmunoassay in 10 of 13 rabbits fed diphtheria toxoid for 10 days and 11 of 13 rabbits fed bovin...

  14. Drug: D05187 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D05187 Drug Gas gangrene antitoxin, equine (JP16); Gas gangrene antitoxin, pentaval...ent; Freeze-dried gas gangrene antitoxin, equine (TN) Therapeutic category: 6331 Therapeutic category of dru...Antitoxins, anti-leptospiral sera 6331 Antitoxins D05187 Gas gangrene antitoxin, equine (JP16) PubChem: 17398252 ...

  15. Anti-toxin antibodies in prophylaxis and treatment of inhalation anthrax

    OpenAIRE

    Schneemann, Anette; Manchester, Marianne

    2009-01-01

    The CDC recommend 60 days of oral antibiotics combined with a three-dose series of the anthrax vaccine for prophylaxis after potential exposure to aerosolized Bacillus anthracis spores. The anthrax vaccine is currently not licensed for anthrax postexposure prophylaxis and has to be made available under an Investigational New Drug protocol. Postexposure prophylaxis based on antibiotics can be problematic in cases where the use of antibiotics is contraindicated. Furthermore, there is a concern ...

  16. A Viral Nanoparticle with Dual Function as an Anthrax Antitoxin and Vaccine

    OpenAIRE

    Darly J Manayani; Diane Thomas; Dryden, Kelly A; Vijay Reddy; Marc E Siladi; Marlett, John M; Rainey, G. Jonah A.; Pique, Michael E.; Heather M Scobie; Mark Yeager; Young, John A. T.; Marianne Manchester; Anette Schneemann

    2007-01-01

    Author Summary Anthrax is caused by the spore-forming, Gram-positive bacterium Bacillus anthracis. The toxic effects of B. anthracis are predominantly due to an AB-type toxin made up of the receptor-binding subunit protective antigen (PA) and two enzymatic subunits called lethal factor and edema factor. Protective immunity to B. anthracis infection is conferred by antibodies against PA, which is the primary component of the current anthrax vaccine. Although the vaccine is safe and effective, ...

  17. The origin of the production of diphtheria antitoxin in France, between philanthropy and commerce

    OpenAIRE

    Simon, Jonathan

    2007-01-01

    Serotherapy for the treatment of diphtheria represented a major therapeutic innovation at the end of the nineteenth century. The manner in which large-scale production of this medicament was undertaken and the regulations that governed its production and distribution were important elements of public health policy in France as in other European countries. This paper describes the dominance of the Pasteur Institute in this field and, starting from this observation, explores what this event in ...

  18. Rapid Curtailing of the Stringent Response by Toxin-Antitoxin Encoded mRNases

    DEFF Research Database (Denmark)

    Tian, Chengzhe; Roghanian, Mohammad; Jørgensen, Mikkel Girke;

    2016-01-01

    Escherichia coli regulates its metabolism to adapt to changes in the environment, in particular to stressful downshifts in nutrient quality. Such shifts elicit the so-called stringent response coordinated by the alarmone guanosine tetra- and pentaphosphate [(p)ppGpp]. At sudden amino-acid (aa) st...... short time scale, and the activated mRNases negatively affects the (p)ppGpp level. The proposed mathematical model of (p)ppGpp regulation through mRNA level highlights the importance of several feedback loops in the early (p)ppGpp regulation.......Escherichia coli regulates its metabolism to adapt to changes in the environment, in particular to stressful downshifts in nutrient quality. Such shifts elicit the so-called stringent response coordinated by the alarmone guanosine tetra- and pentaphosphate [(p)ppGpp]. At sudden amino-acid (aa......) starvation, RelA [(p)ppGpp synthetase I] activity is stimulated by binding of uncharged tRNAs to a vacant ribosomal site; the (p)ppGpp level increases dramatically and peaks within the time scale of a few minutes. The decrease of (p)ppGpp level after the peak is mediated by the decreased production of m...

  19. Functional Analysis of the Role of Toxin-Antitoxin (TA) Loci in Bacterial Persistence.

    Science.gov (United States)

    Butt, Aaron T; Titball, Richard W

    2016-01-01

    We have developed a method to analyze the functionality of putative TA loci by expressing them in Escherichia coli. Here, we describe the procedure for cloning recombinant TA genes into inducible plasmids and expressing these in E. coli. Following expression, toxicity, resuscitation of growth, and changes in persister cell formation are assayed. This can confirm whether predicted TA loci are active in E. coli and whether expression can affect persister cell formation. PMID:26468105

  20. Overview of Antibacterial, Antitoxin, Antiviral, and Antifungal Activities of Tea Flavonoids and Teas

    Science.gov (United States)

    Tea leaves produce secondary metabolites, organic compounds that are involved in the defense of the plants against invading pathogens including insects, bacteria, fungi, and viruses. These metabolites include polyphenolic compounds, the six so-called catechins, and the methyl-xanthine alkaloids caf...

  1. Drug: D03925 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D03925 Drug Freeze-dried diphtheria antitoxin, equine (JP16); Freeze-dried diphther...iphtheria antitoxin D03925 Freeze-dried diphtheria antitoxin, equine (JP16) PubChem: 47205914 ...

  2. Plasmid vectors for Xylella fastidiosa utilizing a toxin-antitoxin system for plasmid stability in the absence of antibiotic selection

    Science.gov (United States)

    The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacte...

  3. Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

    OpenAIRE

    Goldman Ellen R; Anderson George P; Liu Jinny L

    2007-01-01

    Abstract Background Shark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct a...

  4. Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

    Directory of Open Access Journals (Sweden)

    Goldman Ellen R

    2007-11-01

    Full Text Available Abstract Background Shark heavy chain antibody, also called new antigen receptor (NAR, consists of one single Variable domain (VH, containing only two complementarity-determining regions (CDRs. The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. Results A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB, ricin, and botulinum toxin A (BoNT/A complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. Conclusion We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.

  5. Liposomes loaded with P. falciparum merozoite-derived proteins are highly immunogenic and produce invasion-inhibiting and anti-toxin antibodies.

    Science.gov (United States)

    Fotoran, Wesley L; Santangelo, Rachele M; Medeiros, Márcia M; Colhone, Marcelle; Ciancaglini, Pietro; Barboza, Renato; Marinho, Claudio Romero Farias; Stábeli, Rodrigo Guerino; Wunderlich, Gerhard

    2015-11-10

    The formulation of an effective vaccine against malaria is still a significant challenge and the induction of high anti-parasite antibody titers plus a sustained T cell response is mandatory for the success of such a vaccine. We have developed a nanoliposome-based structure which contains plasma membrane-associated proteins (PfMNP) of Plasmodium falciparum merozoites on its surface. Incorporation of parasite-derived proteins led to a significant increase in the size and dispersity of particles. Immunization of particles in BalbC and C57BL/6 mice led to high anti-MSP119 IgG titers (10(4)) after the first dose and reached a plateau (>10(6)) after the third dose. While very high titers were observed against the C-terminal domain of the vaccine candidate MSP1, only modest titers (≤10(3)) were detected against MSP2. The induced antibodies showed also a strong growth-inhibiting effect in reinvasion assays. In addition, PfMNP immunization generated antibodies which partially blocked the inflammatory response, probably by blocking TLR-induced activation of macrophages by malarial toxins such as GPI anchors. The results underline the potential of nanoliposome-based formulations as anti-malarial vaccines. PMID:26334481

  6. Crystal Structure of the VapBC Toxin–Antitoxin Complex from Shigella flexneri Reveals a Hetero-Octameric DNA-Binding Assembly

    DEFF Research Database (Denmark)

    Dienemann, Christian; Bøggild, Andreas; Winther, Kristoffer S.; Gerdes, Kenn; Brodersen, Ditlev

    2011-01-01

    the crystal structure of the intact Shigella flexneri VapBC TA complex, determined to 2.7 Å resolution. Both in solution and in the crystal structure, four molecules of each protein combine to form a large and globular hetero-octameric assembly with SpoVT/AbrB-type DNA-binding domains at each end and...

  7. Drug: D06088 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D06088 Drug Tetanus ... antitoxin; TAT Immunizing agent [passive] [DS:H00337] ATC code: J06AA02 Anat ... OBULINS J06A IMMUNE SERA J06AA Immune sera J06AA02 Tetanus ... antitoxin D06088 Tetanus ... antitoxin CAS: 93384-51-1 ...

  8. Drug: D06513 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D06513 Drug Freeze-dried tetanus antitoxin, equine (JP16) Antitoxin [DS:H00337] ATC...IC USE J06 IMMUNE SERA AND IMMUNOGLOBULINS J06A IMMUNE SERA J06AA Immune sera J06AA02 Tetanus antitoxin D06513 Freeze-dried tetanus

  9. Possível interferência da antitoxina tetânica pré-existente, na vacinação antitetânica Possible interference of pre-existing tetanus antitoxin in antitetanus immunization

    Directory of Open Access Journals (Sweden)

    Rosalvo Guidolin

    1981-04-01

    Full Text Available Estudou-se qual a possível interferência no desenvolvimento da imunidade antitetânica ativa em cobaias e camundongos, filhos de fêmeas vacinadas contra o tétano em diferentes épocas durante o período da prenhês. Verificou-se que a vacinação das fêmeas, em gestação não interferiu, negativamente, no desenvolvimento da imunidade ativa dos animais filhos, quando submetidos à vacinação ao redor de 60 dias após o nascimento. A presença de baixos níveis de anticorpos circulantes, recebidos congenitamente, parece ter, em determinadas condições, estimulado a resposta imunitária quando, posteriormente, os animais filhos foram vacinados contra o tétano. Sugere-se que o complexo antígeno-anticorpo formado seja capaz de melhorar a resposta imunitária induzida pelo toxóide.Possibility of interference in the development of active antitetanic immunity was studied in the offspring of guinea pigs and mice whose mothers had received antitetanic vaccine during different periods of pregnancy. Vaccination of the females during pregnancy did not interfere negatively in the development of active immunity in their offspring when the latter were vaccinated about 60 days after birth. The presence of small amounts of circulating antibodies, congenitally received, seems, under certain conditions, to stimulate the immune response upon posterior vaccination against tetanus. This suggests that the acquired antigen-antibody complex heightens the immune response induced by the toxoid.

  10. Modeling sRNA-regulated Plasmid Maintenance

    CERN Document Server

    Gong, Chen Chris

    2016-01-01

    We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin's mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, ...

  11. Drug: D05372 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D05372 Drug Freeze-dried mamushi antivenom, equine (JP16) Therapeutic category: 633... Biological preparations 633 Antitoxins, anti-leptospiral sera 6331 Antitoxins D05372 Freeze-dried mamushi antivenom, equine (JP16) PubChem: 17398294 ...

  12. Drug: D05317 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D05317 Drug Freeze-dried habu antivenom, equine (JP16) Therapeutic category: 6331 T...ological preparations 633 Antitoxins, anti-leptospiral sera 6331 Antitoxins D05317 Freeze-dried habu antivenom, equine (JP16) PubChem: 17398276 ...

  13. Tetanus in the dog: review and a case-report of concurrent tetanus with hiatal hernia

    OpenAIRE

    Acke Els; Jones Boyd R; Breathnach Rory; McAllister Hester; Mooney Carmel T

    2004-01-01

    Tetanus with hiatal hernia was diagnosed in a four-month-old female sheepdog pup. The animal was treated with tetanus antitoxin, antibiotics, fluids and intensive nursing care for three weeks and subsequently made a full recovery.

  14. Postsegregational killing does not increase plasmid stability but acts to mediate the exclusion of competing plasmids

    OpenAIRE

    Cooper, Tim F; Heinemann, Jack A.

    2000-01-01

    Postsegregational killing (PSK) systems consist of a tightly linked toxin–antitoxin pair. Antitoxin must be continually produced to prevent the longer lived toxin from killing the cell. PSK systems on plasmids are widely believed to benefit the plasmid by ensuring its stable vertical inheritance. However, experimental tests of this “stability” hypothesis were not consistent with its predictions. We suggest an alternative hypothesis to explain the evolution of PSK: ...

  15. Linking bacterial type I toxins with their actions

    OpenAIRE

    Brielle, Régine; Pinel-Marie, Marie-Laure; Felden, Brice

    2016-01-01

    Bacterial type I toxin–antitoxin systems consist of stable toxin-encoding mRNAs whose expression is counteracted by unstable RNA antitoxins. Accumulating evidence suggests that these players belong to broad regulatory networks influencing overall bacterial physiology. The majority of known transmembrane type I toxic peptides have conserved structural characteristics. However, recent studies demonstrated that their mechanisms of toxicity are diverse and complex. To better assess the current st...

  16. Cluster of Botulism among dutch tourists in Turkey, june 2008

    OpenAIRE

    Swaan, C.M.; Ouwerkerk, van, E.N.J.; Roest, H.I.J.

    2010-01-01

    In June 2008, three Dutch tourists participating in a mini-cruise in Turkey needed urgent repatriation for antitoxin treatment because of symptoms of botulism. Because there was a shortage of antitoxin in the Netherlands, an emergency delivery was requested from the manufacturer in Germany. An outbreak investigation was initiated into all nine cruise members, eight of whom developed symptoms. C. botulinum type B was isolated in stool culture from four of them. No other patients were notified ...

  17. Phylogenetic identification of bacterial MazF toxin protein motifs among probiotic strains and foodborne pathogens and potential implications of engineered probiotic intervention in food

    OpenAIRE

    Yan Xianghe; Gurtler Joshua B; Fratamico Pina M; Hu Jing; Juneja Vijay K

    2012-01-01

    Abstract Background Toxin-antitoxin (TA) systems are commonly found in bacteria and Archaea, and it is the most common mechanism involved in bacterial programmed cell death or apoptosis. Recently, MazF, the toxin component of the toxin-antitoxin module, has been categorized as an endoribonuclease, or it may have a function similar to that of a RNA interference enzyme. Results In this paper, with comparative data and phylogenetic analyses, we are able to identify several potential MazF-conserv...

  18. PCR-based plasmid typing in Enterococcus faecium strains reveals widely distributed pRE25-, pRUM-, pIP501-and pHT beta-related replicons associated with glycopeptide resistance and stabilizing toxin-antitoxin systems

    DEFF Research Database (Denmark)

    Rosvoll, T.C.S.; Pedersen, T.; Sletvold, H.;

    2010-01-01

    AM373, pMBB1 or pEF418 were not detected. Furthermore, 61% of the strains contained the axe-txe (n=42) or/and the omega-epsilon-zeta (n=18) plasmid stabilization loci. Sequence analyses divided the omega-epsilon-zeta operon into two distinct phylogenetic groups. The present typing scheme accounted for......A PCR-based typing scheme was applied to identify plasmids in an epidemiologically and geographically diverse strain collection of Enterococcus faecium (n=93). Replicon types of pRE25 (n=56), pRUM (n=41), pIP501 (n=17) and pHT beta (n=14) were observed in 83% of the strains, while pS86, pCF10, p...... about 60% of the total number of plasmids detected by S1 nuclease analyses, which revealed zero to seven plasmids (10 kb to > 200 kb) per isolate. Interestingly, strains belonging to the clinically important clonal complex 17 (CC17) yielded a significantly higher number of plasmids (3.1) and p...

  19. Evolving new protein-protein interaction specificity through promiscuous intermediates.

    Science.gov (United States)

    Aakre, Christopher D; Herrou, Julien; Phung, Tuyen N; Perchuk, Barrett S; Crosson, Sean; Laub, Michael T

    2015-10-22

    Interacting proteins typically coevolve, and the identification of coevolving amino acids can pinpoint residues required for interaction specificity. This approach often assumes that an interface-disrupting mutation in one protein drives selection of a compensatory mutation in its partner during evolution. However, this model requires a non-functional intermediate state prior to the compensatory change. Alternatively, a mutation in one protein could first broaden its specificity, allowing changes in its partner, followed by a specificity-restricting mutation. Using bacterial toxin-antitoxin systems, we demonstrate the plausibility of this second, promiscuity-based model. By screening large libraries of interface mutants, we show that toxins and antitoxins with high specificity are frequently connected in sequence space to more promiscuous variants that can serve as intermediates during a reprogramming of interaction specificity. We propose that the abundance of promiscuous variants promotes the expansion and diversification of toxin-antitoxin systems and other paralogous protein families during evolution. PMID:26478181

  20. Prevalence of diphtheria toxin antibodies in human sera from a cross-section of the Italian population.

    Science.gov (United States)

    Bergamini, M; Comodo, N; Gasparini, R; Gabutti, G; Fabrizi, P; Severini, R; Ajello, F; Bonanni, P; Castagnari, L; Cocchioni, M; Della Pietra, P; Fragapane, E; Grilli, A; Liberatore, S; Lo Nostro, A; Moiraghi-Ruggenini, A; Pellegrini, M G; Pozzi, T; Tarsitani, G; Zotti, C; Crovari, P

    1999-01-21

    A polycentric study was carried out between 1993 and 1995 in order to evaluate diphtheria immunity on a representative sample of population from different areas of Italy. To determine diphtheria antitoxin, sera from 5187 apparently healthy subjects, divided according to sex and age groups, were titrated using an ELISA indirect method. A basic protective titre of diphtheria antitoxin (> 0.01 IU ml-1) was found in 4080 (78.6%) subjects. No statistically significant differences between males and females were observed. Our findings show that the proportion of susceptibles increases with age and a high proportion of adults no longer has diphtheria antitoxin at protective levels since toxigenic C. diphtheriae circulation is presently lacking in Italy. PMID:9987165

  1. AAU-Specific RNA Cleavage Mediated by MazF Toxin Endoribonuclease Conserved in Nitrosomonas europaea

    Directory of Open Access Journals (Sweden)

    Tatsuki Miyamoto

    2016-06-01

    Full Text Available Nitrosomonas europaea carries numerous toxin-antitoxin systems. However, despite the abundant representation in its chromosome, studies have not surveyed the underlying molecular functions in detail, and their biological roles remain enigmatic. In the present study, we found that a chromosomally-encoded MazF family member, predicted at the locus NE1181, is a functional toxin endoribonuclease, and constitutes a toxin-antitoxin system, together with its cognate antitoxin, MazE. Massive parallel sequencing provided strong evidence that this toxin endoribonuclease exhibits RNA cleavage activity, primarily against the AAU triplet. This sequence-specificity was supported by the results of fluorometric assays. Our results indicate that N. europaea alters the translation profile and regulates its growth using the MazF family of endoribonuclease under certain stressful conditions.

  2. RNA decay by messenger RNA interferases

    DEFF Research Database (Denmark)

    Christensen-Dalsgaard, Mikkel; Overgaard, Martin; Winther, Kristoffer Skovbo; Gerdes, Kenn

    2008-01-01

    Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mRNA...... cleaving enzymes such as RelE of Escherichia coli and the corresponding antitoxin RelB. In particular, we describe a set of plasmid vectors useful for the detailed analysis of cleavage sites in model mRNAs....

  3. 21 CFR 610.53 - Dating periods for licensed biological products.

    Science.gov (United States)

    2010-04-01

    ... years 5 years. Blood Group Substance AB ......do ......do 2 years. Blood Group Substance A ......do ......do Do. Blood Group Substance B ......do ......do Do. Botulism Antitoxin ......do Not applicable 5... blood cells ......do ......do Do. 3. Enzyme conjugated products ......do ......do Do. Iodinated...

  4. Genome Sequences of Gordonia terrae Phages Attis and SoilAssassin

    Science.gov (United States)

    Biery, Daniel N.; Huff, Zachary T.; Huynh, Amy B.; McFadden, William M.; Mouat, Julia S.; Schneiderman, Scott E.; Song, Hannah; Szpak, Leah E.; Umbaugh, Melanie S.; German, Brian A.; McDonnell, Jill E.; Mezghani, Nadia; Schafer, Claire E.; Thompson, Paige K.; Ulbrich, Megan C.; Yu, Victor J.; Furbee, Emily C.; Grubb, Sarah R.; Warner, Marcie H.; Montgomery, Matthew T.; Garlena, Rebecca A.; Russell, Daniel A.; Jacobs-Sera, Deborah; Hatfull, Graham F.

    2016-01-01

    Attis and SoilAssassin are two closely related bacteriophages isolated on Gordonia terrae 3612 from separate soil samples in Pittsburgh, PA. The Attis and SoilAssassin genomes are 47,881 bp and 47,880 bp, respectively, and have 74 predicted protein-coding genes, including toxin-antitoxin systems, but no tRNAs. PMID:27365347

  5. Phylogenetic identification of bacterial MazF toxin protein motifs among probiotic strains and foodborne pathogens and potential implications of engineered probiotic intervention in food

    Science.gov (United States)

    The most common mechanism involved in bacterial programmed cell death or apoptosis is through toxin-antitoxin (TA) modules, which exist in many bacterial species. An experimental procedure or method that provides novel insights into the molecular basis for the development of engineered/synthetic pr...

  6. Impairment of antitoxic antitetanus immunity under the combined effect of radiation and heat

    International Nuclear Information System (INIS)

    In experiments with mice a delay was noted in the development of secondary immune response to revaccination with a tetanic anatoxin after the combined effect of radiation and heat; the maximum antibody formation occured at later times. The low level of antitoxins within the first 10 days after the combined effect of radiation and heat correlated with the low tetanus resistance of animals

  7. Diphtheria with polyneuropathy in a closed community despite receiving recent booster vaccination

    OpenAIRE

    Krumina, A; Logina, I; Donaghy, M.; Rozentale, B; Kravale, I; Griskevica, A; Viksna, L

    2005-01-01

    Introduction and Methods: We report 20 patients aged 18–24 years from Latvia with diphtheritic polyneuropathy. All lived in a closed community and 80% were known to have been fully vaccinated against diphtheria until at least 14 years old. Diphtheria antitoxin had been administered within 3 days of the onset of upper respiratory tract infection in 16 patients and 15 received antibiotics.

  8. Translation affects YoeB and MazF messenger RNA interferase activities by different mechanisms

    DEFF Research Database (Denmark)

    Christensen-Dalsgaard, Mikkel; Gerdes, Kenn

    2008-01-01

    Prokaryotic toxin-antitoxin loci encode mRNA cleaving enzymes that inhibit translation. Two types are known: those that cleave mRNA codons at the ribosomal A site and those that cleave any RNA site specifically. RelE of Escherichia coli cleaves mRNA at the ribosomal A site in vivo and in vitro but...

  9. Conjugative plasmids of Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Emilia Pachulec

    Full Text Available Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones and with and without different tetM determinants (Dutch and American type tetM determinants have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233 or containing Dutch (pEP5289 or American (pEP5050 type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1alpha, beta, gamma, delta and epsilon subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids

  10. A New Selectable Marker System for Genetic Studies of Bacteria: Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Parsons, D; Tolmasky, M; Chain, P; Segelke, B W

    2011-03-18

    Genetic manipulations in bacteria currently rely on the introduction of antibiotic resistance genes into a bacterial strain; for those organisms that will be used for commercial or industrial applications, the genetic cassette encoding the antibiotic resistance is sometimes removed after selection. it is clear that if alternative technologies could obviate the need to introduce antibiotic resistance into bacteria, they would most certainly become a standard tool in molecular micriobiology for commercial, industrial as well as research applications. Here, they present the development of a novel genetic engineering technology based on toxin-antitoxin systems to modify bacterial genomes without the use of antibiotic resistance in the mutagenesis process. The primary goal is to develop antibiotic-free selection for genetically altered select agent pathogens. They are adapting the toxinc-antitoxin system to enable gene replacement in select agent pathogens since the NIH restrictions introducing antibiotic resistance into select agent pathogens have hindered research with select agent pathogens.

  11. [Clostridia: toxin masters. Botulism: from botox to sausages?].

    Science.gov (United States)

    Buzzi, Marta; Rossel, Anne; Coen, Matteo; Kaiser, Laurent; Abbas, Mohamed

    2016-04-13

    Clostridia are ubiquitous Gram-positive bacteria whose toxins are responsible for serious diseases. In this article we report a case of foodborne botulism we have recently managed. Moreover, we briefly describe the major clinical syndromes caused by different species of Clostridium (except for C. difficile infections, as this subject has been previously extensively reviewed in this journal). Botulism causes a flaccid paralysis starting with cranial nerves. Administration of botulism anti-toxin should be rapidly considered as soon as botulism is suspected, as prognosis is largely dependent on timely treatment; alerting the public health authorities is equally important. In Switzerland botulinum antitoxin can be obtained from the pharmacy of the Swiss Army. PMID:27263152

  12. Sera-therapy, exile and Franco's regime. The survival strategy of the Ravetllat-Pla Institute in postwar Spain.

    Science.gov (United States)

    Estapé Egea, Marc

    2014-01-01

    The aim of this paper is to analyse the scientific and commercial survival strategy of the Ravetllat-Pla Institute after the Spanish Civil War. Founded in 1923 by Ramon Pla (1880-1956) and Joaquim Ravetllat (1872-1923), it produced two sera: 'Hemo-antitoxin' and the 'Ravetllat-Pla serum'. When the Civil War ended and Ramon Pla was forced into exile, management of this laboratory was taken over by his daughter, Núia Pla Monseny (1918- 2011) who had to deal with an extremely difficult economic, political and commercial situation. In this paper, I analyse the means by which the Institute survived. These involved the Institute's ability to construct different political symbols from 'Hemo-antitoxin'. I study how Franco's repression influenced this survival strategy and Ramon Pla's role in exile. I also analyse the part played in this scientific and commercial process by the Institute's scientific and commercial network in Chile. PMID:26054212

  13. Linking bacterial type I toxins with their actions.

    Science.gov (United States)

    Brielle, Régine; Pinel-Marie, Marie-Laure; Felden, Brice

    2016-04-01

    Bacterial type I toxin-antitoxin systems consist of stable toxin-encoding mRNAs whose expression is counteracted by unstable RNA antitoxins. Accumulating evidence suggests that these players belong to broad regulatory networks influencing overall bacterial physiology. The majority of known transmembrane type I toxic peptides have conserved structural characteristics. However, recent studies demonstrated that their mechanisms of toxicity are diverse and complex. To better assess the current state of the art, type I toxins can be grouped into two classes according to their location and mechanisms of action: membrane-associated toxins acting by pore formation and/or by nucleoid condensation; and cytosolic toxins inducing nucleic acid cleavage. This classification will evolve as a result of future investigations. PMID:26874964

  14. Outbreak of wound botulism in people who inject drugs, Norway, October to November 2013.

    Science.gov (United States)

    MacDonald, E; Arnesen, T M; Brantsaeter, A B; Gerlyng, P; Grepp, M; Hansen, B Å; Jonsrud, K; Lundgren, B; Mellegård, H; Møller-Stray, J; Rønning, K; Vestrheim, D F; Vold, L

    2013-01-01

    In October and November 2013, four cases of wound botulism were confirmed in people who inject drugs (PWID) in Norway. Two additional cases are suspected. Because of the international distribution pathways for heroin – the likely source of the outbreak – healthcare workers and public health authorities in other countries should remain vigilant for wound botulism in PWID. This outbreak serves as a reminder that countries should ensure access to botulinum antitoxin in case of outbreak situations. PMID:24229788

  15. Enterotoxemia in the goat: the humoral response and local tissue reaction following vaccination with two different bacterin-toxoids.

    OpenAIRE

    Blackwell, T. E.; Butler, D G; Bell, J A

    1983-01-01

    A vaccination trial involving 72 goats was designed to compare the epsilon antitoxin titres and local reactions at the injection sites, of two commercial enterotoxemia vaccines. Three dosage regimens were used for each vaccine (12 goats per group). Although no significant differences were noted in humoral immune response between the two vaccines (P = 0.05), one vaccine regime resulted in low titres (P = 0.05) on two occasions. Local tissue reactions at injection sites persisted for six months...

  16. La gestión de información como herramienta fundamental en el desarrollo de los centros toxicológicos

    OpenAIRE

    2003-01-01

    Information management is the process aimed at supplying the resources required for decision making, as well as to improve the process, products and services related to the organization. For the purpose of showing the influence of information management in the development and strengthening of the toxicological centers in the American continent, a review was carried out on the application of this tool in toxicological centers and toxicology networks. Due to the importance of the anti-toxin cen...

  17. THE EFFECT OF A NEW SALICYLATE SYNTHESIS PRODUCT ON BLOOD GSH VALUES IN RATS

    OpenAIRE

    CORINA GRĂVILĂ; LETIŢIA STANA; MIRABELA PĂDURE; F. MUSELIN

    2013-01-01

    GSH (γ-glutamylcysteinylglycine) is a sulfhydril (-SH) antioxidant, antitoxin and enzyme cofactor which is an important component of the cellular detoxification of reactive oxygen species (ROS). Being water soluble it is found mainly in the cytosol and other aqueous phases of the living system and thus constitute one of the most important intracellular antioxidants (10,7,9). GSH plays a role in removing various toxic chemicals and drugs from the body. As a result glutathione levels in the bod...

  18. Comparative genomics of defense systems in archaea and bacteria

    OpenAIRE

    Makarova, Kira S.; Wolf, Yuri I.; Koonin, Eugene V.

    2013-01-01

    Our knowledge of prokaryotic defense systems has vastly expanded as the result of comparative genomic analysis, followed by experimental validation. This expansion is both quantitative, including the discovery of diverse new examples of known types of defense systems, such as restriction-modification or toxin-antitoxin systems, and qualitative, including the discovery of fundamentally new defense mechanisms, such as the CRISPR-Cas immunity system. Large-scale statistical analysis reveals that...

  19. Molecular Structure of Endotoxins from Gram-negative Marine Bacteria: An Update

    OpenAIRE

    Antonio Molinaro; Michelangelo Parrilli; Rosa Lanzetta; Nazarenko, Evgeny L.; Alba Silipo; Serena Leone

    2007-01-01

    Marine bacteria are microrganisms that have adapted, through millions of years, to survival in environments often characterized by one or more extreme physical or chemical parameters, namely pressure, temperature and salinity. The main interest in the research on marine bacteria is due to their ability to produce several biologically active molecules, such as antibiotics, toxins and antitoxins, antitumor and antimicrobial agents. Nonetheless, lipopolysaccharides (LPSs), or their portions, fro...

  20. Botulisme hos spaedbørn

    DEFF Research Database (Denmark)

    Hoffmann, Thomas; Mølbak, Kåre; Paerregaard, Anders

    2010-01-01

    Infant botulism is a rare disease that affects infants below the age of 12 months following absorption of neurotoxins produced by ingested Clostridium botulinum spores. The clinical manifestations are caused by symmetrical cranial nerve palsies followed by descending, symmetric flaccid paralysis of...... voluntary muscles. Presenting symptoms include constipation, lethargy, mydriasis and ptosis. The diagnosis is made on the basis of clinical examination and confirmed by isolating the toxin in serum or stools. Treatment consists of supportive intensive care and treatment with antitoxins....

  1. Cholera toxin-like toxin released by Salmonella species in the presence of mitomycin C.

    OpenAIRE

    Molina, N C; Peterson, J W

    1980-01-01

    Several serotypes of Salmonella were shown to release increased amounts of a cholera toxin-like toxin during culture in vitro with mitomycin C (MTC). Filter-sterilized culture supernatants containing the toxin caused elongation of Chinese hamster ovary cells, which could be blocked by heating the supernatants at 100 degrees C for 15 min or by adding mixed gangliosides or monospecific cholera antitoxin. When MTC was not added to the Salmonella cultures, little or no toxin was detected in crude...

  2. Antibodies against the light chain of tetanus toxin in human sera.

    OpenAIRE

    Lin, C S.; Habig, W H; Hardegree, M C

    1985-01-01

    Tetanus toxoid elicits protective antibodies against tetanus toxin in humans and animals. It has been reported that antitoxin from immunized humans contains no anti-light chain antibodies, based on immunodiffusion and quantitative precipitin analyses. We confirmed the absence of precipitating anti-light chain antibodies in tetanus immune globulin. However, the presence of antibodies against the light chain of the toxin was shown by direct binding and inhibition analyses, using enzyme-linked i...

  3. The statistical analysis of dilution series by maximum likelihood: an application to in vitro bioassays estimating the potency of the diphteria component in vaccines by serology

    OpenAIRE

    Slob W; Hendriksen CFM

    1989-01-01

    Dit rapport bespreekt de analyse van verdunningsreeksen met maximum likelihood, met als toepassing het in vitro serologisch toetsen van de werkzaamheid van bacteriele vaccins voor de mens. Met computersimulaties wordt aangetoond dat de maximum likelihood methode adequaat is voor de in het werkzaamheidsonderzoek gebruikelijke steekproefomvang. De relatie tussen de antitoxine respons en vaccinverdunning wordt goed beschreven met een rechte lijn op dubbele log-schaal binnen de gebruikelijke expe...

  4. Seroprevalence of diphtheria toxoid IgG antibodies in children, adolescents and adults in Poland

    OpenAIRE

    Aleksandra A. Zasada; Rastawicki, Waldemar; Rokosz, Natalia; Jagielski, Marek

    2013-01-01

    Background Recommendations for diphtheria immunization are to apply an effective primary immunization in infancy and to maintain immunity throughout life. Immunity against diphtheria depends primarily on antibody to the diphtheria toxin. This study evaluated the seroprevalence of IgG diphtheria antitoxin in sera of healthy children, adolescents and adults in Poland. Methods A total of 1387 serum samples collected between 2010 and 2012 from individuals with ages ranging from 1 month to 85 year...

  5. Immunity to diphtheria in a sample of the Canadian adult population

    OpenAIRE

    Pelletier, Louise; Duclos, Philippe; Gill, Peter; Deforest, Adamedia

    1998-01-01

    OBJECTIVE: To assess immunity to diphtheria in a sample of Canadian adults.DESIGN: A seroprevalence study of a group of plasmapheresis donors was performed over a four-month period in 1996. A convenience sample of 1619 sera was collected to obtain a good distribution by age groups and centres. The determination of diphtheria antitoxin concentrations was performed by neutralization of diphtheria toxin in cell culture.SUBJECTS: A total of 1619 plasmapheresis donors from Halifax, Quebec City, Lo...

  6. Coxofemoral luxation in a border collie as a complication of a Clostridium tetani infection.

    Science.gov (United States)

    Goldhammer, M A; Chapman, P S; Grierson, J M

    2008-03-01

    A four-month-old male, entire, border collie was presented to the Queen Mother Hospital for Animals with a two day history of muscular spasms and "Risus sardonicus". Tetanus was diagnosed, and the dog was treated with tetanus antitoxin, antibiotics and supportive therapy. Coxofemoral luxation resulted as a complication of the tetanus and was successfully managed by performing a femoral head and neck excision. This is the first report of joint luxation associated with Clostridium tetani infection in a dog. PMID:18005106

  7. Sir Charles James Martin MB FRS: Australian serpents and Indian plague, one-hundred years ago.

    Science.gov (United States)

    Hawgood, B J

    1997-07-01

    In 1891 as Demonstrator in Physiology at the University of Sydney, Charles Martin began the first systematic study of the chemical and physiological properties of the venoms of the Australian elapid species, Pseudechis porphyriacus and Notechis scutatus. Two major constituents were detected: a large coagulable protein which was associated with intravascular clotting, and a small proteinaceous molecule, an albumose, associated with neurotoxicity. Martin designed and constructed a high-pressure gelatin membrane ultrafilter for fractionation of venom. His studies indicated that certain physiological actions and clinical symptoms were related to the faster rate of diffusion within the tissue space of a neurotoxic constituent relative to a clotting constituent. Extending this work to toxin-antitoxin relationships, Martin provided evidence that antitoxin was a large molecule with slow diffusibility in tissue and advised the administration of curative serum (including diphtheria antitoxin) by intravenous injection. In 1903, Martin returned to London as Director of the Lister Institute of Preventive Medicine. He was soon involved in the planning of scientific work to be undertaken by the Commission for Investigation of Plague in India as the disease continued to ravage the subcontinent. Detailed epidemiological studies of possible factors involved in the spread of Pasteurella pestis showed, unequivocally, that infected rat fleas were the vector of transmission from rats to humans. PMID:9247999

  8. Heat-shock-induced refolding entails rapid degradation of bsrG toxin mRNA by RNases Y and J1.

    Science.gov (United States)

    Jahn, Natalie; Brantl, Sabine

    2016-02-01

    Gene regulation accomplished by alternative folding of an mRNA is a widely used mechanism. Classical examples are the various transcriptional attenuation mechanisms that employ, for example, leader peptide translation, or binding of a modified protein, an uncharged tRNA or an antisense RNA to the 5' untranslated region of an mRNA. With the discovery of transcriptional and translational riboswitches, it became clear that small metabolites or even metal ions can also alter RNA secondary structures and, hence, gene expression. In addition, biophysical factors like temperature can affect RNA folding, as exemplified by RNA thermometers. We have investigated in detail the type I toxin-antitoxin system bsrG/SR4 from Bacillus subtilis. The antitoxin SR4 is a cis-encoded regulatory RNA that neutralizes BsrG toxin action. SR4 prevents toxin expression by promoting degradation of the toxin mRNA and inhibiting its translation. In addition, upon temperature shock the amount of toxin mRNA decreases significantly. Here, we demonstrate that heat shock induces a refolding in the central region of the toxin mRNA that makes it more accessible to degradation by RNases Y and J1. Furthermore, we show that BsrG might play a role at the onset of stationary phase, when the antitoxin SR4 can no longer prevent toxin synthesis. PMID:26802042

  9. Cluster of botulism among Dutch tourists in Turkey, June 2008.

    Science.gov (United States)

    Swaan, C M; van Ouwerkerk, I M; Roest, H J

    2010-01-01

    In June 2008, three Dutch tourists participating in a mini-cruise in Turkey needed urgent repatriation for antitoxin treatment because of symptoms of botulism. Because there was a shortage of antitoxin in the Netherlands, an emergency delivery was requested from the manufacturer in Germany. An outbreak investigation was initiated into all nine cruise members, eight of whom developed symptoms. C. botulinum type B was isolated in stool culture from four of them. No other patients were notified locally. Food histories revealed locally purchased unprocessed black olives, consumed on board of the ship, as most likely source, but no left-overs were available for investigation. C. botulinum type D was detected in locally purchased canned peas, and whilst type D is not known to be a cause of human intoxication, its presence in a canned food product indicates an inadequate preserving process. With increasing tourism to areas where food-borne botulism is reported regularly special requests for botulism antitoxin may become necessary. Preparing an inventory of available reserve stock in Europe would appear to be a necessary and valuable undertaking. PMID:20394717

  10. [Botulism after intake of half-fermented fish].

    Science.gov (United States)

    Jensen, T; Jacobsen, D; von der Lippe, E; Yndestad, M

    1998-11-20

    From 1975 to 1997, 21 cases of foodborn botulism have been reported in Norway. Half-fermented fish is the major cause. We describe one patient with botulism following intake of home-prepared half-fermented fish. Seven people had eaten fish from the same bucket, but only two developed symptoms. The fish was initially stored at 13 degrees C; this probably explains why toxin developed. Type E toxin in moderate concentrations was found in fish samples. The patient was treated with specific antitoxin and made a gradual recovery. He returned to work after eight months. PMID:9889609

  11. Physico-Chemical-Managed Killing of Penicillin-Resistant Static and Growing Gram-Positive and Gram-Negative Vegetative Bacteria

    Science.gov (United States)

    Richmond, Robert Chaffee (Inventor); Schramm, Jr., Harry F. (Inventor); Defalco, Francis G. (Inventor); Farris, III, Alex F. (Inventor)

    2012-01-01

    Systems and methods for the use of compounds from the Hofmeister series coupled with specific pH and temperature to provide rapid physico-chemical-managed killing of penicillin-resistant static and growing Gram-positive and Gram-negative vegetative bacteria. The systems and methods represent the more general physico-chemical enhancement of susceptibility for a wide range of pathological macromolecular targets to clinical management by establishing the reactivity of those targets to topically applied drugs or anti-toxins.

  12. Complete Sequences of IncU Plasmids Harboring Quinolone Resistance Genes qnrS2 and aac(6')-Ib-cr in Aeromonas spp. from Ornamental Fish.

    Science.gov (United States)

    Dobiasova, Hana; Videnska, Petra; Dolejska, Monika

    2016-01-01

    The nucleotide sequences of three IncU plasmids from Aeromonas spp. isolated from ornamental fish are described. They had a typical IncU backbone for plasmid replication and maintenance functions, but conjugative transfer modules were disrupted. The gene qnrS2 was inserted into mpR as a mobile insertion cassette. Novel Tn3 family transposons carrying putative toxin-antitoxin and plasmid stability genes were identified. The study demonstrates high plasticity of IncU plasmids from aquatic environments. PMID:26525788

  13. Enterotoxemia in the goat: the humoral response and local tissue reaction following vaccination with two different bacterin-toxoids.

    Science.gov (United States)

    Blackwell, T E; Butler, D G; Bell, J A

    1983-04-01

    A vaccination trial involving 72 goats was designed to compare the epsilon antitoxin titres and local reactions at the injection sites, of two commercial enterotoxemia vaccines. Three dosage regimens were used for each vaccine (12 goats per group). Although no significant differences were noted in humoral immune response between the two vaccines (P = 0.05), one vaccine regime resulted in low titres (P = 0.05) on two occasions. Local tissue reactions at injection sites persisted for six months in 53% of the goats regardless of vaccine used or dosage administered. No immunological basis for the reported differences in vaccine efficacy between sheep and goats was observed in this trial. PMID:6309346

  14. Kid cleaves specific mRNAs at UUACU sites to rescue the copy number of plasmid R1

    OpenAIRE

    Pimentel, Belén; Madine, Mark A.; de la Cueva-Méndez, Guillermo

    2005-01-01

    Stability and copy number of extra-chromosomal elements are tightly regulated in prokaryotes and eukaryotes. Toxin Kid and antitoxin Kis are the components of the parD stability system of prokaryotic plasmid R1 and they can also function in eukaryotes. In bacteria, Kid was thought to become active only in cells that lose plasmid R1 and to cleave exclusively host mRNAs at UA(A/C/U) trinucleotide sites to eliminate plasmid-free cells. Instead, we demonstrate here that Kid becomes active in plas...

  15. Plasmid Evolution and Interaction between the Plasmid Addiction Stability Systems of Two Related Broad-Host-Range IncQ-Like Plasmids

    OpenAIRE

    Deane, Shelly M.; Rawlings, Douglas E

    2004-01-01

    Plasmid pTC-F14 contains a plasmid stability system called pas (plasmid addiction system), which consists of two proteins, a PasA antitoxin and a PasB toxin. This system is closely related to the pas of plasmid pTF-FC2 (81 and 72% amino acid identity for PasA and PasB, respectively) except that the pas of pTF-FC2 contains a third protein, PasC. As both pTC-F14 and pTF-FC2 are highly promiscuous broad-host-range plasmids isolated from bacteria that share a similar ecological niche, the plasmid...

  16. Involvement of a cell wall receptor in the mode of action of an anti-Candida toxin of Pichia anomala.

    OpenAIRE

    Sawant, A D; Ahearn, D G

    1990-01-01

    Hanes-Woolf, Dixon, and Hill plots of growth rates of Candida albicans RC1 grown in various concentrations of glucose and a Pichia anomala WC65 toxin suggested the presence of toxin-binding sites. Indirect immunofluorescence microscopy with antitoxin antibodies demonstrated binding of the toxin to the cell wall. Resistance to the toxin of a mutant Saccharomyces cerevisiae deficient in cell wall beta-1-6-D-glucan suggests that the glucan either served as the receptor or influenced the number o...

  17. VapC20 of Mycobacterium tuberculosis cleaves the Sarcin-Ricin loop of 23S rRNA

    OpenAIRE

    Winther, Kristoffer Skovbo; Brodersen, Ditlev E.; Brown, Alistair K.; Gerdes, Kenn

    2013-01-01

    The highly persistent and often lethal human pathogen, Mycobacterium tuberculosis contains at least 88 toxin–antitoxin genes. More than half of these encode VapC PIN domain endoribonucleases that inhibit cell growth by unknown mechanisms. Here we show that VapC20 of M. tuberculosis inhibits translation by cleavage of the Sarcin–Ricin loop (SRL) of 23S ribosomal RNA at the same position where Sarcin and other eukaryotic ribotoxins cleave. Toxin-inhibited cells can be rescued by the expression ...

  18. Experimental investigations on the immunity development in irradiated mice after local and parenteral immunization with tetanus-toxoid

    International Nuclear Information System (INIS)

    Using the 'tetanus-mouse' model the differences in immunity development between animals exposed to irradiation (300 R and 400 R) and animals not exposed to irradiation after nasal and subcutaneous vaccination was investigated. Immunization was carried out 1, 3, 6 and 10 days before and after exposure to irradiation. Efficacy of immunization was tested by challenge with 10 x LD50 tetanus-toxin and by antitoxin determination with L+-method. The degree of the immune response was dependent on 1) the irradiation dose, 2) the interval between active immunization and the ensuing or preceding irradiation, 3) the mode of vaccination (local or parenteral) and 4) the vaccination dose. (orig.)

  19. A micro-enzyme-linked immunosorbent assay (ELISA) and radioimmunosorbent technique (RIST) for the detection of immunity to clinical tetanus

    International Nuclear Information System (INIS)

    Enzyme-linked immunosorbent assay (ELISA), and radioimmunosorbent assay (RIST) techniques for the detection of tetanus toxin antibodies are described. Both methods proved to be highly sensitive, and allowed the measurement of 5 x 10-3 units/ml tetanus antitoxin in human serum or plasma, sensitivity and reproducibility comparing well with other techniques previously described, and being superior to haemagglutination and latex agglutination tests. Results of the two methods correlated well, and reflected the immunization histories obtained. Micro ELISA and micro RIST would seem to be suitable for the detection of immunity, or non-immunity to clinical tetanus. (author)

  20. A bacterial toxin inhibits DNA replication elongation through a direct interaction with the β sliding clamp.

    Science.gov (United States)

    Aakre, Christopher D; Phung, Tuyen N; Huang, David; Laub, Michael T

    2013-12-12

    Toxin-antitoxin (TA) systems are ubiquitous on bacterial chromosomes, yet the mechanisms regulating their activity and the molecular targets of toxins remain incompletely defined. Here, we identify SocAB, an atypical TA system in Caulobacter crescentus. Unlike canonical TA systems, the toxin SocB is unstable and constitutively degraded by the protease ClpXP; this degradation requires the antitoxin, SocA, as a proteolytic adaptor. We find that the toxin, SocB, blocks replication elongation through an interaction with the sliding clamp, driving replication fork collapse. Mutations that suppress SocB toxicity map to either the hydrophobic cleft on the clamp that binds DNA polymerase III or a clamp-binding motif in SocB. Our findings suggest that SocB disrupts replication by outcompeting other clamp-binding proteins. Collectively, our results expand the diversity of mechanisms employed by TA systems to regulate toxin activity and inhibit bacterial growth, and they suggest that inhibiting clamp function may be a generalizable antibacterial strategy. PMID:24239291

  1. KEBUTUHAN VAKSINASI ULANG TETANUS PADA WANITA USIA SUBUR DI YOGYAKARTA

    Directory of Open Access Journals (Sweden)

    Eko Suprijanto

    2012-09-01

    Full Text Available To enlarge the coverage of tetanus immunization program in preventing neonatal tetanus, there has been a change of the target group from pregnant women to child-bearing age women disregarding their pregnancy status. This change calls for administration of a booster dose to enhance the long-lasting immunity among the child-bearing age women in the community. To determine the proper time to give the booster dose, tetanus antitoxin levels of 21-39 years old women who had received primary immunization at 2-7 years before were measured by passive haemagglutination assay. The women with antitoxin levels of less than 0.01 HAU/ml were assumed to need a booster injection. Regression analysis clearly showed the demand of booster dose was increasing linearly from 40% up to 60% as measured at 2-7 years of post-vaccination period. Geometric mean titres were maintained in between 0.02 - 0.03 HAU/ml up to 5 years of post-vaccination period, then decreased, to 0.015 HAU/ml and 0.0058 HAU/ml in 1 and 2 years later, respectively. Based on the results, and considering some other advantages, it was suggested to administer the booster dose of tetanus toxoid at an earliest opportunity, say at 1 or 2 years after administration of pri­mary immunization.

  2. On the genetic architecture of cytoplasmic incompatibility: inference from phenotypic data.

    Science.gov (United States)

    Nor, Igor; Engelstädter, Jan; Duron, Olivier; Reuter, Max; Sagot, Marie-France; Charlat, Sylvain

    2013-07-01

    Numerous insects carry intracellular bacteria that manipulate the insects' reproduction and thus facilitate their own spread. Cytoplasmic incompatibility (CI) is a common form of such manipulation, where a (currently uncharacterized) bacterial modification of male sperm induces the early death of embryos unless the fertilized eggs carry the same bacteria, inherited from the mother. The death of uninfected embryos provides an indirect selective advantage to infected ones, thus enabling the spread of the bacteria. Here we use and expand recently developed algorithms to infer the genetic architecture underlying the complex incompatibility data from the mosquito Culex pipiens. We show that CI requires more genetic determinants than previously believed and that quantitative variation in gene products potentially contributes to the observed CI patterns. In line with population genetic theory of CI, our analysis suggests that toxin factors (those inducing embryo death) are present in fewer copies in the bacterial genomes than antitoxin factors (those ensuring that infected embryos survive). In combination with comparative genomics, our approach will provide helpful guidance to identify the genetic basis of CI and more generally of other toxin/antitoxin systems that can be conceptualized under the same framework. PMID:23778233

  3. Toxin ζ Reversible Induces Dormancy and Reduces the UDP-N-Acetylglucosamine Pool as One of the Protective Responses to Cope with Stress

    Directory of Open Access Journals (Sweden)

    Mariangela Tabone

    2014-09-01

    Full Text Available Toxins of the ζ/PezT family, found in the genome of major human pathogens, phosphorylate the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG leading to unreactive UNAG-3P. Transient over-expression of a PezT variant impairs cell wall biosynthesis and triggers autolysis in Escherichia coli. Conversely, physiological levels of ζ reversibly induce dormancy produce a sub-fraction of membrane-compromised cells, and a minor subpopulation of Bacillus subtilis cells become tolerant of toxin action. We report here that purified ζ is a strong UNAG-dependent ATPase, being GTP a lower competitor. In vitro, ζ toxin phosphorylates a fraction of UNAG. In vivo, ζ-mediated inactivation of UNAG by phosphorylation does not deplete the active UNAG pool, because expression of the toxin enhances the efficacy of genuine cell wall inhibitors (fosfomycin, vancomycin or ampicillin. Transient ζ expression together with fosfomycin treatment halt cell proliferation, but ε2 antitoxin expression facilitates the exit of ζ-induced dormancy, suggesting that there is sufficient UNAG for growth. We propose that ζ induces diverse cellular responses to cope with stress, being the reduction of the UNAG pool one among them. If the action of ζ is not inhibited, e.g., by de novo ε2 antitoxin synthesis, the toxin markedly enhances the efficacy of antimicrobial treatment without massive autolysis in Firmicutes.

  4. Toxin ζ reversible induces dormancy and reduces the UDP-N-acetylglucosamine pool as one of the protective responses to cope with stress.

    Science.gov (United States)

    Tabone, Mariangela; Ayora, Silvia; Alonso, Juan C

    2014-09-01

    Toxins of the ζ/PezT family, found in the genome of major human pathogens, phosphorylate the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) leading to unreactive UNAG-3P. Transient over-expression of a PezT variant impairs cell wall biosynthesis and triggers autolysis in Escherichia coli. Conversely, physiological levels of ζ reversibly induce dormancy produce a sub-fraction of membrane-compromised cells, and a minor subpopulation of Bacillus subtilis cells become tolerant of toxin action. We report here that purified ζ is a strong UNAG-dependent ATPase, being GTP a lower competitor. In vitro, ζ toxin phosphorylates a fraction of UNAG. In vivo, ζ-mediated inactivation of UNAG by phosphorylation does not deplete the active UNAG pool, because expression of the toxin enhances the efficacy of genuine cell wall inhibitors (fosfomycin, vancomycin or ampicillin). Transient ζ expression together with fosfomycin treatment halt cell proliferation, but ε2 antitoxin expression facilitates the exit of ζ-induced dormancy, suggesting that there is sufficient UNAG for growth. We propose that ζ induces diverse cellular responses to cope with stress, being the reduction of the UNAG pool one among them. If the action of ζ is not inhibited, e.g., by de novo ε2 antitoxin synthesis, the toxin markedly enhances the efficacy of antimicrobial treatment without massive autolysis in Firmicutes. PMID:25238046

  5. VapC20 of Mycobacterium tuberculosis cleaves the sarcin-ricin loop of 23S rRNA.

    Science.gov (United States)

    Winther, Kristoffer S; Brodersen, Ditlev E; Brown, Alistair K; Gerdes, Kenn

    2013-01-01

    The highly persistent and often lethal human pathogen, Mycobacterium tuberculosis contains at least 88 toxin-antitoxin genes. More than half of these encode VapC PIN domain endoribonucleases that inhibit cell growth by unknown mechanisms. Here we show that VapC20 of M. tuberculosis inhibits translation by cleavage of the Sarcin-Ricin loop (SRL) of 23S ribosomal RNA at the same position where Sarcin and other eukaryotic ribotoxins cleave. Toxin-inhibited cells can be rescued by the expression of the antitoxin, thereby raising the possibility that vapC20 contributes to the extreme persistence exhibited by M. tuberculosis. VapC20 cleavage is inhibited by mutations in the SRL that flank the cleavage site but not by changes elsewhere in the loop. Disruption of the SRL stem abolishes cleavage; however, further mutations that restore the SRL stem structure restore cleavage, revealing that the structure rather than the exact sequence of the SRL is important for this activity. PMID:24225902

  6. Analysis of defence systems and a conjugative IncP-1 plasmid in the marine polyaromatic hydrocarbons-degrading bacterium Cycloclasticus sp. 78-ME.

    Science.gov (United States)

    Yakimov, Michail M; Crisafi, Francesca; Messina, Enzo; Smedile, Francesco; Lopatina, Anna; Denaro, Renata; Pieper, Dietmar H; Golyshin, Peter N; Giuliano, Laura

    2016-08-01

    Marine prokaryotes have evolved a broad repertoire of defence systems to protect their genomes from lateral gene transfer including innate or acquired immune systems and infection-induced programmed cell suicide and dormancy. Here we report on the analysis of multiple defence systems present in the genome of the strain Cycloclasticus sp. 78-ME isolated from petroleum deposits of the tanker 'Amoco Milford Haven'. Cycloclasticus are ubiquitous bacteria globally important in polyaromatic hydrocarbons degradation in marine environments. Two 'defence islands' were identified in 78-ME genome: the first harbouring CRISPR-Cas with toxin-antitoxin system, while the second was composed by an array of genes for toxin-antitoxin and restriction-modification proteins. Among all identified spacers of CRISPR-Cas system only seven spacers match sequences of phages and plasmids. Furthermore, a conjugative plasmid p7ME01, which belongs to a new IncP-1θ ancestral archetype without any accessory mobile elements was found in 78-ME. Our results provide the context to the co-occurrence of diverse defence mechanisms in the genome of Cycloclasticus sp. 78-ME, which protect the genome of this highly specialized PAH-degrader. This study contributes to the further understanding of complex networks established in petroleum-based microbial communities. PMID:27345842

  7. Modeling suggests that gene circuit architecture controls phenotypic variability in a bacterial persistence network

    Directory of Open Access Journals (Sweden)

    Koh Rachel S

    2012-05-01

    Full Text Available Abstract Background Bacterial persistence is a non-inherited bet-hedging mechanism where a subpopulation of cells enters a dormant state, allowing those cells to survive environmental stress such as treatment with antibiotics. Persister cells are not mutants; they are formed by natural stochastic variation in gene expression. Understanding how regulatory architecture influences the level of phenotypic variability can help us explain how the frequency of persistence events can be tuned. Results We present a model of the regulatory network controlling the HipBA toxin-antitoxin system from Escherichia coli. Using a biologically realistic model we first determine that the persistence phenotype is not the result of bistability within the network. Next, we develop a stochastic model and show that cells can enter persistence due to random fluctuations in transcription, translation, degradation, and complex formation. We then examine alternative gene circuit architectures for controlling hipBA expression and show that networks with more noise (more persisters and less noise (fewer persisters are straightforward to achieve. Thus, we propose that the gene circuit architecture can be used to tune the frequency of persistence, a trait that can be selected for by evolution. Conclusions We develop deterministic and stochastic models describing how the regulation of toxin and antitoxin expression influences phenotypic variation within a population. Persistence events are the result of stochastic fluctuations in toxin levels that cross a threshold, and their frequency is controlled by the regulatory topology governing gene expression.

  8. Raxibacumab: potential role in the treatment of inhalational anthrax

    Directory of Open Access Journals (Sweden)

    Kummerfeldt CE

    2014-04-01

    Full Text Available Carlos E KummerfeldtDivision of Pulmonary, Critical Care, Allergy and Sleep Medicine, Medical University of South Carolina, Charleston, SC, USAAbstract: Anthrax is a highly contagious and potentially fatal human disease caused by Bacillus anthracis, an aerobic, Gram-positive, spore-forming rod-shaped bacterium with worldwide distribution as a zoonotic infection in herbivore animals. Bioterrorist attacks with inhalational anthrax have prompted the development of more effective treatments. Antibodies against anthrax toxin have been shown to decrease mortality in animal studies. Raxibacumab is a recombinant human monoclonal antibody developed against inhalational anthrax. The drug received approval after human studies showed its safety and animal studies demonstrated its efficacy for treatment as well as prophylaxis against inhalational anthrax. It works by preventing binding of the protective antigen component of the anthrax toxin to its receptors in host cells, thereby blocking the toxin's deleterious effects. Recently updated therapy guidelines for Bacillus anthracis recommend the use of antitoxin treatment. Raxibacumab is the first monoclonal antitoxin antibody made available that can be used with the antibiotics recommended for treatment of the disease. When exposure is suspected, raxibacumab should be given with anthrax vaccination to augment immunity. Raxibacumab provides additional protection against inhalational anthrax via a mechanism different from that of either antibiotics or active immunization. In combination with currently available and recommended therapies, raxibacumab should reduce the morbidity and mortality of inhalational anthrax.Keywords: anthrax, monoclonal antibody, protective antigen, raxibacumab

  9. Comparative seroepidemiology of diphtheria in six European countries and Israel.

    Science.gov (United States)

    di Giovine, P; Kafatos, G; Nardone, A; Andrews, N; Ölander, R M; Alfarone, G; Broughton, K; Cohen, D; Kriz, B; Mikova, I; O'Flanagan, D; Schneider, F; Selga, I; Valinsky, L; Velicko, I; Karacs, I; Pebody, R; von Hunolstein, C

    2013-01-01

    Serological surveys for diphtheria were conducted in six European countries including Czech Republic, Hungary, Ireland, Latvia, Luxembourg, Slovakia and one country outside Europe, Israel. For each country, a nationally representative population sample was collected across the entire age range and was tested for antibodies to diphtheria toxin. Although each national laboratory used its preferred assay, the results were all standardized to those of the in vitro neutralization test and expressed in international units (IU) which allowed comparative analyses to be performed. The results showed that increasing age is related to a gradual increase in seronegative subjects (<0·01 IU/ml of diphtheria antitoxin antibodies). This may reflect waning immunity following childhood vaccination without repeated booster vaccinations in adults. Differences in seronegativity were also found according to gender. In subjects aged 1-19 years, geometric mean titres of antitoxin are clearly related to the different vaccination schedules used in the participating countries. Although clinical disease remains rare, the susceptibility to diphtheria observed in these serosurveys highlights the importance of strengthened surveillance. PMID:22361223

  10. Minocycline resistance in an oral Streptococcus infantis isolate is encoded by tet(S) on a novel small, low copy number plasmid

    Science.gov (United States)

    Ciric, Lena; Brouwer, Michael S M; Mullany, Peter; Roberts, Adam P

    2014-01-01

    We have determined the genetic basis of minocycline resistance in a strain of Streptococcus infantis isolated from a healthy human oral cavity. We demonstrate that tet(S), identical to tet(S) found on the enterococcal conjugative transposon Tn6000, is responsible for the observed resistance. The gene is located on a small, low copy number plasmid and is flanked by IS1216 elements. The tet(S) gene is capable of excising from the plasmid together with one of the IS1216 elements. The plasmid contains a putative toxin/antitoxin system related to relBE. Deletion of the toxin, relE, did not result in plasmid instability but did increase the fitness of the mutant compared to the wild-type strain. PMID:24605990

  11. [Shiga toxin and tetanus toxin as a potential biologic weapon].

    Science.gov (United States)

    Toczyska, Izabela; Płusa, Tadeusz

    2015-09-01

    Toxins produced by the bacteria are of particular interest as potential cargo combat possible for use in a terrorist attack or war. Shiga toxin is usually produced by shiga toxigenic strains of Escherichia coli (STEC - shigatoxigenic Escherichia coli). To infection occurs mostly after eating contaminated beef. Clinical syndromes associated with Shiga toxin diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS - hemolytic uremic syndrome) or thrombotic thrombocytopenic purpura. Treatment is symptomatic. In HUS, in which mortality during an epidemic reaches 20%, extending the kidney injury dialysis may be necessary. Exposure to tetanus toxin produced by Clostridium tetani, resulting in the most generalized tetanus, characterized by increased muscle tension and painful contractions of individual muscle groups. In the treatment beyond symptomatic behavior (among others spasticity medications, anticonvulsants, muscle relaxants) is used tetanus antitoxin and antibiotics (metronidazole choice). A common complication is acute respiratory failure - then it is necessary to implement mechanical ventilation. PMID:26449578

  12. Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon

    DEFF Research Database (Denmark)

    Jaubert, Carole; Danioux, Chloë; Oberto, Jacques;

    2013-01-01

    common core genome of approximately 2 Mb and a long hyperplastic region containing most of the strain-specific genes. In LAL14/1, the latter region is enriched in insertion sequences, CRISPR (clustered regularly interspaced short palindromic repeats), glycosyl transferase genes, toxin-antitoxin genes and...... MITE (miniature inverted-repeat transposable elements). The tRNA genes of LAL14/1 are preferential targets for the integration of mobile elements but clusters of atypical genes (CAG) are also integrated elsewhere in the genome. LAL14/1 carries five CRISPR loci with 10 per cent of spacers matching...... perfectly or imperfectly the genomes of archaeal viruses and plasmids found in the Icelandic hot springs. Strikingly, the CRISPR_2 region of LAL14/1 carries an unusually long 1.9 kb spacer interspersed between two repeat regions and displays a high similarity to pING1-like conjugative plasmids. Finally, we...

  13. Transcriptome changes in STSV2-infected Sulfolobus islandicus REY15A undergoing continuous CRISPR spacer acquisition

    DEFF Research Database (Denmark)

    León-Sobrino, Carlos; Kot, Witold P; Garrett, Roger A

    2016-01-01

    A transcriptome study was performed on Sulfolobus islandicus REY15A actively undergoing CRISPR spacer acquisition from the crenarchaeal monocaudavirus STSV2 in rich and basal media over a 6 day period. Spacer acquisition preceded strong host growth retardation, altered transcriptional activity of...... four different CRISPR-Cas modules and changes in viral copy numbers, and with significant differences in the two media. Transcript levels of proteins involved in the cell cycle were reduced, while those of DNA replication, DNA repair, transcriptional regulation, and some antitoxin-toxin pairs and...... transposases were unchanged or enhanced. Antisense RNAs were implicated in the transcriptional regulation of adaptation and interference modules of the type I-A CRISPR-Cas system and evidence was found for the occurrence of functional coordination between the single CRISPR-Cas adaptation module and the...

  14. Transcriptome changes in STSV2-infected Sulfolobus islandicus REY15A undergoing continuous CRISPR spacer acquisition

    DEFF Research Database (Denmark)

    León Sobrino, Carlos; Kot, W.P.; Garrett, Roger Antony

    2015-01-01

    transposases were unchanged or enhanced. Antisense RNAs were implicated in the transcriptional regulation of adaptation and interference modules of the type I-A CRISPR-Cas system and evidence was found for the occurrence of functional coordination between the single CRISPR-Cas adaptation module and the......A transcriptome study was performed on Sulfolobus islandicus REY15A actively undergoing CRISPR spacer acquisition from the crenarchaeal monocaudavirus STSV2 in rich and basal media over a 6 day period. Spacer acquisition preceded strong host growth retardation, altered transcriptional activity of...... four different CRISPR-Cas modules and changes in viral copy numbers, and with significant differences in the two media. Transcript levels of proteins involved in the cell cycle were reduced, while those of DNA replication, DNA repair, transcriptional regulation, and some antitoxin-toxin pairs and...

  15. The Nobel Prize Highlights in Immunologic History for a Century%诺贝尔奖与免疫学的百年渊源

    Institute of Scientific and Technical Information of China (English)

    刘伯宁

    2012-01-01

    2011 Nobel Prize in physiology or medicine was shared a-mong Ralph Steinman, Jules Hoffman and Bruce beutler, who dis covered biological function of Dendritic Cells and Toll like receptor respectively. Since 1901 the first-ever Nobel Prize was award to Emil von Behring for the funding of antitoxin, overall 17 Nobel pri zes in physiology and medicine were given for achievements in im munologic fields. In a historical perspective, the Nobel laureates have witnessed the whole development course of immunology as a discipline. After originated from bacteriology, immunology has un dergone major shift from immunochemistry to immunobiology, and become an important frontier discipline of the life sciences eventu ally. The review summarizes all immunologic funding awarded to Nobel Prize in physiology or medicine retrospective, and discusses the contribution of those achievements to immunologic theory de velopment and clinic medicine.

  16. Engineered nanoparticles mimicking cell membranes for toxin neutralization.

    Science.gov (United States)

    Fang, Ronnie H; Luk, Brian T; Hu, Che-Ming J; Zhang, Liangfang

    2015-08-01

    Protein toxins secreted from pathogenic bacteria and venomous animals rely on multiple mechanisms to overcome the cell membrane barrier to inflict their virulence effect. A promising therapeutic concept toward developing a broadly applicable anti-toxin platform is to administer cell membrane mimics as decoys to sequester these virulence factors. As such, lipid membrane-based nanoparticulates are an ideal candidate given their structural similarity to cellular membranes. This article reviews the virulence mechanisms employed by toxins at the cell membrane interface and highlights the application of cell-membrane mimicking nanoparticles as toxin decoys for systemic detoxification. In addition, the implication of particle/toxin nanocomplexes in the development of toxoid vaccines is discussed. PMID:25868452

  17. High Persister Mutants in Mycobacterium tuberculosis

    Science.gov (United States)

    Torrey, Heather L.; Keren, Iris; Via, Laura E.; Lee, Jong Seok; Lewis, Kim

    2016-01-01

    Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip) mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection. PMID:27176494

  18. VapC20 of Mycobacterium tuberculosis Cleaves the Sarcin Ricin Loop of 23S rRNA

    DEFF Research Database (Denmark)

    Winther, Kristoffer Skovbo; Brodersen, Ditlev E.; Brown, Alistair K;

    2013-01-01

    exhibited by M. tuberculosis. VapC20 cleavage is inhibited by mutations in the SRL that flank the cleavage site but not by changes elsewhere in the loop. Disruption of the SRL stem abolishes cleavage; however, further mutations that restore the SRL stem structure restore cleavage, revealing that the...... translation by cleavage of the Sarcin–Ricin loop (SRL) of 23S ribosomal RNA at the same position where Sarcin and other eukaryotic ribotoxins cleave. Toxin-inhibited cells can be rescued by the expression of the antitoxin, thereby raising the possibility that vapC20 contributes to the extreme persistence...... structure rather than the exact sequence of the SRL is important for this activity...

  19. (Meta)genomic insights into the pathogenome of Cellulosimicrobium cellulans.

    Science.gov (United States)

    Sharma, Anukriti; Gilbert, Jack A; Lal, Rup

    2016-01-01

    Despite having serious clinical manifestations, Cellulosimicrobium cellulans remain under-reported with only three genome sequences available at the time of writing. Genome sequences of C. cellulans LMG16121, C. cellulans J36 and Cellulosimicrobium sp. strain MM were used to determine distribution of pathogenicity islands (PAIs) across C. cellulans, which revealed 49 potential marker genes with known association to human infections, e.g. Fic and VbhA toxin-antitoxin system. Oligonucleotide composition-based analysis of orthologous proteins (n = 791) across three genomes revealed significant negative correlation (P metagenomic islands using strain MM's genome with environmental data from the site of isolation (hot-spring biofilm) revealed (an)aerobic respiration as population segregation factor across the in situ cohorts. Using reference genomes and metagenomic data, our results highlight the emergence and evolution of PAIs in the genus Cellulosimicrobium. PMID:27151933

  20. Immunity to tetanus and diphtheria in rural Africa

    DEFF Research Database (Denmark)

    Kurtzhals, J A; Kjeldsen, K; Hey, A S;

    1997-01-01

    equal in the two populations, whereas protection in fertile women was significantly lower in the population living a long distance from a health center. Diphtheria anti-toxin was measured in the samples from one sublocation, and 70 of 84 individuals (83%) had antibody levels greater than the protective...... level. No age or sex difference could be found, and there was no correlation between response levels to diphtheria and tetanus. This implicates natural infections as an important source of diphtheria antibodies. Our findings demonstrate a need for better coverage of the adult population against tetanus....... Furthermore, diphtheria transmission still appears to take place, underscoring the importance of diphtheria vaccination of travelers to rural Africa....

  1. An update on antibody-based immunotherapies for Clostridium difficile infection

    Science.gov (United States)

    Hussack, Greg; Tanha, Jamshid

    2016-01-01

    Clostridium difficile continues to be one of the most prevalent hospital-acquired bacterial infections in the developed world, despite the recent introduction of a novel and effective antibiotic agent (fidaxomicin). Alternative approaches under investigation to combat the anaerobic Gram-positive bacteria include fecal transplantation therapy, vaccines, and antibody-based immunotherapies. In this review, we catalog the recent advances in antibody-based approaches under development and in the clinic for the treatment of C. difficile infection. By and large, inhibitory antibodies that recognize the primary C. difficile virulence factors, toxin A and toxin B, are the most popular passive immunotherapies under investigation. We provide a detailed summary of the toxin epitopes recognized by various antitoxin antibodies and discuss general trends on toxin inhibition efficacy. In addition, antibodies to other C. difficile targets, such as surface-layer proteins, binary toxin, motility factors, and adherence and colonization factors, are introduced in this review. PMID:27536153

  2. Autoproteolytic Activation of Bacterial Toxins

    Directory of Open Access Journals (Sweden)

    Aimee Shen

    2010-05-01

    Full Text Available Protease domains within toxins typically act as the primary effector domain within target cells. By contrast, the primary function of the cysteine protease domain (CPD in Multifunctional Autoprocessing RTX-like (MARTX and Clostridium sp. glucosylating toxin families is to proteolytically cleave the toxin and release its cognate effector domains. The CPD becomes activated upon binding to the eukaryotic-specific small molecule, inositol hexakisphosphate (InsP6, which is found abundantly in the eukaryotic cytosol. This property allows the CPD to spatially and temporally regulate toxin activation, making it a prime candidate for developing anti-toxin therapeutics. In this review, we summarize recent findings related to defining the regulation of toxin function by the CPD and the development of inhibitors to prevent CPD-mediated activation of bacterial toxins.

  3. Generation and characterization of recombinant bivalent fusion protein r-Cpib for immunotherapy against Clostridium perfringens beta and iota toxemia.

    Science.gov (United States)

    Das, Shreya; Majumder, Saugata; Kingston, Joseph J; Batra, Harsh V

    2016-02-01

    Clostridium perfringens beta (CPB) and iota (CPI) toxaemias result in some of the most lethal forms of haemorrhagic and necrotic enteritis and sudden death syndrome affecting especially neonates. While CPB enterotoxemia is one of the most common forms of clostridial enterotoxemia, CPI enterotoxemia though putatively considered to be rare is an emerging cause of concern. The similarities in clinical manifestation, gross and histopathology findings of both types of toxaemias coupled to the infrequency of CPI toxaemia might lead to symptomatic misidentification with Type C resulting in therapeutic failure due to habitual administration of CPB anti-toxin which is ineffective against CPI. Therefore in the present study, to generate a composite anti-toxin capable of neutralizing both toxaemias, a novel bivalent chimera r-Cpib was constructed by splicing the non-toxic C terminal binding regions of CPB and CPI, via a flexible glycine linker (G4S) by overlap-extension PCR. The fusion protein was characterized for its therapeutic abilities toward CPI and CPB toxin neutralizations. The r-Cpib was found to be non-toxic and could competitively inhibit binding of CPB to host cell receptors thereby reducing its cytotoxicity. Immunization of mice with r-Cpib generated specific antibodies capable of neutralizing the above toxaemias both in vitro and in vivo. Caco-2 cells exposed to a mixture of anti-r-Cpib sera and native CPI or CPB, displayed significantly superior protection against the respective toxins while passive challenge of mice with a similar mixture resulted in 83 and 91% protection against CPI and CPB respectively. Alternatively, mice exposed to a mixture of sham sera and native toxins died within 2-3 days. This work thus demonstrates r-Cpib as a novel bivalent fusion protein capable of efficient immunotherapy against C. perfringens CPI and CPB toxaemia. PMID:26774054

  4. Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines

    Directory of Open Access Journals (Sweden)

    Angela Buys

    2014-02-01

    Full Text Available Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT. Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL. These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required.

  5. GeneGuard: A modular plasmid system designed for biosafety.

    Science.gov (United States)

    Wright, Oliver; Delmans, Mihails; Stan, Guy-Bart; Ellis, Tom

    2015-03-20

    Synthetic biology applications in biosensing, bioremediation, and biomining envision the use of engineered microbes beyond a contained laboratory. Deployment of such microbes in the environment raises concerns of unchecked cellular proliferation or unwanted spread of synthetic genes. While antibiotic-resistant plasmids are the most utilized vectors for introducing synthetic genes into bacteria, they are also inherently insecure, acting naturally to propagate DNA from one cell to another. To introduce security into bacterial synthetic biology, we here took on the task of completely reformatting plasmids to be dependent on their intended host strain and inherently disadvantageous for others. Using conditional origins of replication, rich-media compatible auxotrophies, and toxin-antitoxin pairs we constructed a mutually dependent host-plasmid platform, called GeneGuard. In this, replication initiators for the R6K or ColE2-P9 origins are provided in trans by a specified host, whose essential thyA or dapA gene is translocated from a genomic to a plasmid location. This reciprocal arrangement is stable for at least 100 generations without antibiotic selection and is compatible for use in LB medium and soil. Toxin genes ζ or Kid are also employed in an auxiliary manner to make the vector disadvantageous for strains not expressing their antitoxins. These devices, in isolation and in concert, severely reduce unintentional plasmid propagation in E. coli and B. subtilis and do not disrupt the intended E. coli host's growth dynamics. Our GeneGuard system comprises several versions of modular cargo-ready vectors, along with their requisite genomic integration cassettes, and is demonstrated here as an efficient vector for heavy-metal biosensors. PMID:24847673

  6. Purification and crystallization of Vibrio fischeri CcdB and its complexes with fragments of gyrase and CcdA

    International Nuclear Information System (INIS)

    A CcdB homologue from V. fischeri was overexpressed in E. coli and purified. The free protein was crystallized, as were its complexes with fragments of E. coli and V. fischeri gyrase and with the F-plasmid CcdA C-terminal domain. The ccd toxin–antitoxin module from the Escherichia coli F plasmid has a homologue on the Vibrio fischeri integron. The homologue of the toxin (CcdBVfi) was crystallized in two different crystal forms. The first form belongs to space group I23 or I213, with unit-cell parameter a = 84.5 Å, and diffracts to 1.5 Å resolution. The second crystal form belongs to space group C2, with unit-cell parameters a = 58.5, b = 43.6, c = 37.5 Å, β = 110.0°, and diffracts to 1.7 Å resolution. The complex of CcdBVfi with the GyrA14Vfi fragment of V. fischeri gyrase crystallizes in space group P212121, with unit-cell parameters a = 53.5, b = 94.6, c = 58.1 Å, and diffracts to 2.2 Å resolution. The corresponding mixed complex with E. coli GyrA14Ec crystallizes in space group C2, with unit-cell parameters a = 130.1, b = 90.8, c = 58.1 Å, β = 102.6°, and diffracts to 1.95 Å. Finally, a complex between CcdBVfi and part of the F-plasmid antitoxin CcdAF crystallizes in space group P212121, with unit-cell parameters a = 46.9, b = 62.6, c = 82.0 Å, and diffracts to 1.9 Å resolution

  7. Purification and crystallization of Vibrio fischeri CcdB and its complexes with fragments of gyrase and CcdA

    Energy Technology Data Exchange (ETDEWEB)

    De Jonge, Natalie, E-mail: ndejonge@vub.ac.be; Buts, Lieven; Vangelooven, Joris [Department of Molecular and Cellular Interactions, VIB, Pleinlaan 2, 1050 Brussels (Belgium); Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels (Belgium); Mine, Natacha; Van Melderen, Laurence [Laboratoire de Génétique des Procaryotes, Institut de Biologie et de Médecine, Université Libre de Bruxelles, Gosselies (Belgium); Wyns, Lode; Loris, Remy [Department of Molecular and Cellular Interactions, VIB, Pleinlaan 2, 1050 Brussels (Belgium); Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels (Belgium)

    2007-04-01

    A CcdB homologue from V. fischeri was overexpressed in E. coli and purified. The free protein was crystallized, as were its complexes with fragments of E. coli and V. fischeri gyrase and with the F-plasmid CcdA C-terminal domain. The ccd toxin–antitoxin module from the Escherichia coli F plasmid has a homologue on the Vibrio fischeri integron. The homologue of the toxin (CcdB{sub Vfi}) was crystallized in two different crystal forms. The first form belongs to space group I23 or I2{sub 1}3, with unit-cell parameter a = 84.5 Å, and diffracts to 1.5 Å resolution. The second crystal form belongs to space group C2, with unit-cell parameters a = 58.5, b = 43.6, c = 37.5 Å, β = 110.0°, and diffracts to 1.7 Å resolution. The complex of CcdB{sub Vfi} with the GyrA14{sub Vfi} fragment of V. fischeri gyrase crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.5, b = 94.6, c = 58.1 Å, and diffracts to 2.2 Å resolution. The corresponding mixed complex with E. coli GyrA14{sub Ec} crystallizes in space group C2, with unit-cell parameters a = 130.1, b = 90.8, c = 58.1 Å, β = 102.6°, and diffracts to 1.95 Å. Finally, a complex between CcdB{sub Vfi} and part of the F-plasmid antitoxin CcdA{sub F} crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.9, b = 62.6, c = 82.0 Å, and diffracts to 1.9 Å resolution.

  8. Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines.

    Science.gov (United States)

    Buys, Angela; Macdonald, Raynard; Crafford, Jannie; Theron, Jacques

    2014-01-01

    Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT). Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA) was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA) and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA) tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL). These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required. PMID:24832497

  9. Comparative genomics of Gardnerella vaginalis strains reveals substantial differences in metabolic and virulence potential.

    Directory of Open Access Journals (Sweden)

    Carl J Yeoman

    Full Text Available BACKGROUND: Gardnerella vaginalis is described as a common vaginal bacterial species whose presence correlates strongly with bacterial vaginosis (BV. Here we report the genome sequencing and comparative analyses of three strains of G. vaginalis. Strains 317 (ATCC 14019 and 594 (ATCC 14018 were isolated from the vaginal tracts of women with symptomatic BV, while Strain 409-05 was isolated from a healthy, asymptomatic individual with a Nugent score of 9. PRINCIPAL FINDINGS: Substantial genomic rearrangement and heterogeneity were observed that appeared to have resulted from both mobile elements and substantial lateral gene transfer. These genomic differences translated to differences in metabolic potential. All strains are equipped with significant virulence potential, including genes encoding the previously described vaginolysin, pili for cytoadhesion, EPS biosynthetic genes for biofilm formation, and antimicrobial resistance systems, We also observed systems promoting multi-drug and lantibiotic extrusion. All G. vaginalis strains possess a large number of genes that may enhance their ability to compete with and exclude other vaginal colonists. These include up to six toxin-antitoxin systems and up to nine additional antitoxins lacking cognate toxins, several of which are clustered within each genome. All strains encode bacteriocidal toxins, including two lysozyme-like toxins produced uniquely by strain 409-05. Interestingly, the BV isolates encode numerous proteins not found in strain 409-05 that likely increase their pathogenic potential. These include enzymes enabling mucin degradation, a trait previously described to strongly correlate with BV, although commonly attributed to non-G. vaginalis species. CONCLUSIONS: Collectively, our results indicate that all three strains are able to thrive in vaginal environments, and therein the BV isolates are capable of occupying a niche that is unique from 409-05. Each strain has significant virulence

  10. Clinical Framework and Medical Countermeasure Use During an Anthrax Mass-Casualty Incident.

    Science.gov (United States)

    Bower, William A; Hendricks, Katherine; Pillai, Satish; Guarnizo, Julie; Meaney-Delman, Dana

    2015-12-01

    In 2014, CDC published updated guidelines for the prevention and treatment of anthrax (Hendricks KA, Wright ME, Shadomy SV, et al. Centers for Disease Control and Prevention expert panel meetings on prevention and treatment of anthrax in adults. Emerg Infect Dis 2014;20[2]. Available at http://wwwnc.cdc.gov/eid/article/20/2/13-0687_article.htm). These guidelines provided recommended best practices for the diagnosis and treatment of persons with naturally occurring or bioterrorism-related anthrax in conventional medical settings. An aerosolized release of Bacillus anthracis spores over densely populated areas could become a mass-casualty incident. To prepare for this possibility, the U.S. government has stockpiled equipment and therapeutics (known as medical countermeasures [MCMs]) for anthrax prevention and treatment. However, previously developed, publicly available clinical recommendations have not addressed the use of MCMs or clinical management during an anthrax mass-casualty incident, when the number of patients is likely to exceed the ability of the health care infrastructure to provide conventional standards of care and supplies of MCMs might be inadequate to meet the demand required. To address this gap, in 2013, CDC conducted a series of systematic reviews of the scientific literature on anthrax to identify evidence that could help clinicians and public health authorities set guidelines for intravenous antimicrobial and antitoxin use, diagnosis of anthrax meningitis, and management of common anthrax-specific complications in the setting of a mass-casualty incident. Evidence from these reviews was presented to professionals with expertise in anthrax, critical care, and disaster medicine during a series of workgroup meetings that were held from August 2013 through March 2014. In March 2014, a meeting was held at which 102 subject matter experts discussed the evidence and adapted the existing best practices guidance to a clinical use framework for the

  11. Live virus-free or die: coupling of antivirus immunity and programmed suicide or dormancy in prokaryotes

    Directory of Open Access Journals (Sweden)

    Makarova Kira S

    2012-11-01

    Full Text Available Abstract Background The virus-host arms race is a major theater for evolutionary innovation. Archaea and bacteria have evolved diverse, elaborate antivirus defense systems that function on two general principles: i immune systems that discriminate self DNA from nonself DNA and specifically destroy the foreign, in particular viral, genomes, whereas the host genome is protected, or ii programmed cell suicide or dormancy induced by infection. Presentation of the hypothesis Almost all genomic loci encoding immunity systems such as CRISPR-Cas, restriction-modification and DNA phosphorothioation also encompass suicide genes, in particular those encoding known and predicted toxin nucleases, which do not appear to be directly involved in immunity. In contrast, the immunity systems do not appear to encode antitoxins found in typical toxin-antitoxin systems. This raises the possibility that components of the immunity system themselves act as reversible inhibitors of the associated toxin proteins or domains as has been demonstrated for the Escherichia coli anticodon nuclease PrrC that interacts with the PrrI restriction-modification system. We hypothesize that coupling of diverse immunity and suicide/dormancy systems in prokaryotes evolved under selective pressure to provide robustness to the antivirus response. We further propose that the involvement of suicide/dormancy systems in the coupled antivirus response could take two distinct forms: 1 induction of a dormancy-like state in the infected cell to ‘buy time’ for activation of adaptive immunity; 2 suicide or dormancy as the final recourse to prevent viral spread triggered by the failure of immunity. Testing the hypothesis This hypothesis entails many experimentally testable predictions. Specifically, we predict that Cas2 protein present in all cas operons is a mRNA-cleaving nuclease (interferase that might be activated at an early stage of virus infection to enable incorporation of virus

  12. [Special considerations for the regulation of biological medicinal products in individualised medicine. More than stratified medicine].

    Science.gov (United States)

    Müller-Berghaus, J; Volkers, P; Scherer, J; Cichutek, K

    2013-11-01

    The term individualised medicine, also called personalised medicine, is commonly used as an equivalent to stratified medicine. However, this is erroneous since quite often it is forgotten that especially biological medicinal products have other aspects of individualization that go beyond mere stratification. The principles of stratified medicine have been applied for biological medicinal products for many years. A historical example is diphtheria antitoxin made from horse serum, while current examples are transfusion of red blood cells and the administration of factor VIII in haemophilia A. The stratifying aspects of these medicinal products are given by the following considerations: diphtheria antitoxin is only administered after a diagnosis of diphtheria and not in other forms of tonsillitis, red blood cells should only be transfused once blood group compatibility as been established and factor VIII replacement is only administered in haemophilia A as opposed to other acquired or hereditary disease of the coagulation system. The peculiarities of biological medicinal products, in particular the inherent variability of the drug, are especially important for autologous cellular medicinal products. In addition to the expected variability of the biological source material there is interindividual variability of patients as cell donors, which make definition of specifications and determination of criteria for pharmaceutical quality and potency tests difficult. Therapy with modified autologous cells, a common and important application of advanced therapy medicinal products, is exemplary for the special considerations that must be made when evaluating pharmaceutical quality, mode of action and toxicological properties of the biological medicine. The clinical investigation of advanced therapy medicinal products with the intent of demonstrating safety and efficacy is particularly challenging because of the complexity of therapy, which often involves invasive interventions

  13. Comparative metagenomic analysis of plasmid encoded functions in the human gut microbiome

    Directory of Open Access Journals (Sweden)

    Marchesi Julian R

    2010-01-01

    Full Text Available Abstract Background Little is known regarding the pool of mobile genetic elements associated with the human gut microbiome. In this study we employed the culture independent TRACA system to isolate novel plasmids from the human gut microbiota, and a comparative metagenomic analysis to investigate the distribution and relative abundance of functions encoded by these plasmids in the human gut microbiome. Results Novel plasmids were acquired from the human gut microbiome, and homologous nucleotide sequences with high identity (>90% to two plasmids (pTRACA10 and pTRACA22 were identified in the multiple human gut microbiomes analysed here. However, no homologous nucleotide sequences to these plasmids were identified in the murine gut or environmental metagenomes. Functions encoded by the plasmids pTRACA10 and pTRACA22 were found to be more prevalent in the human gut microbiome when compared to microbial communities from other environments. Among the most prevalent functions identified was a putative RelBE toxin-antitoxin (TA addiction module, and subsequent analysis revealed that this was most closely related to putative TA modules from gut associated bacteria belonging to the Firmicutes. A broad phylogenetic distribution of RelE toxin genes was observed in gut associated bacterial species (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria, but no RelE homologues were identified in gut associated archaeal species. We also provide indirect evidence for the horizontal transfer of these genes between bacterial species belonging to disparate phylogenetic divisions, namely Gram negative Proteobacteria and Gram positive species from the Firmicutes division. Conclusions The application of a culture independent system to capture novel plasmids from the human gut mobile metagenome, coupled with subsequent comparative metagenomic analysis, highlighted the unexpected prevalence of plasmid encoded functions in the gut microbial ecosystem. In

  14. Plasmids as Tools for Containment.

    Science.gov (United States)

    García, José L; Díaz, Eduardo

    2014-10-01

    Active containment systems are a major tool for reducing the uncertainty associated with the introduction of monocultures, genetically engineered or not, into target habitats for a large number of biotechnological applications (e.g., bioremediation, bioleaching, biopesticides, biofuels, biotransformations, live vaccines, etc.). While biological containment reduces the survival of the introduced organism outside the target habitat and/or upon completion of the projected task, gene containment strategies reduce the lateral spread of the key genetic determinants to indigenous microorganisms. In fundamental research, suicide circuits become relevant tools to address the role of gene transfer, mainly plasmid transfer, in evolution and how this transfer contributes to genome plasticity and to the rapid adaptation of microbial communities to environmental changes. Many lethal functions and regulatory circuits have been used and combined to design efficient containment systems. As many new genomes are being sequenced, novel lethal genes and regulatory elements are available, e.g., new toxin-antitoxin modules, and they could be used to increase further the current containment efficiencies and to expand containment to other organisms. Although the current containment systems can increase the predictability of genetically modified organisms in the environment, containment will never be absolute, due to the existence of mutations that lead to the appearance of surviving subpopulations. In this sense, orthogonal systems (xenobiology) appear to be the solution for setting a functional genetic firewall that will allow absolute containment of recombinant organisms. PMID:26104372

  15. Quantification and detoxification of aflatoxin in food items

    International Nuclear Information System (INIS)

    The present study was conducted to quantify and detoxify the antitoxins in food items. For this purpose, total 30 samples of food were collected. The samples were quantified using thin layer chromatography (TLC) for the presence of aflatoxin level in food items. Out of them aflatoxins were not found in 10 samples. Remaining 20 aflatoxins +ve samples were treated with various chemical solutions i.e. 0.1% HCl, 0.3%HCl, 0.5% HCI, 10% citric acid, 30% citric acid, 50% calcium hydroxide, 0.2 and 0.3% NaOCl, 96% ethanol and 99% acetone for detoxification. The aflatoxins were reduced to 55.1%, 90.9%, 28.08% and 80.0% in Super Sella rice, Super Basmati rice, Brown rice and White rice, respectively. The aflatoxin level was reduced in maize grain, damaged wheat, peanut, figs and dates upto 31.3 %, 64.3 %, 63.6%, 42.7% and 19.8%, respectively. Aflatoxins were detoxified in cereals Dal Chana, Dal Mash, Dal Masoor, turmeric (Haldi) and Nigela seeds (Kalwangi) upto 70.5%, 83.0%, 46.2%, 82.09% and 36.9%, respectively. Reduction of aflatoxins was carried out 39.7 %,7.l % 39.5% 82.0% and 62.0% in red chilli, makhana, corn flakes, desert (Kheer Mix) and pistachio. The significant results (p = 0.042) of detoxification of aflatoxins in food items were obtained from present study. (author)

  16. Ca2+ influx and tyrosine kinases trigger Bordetella adenylate cyclase toxin (ACT endocytosis. Cell physiology and expression of the CD11b/CD18 integrin major determinants of the entry route.

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    Kepa B Uribe

    Full Text Available Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3, its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca(2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.

  17. Tracheotomy for infant botulism.

    Science.gov (United States)

    Wolfe, J A; Rowe, L D; Pasquariello, P; Potsic, W P

    1979-01-01

    Botulism is a serious intoxication caused by ingestion of food containing preformed botulinus toxin and characterized by rapidly progressive bulbar paralysis, generalized weakness, and respiratory insufficiency. In 1976 a distinct clinical entity of infant botulism was recognized. The disease apparently results from intraintestinal toxin production which produces a defect in neuromuscular transmission by interfering with release of acetylcholine at cholinergic synapses. Five cases of infant botulism were identified at the Children's Hospital of Philadelphia between 1975 and 1977. Initial symptoms included constipation, slow feeding, lethargy and weak cry. Four of the patients progressed to respiratory insufficiency requiring nasotracheal intubation. Three of the infants with respiratory failure required tracheotomy. Because infants with respiratory failure may require support for months, we recommend that a tracheotomy be performed early in the management to avoid the complications associated with prolonged intubation. The effectiveness of antitoxin or antibiotics to treat infant botulism remains questionable and therefore prolonged respiratory supportive care is the mainstay of therapy. In addition, we offer guidelines for decannulation in cases of infant botulism. None of the patients in our series could be decannulated prior to initial discharge from the hospital. PMID:517932

  18. Noncapsulated toxinogenic Bacillus anthracis presents a specific growth and dissemination pattern in naive and protective antigen-immune mice.

    Science.gov (United States)

    Glomski, Ian J; Corre, Jean-Philippe; Mock, Michèle; Goossens, Pierre L

    2007-10-01

    Bacillus anthracis is a spore-forming bacterium that causes anthrax. B. anthracis has three major virulence factors, namely, lethal toxin, edema toxin, and a poly-gamma-D-glutamic acid capsule. The toxins modulate host immune responses, and the capsule inhibits phagocytosis. With the goal of increasing safety, decreasing security concerns, and taking advantage of mammalian genetic tools and reagents, mouse models of B. anthracis infection have been developed using attenuated bacteria that produce toxins but no capsule. While these models have been useful in studying both toxinogenic infections and antitoxin vaccine efficacy, we questioned whether eliminating the capsule changed bacterial growth and dissemination characteristics. Thus, the progression of infection by toxinogenic noncapsulated B. anthracis was analyzed and compared to that by previously reported nontoxinogenic capsulated bacteria, using in vivo bioluminescence imaging. The influence of immunization with the toxin component protective antigen (PA) on the development of infection was also examined. The toxinogenic noncapsulated bacteria were initially confined to the cutaneous site of infection. Bacteria then progressed to the draining lymph nodes and, finally, late in the infection, to the lungs, kidneys, and frequently the gastrointestinal tract. There was minimal colonization of the spleen. PA immunization reduced bacterial growth from the outset and limited infection to the site of inoculation. These in vivo observations show that dissemination by toxinogenic noncapsulated strains differs markedly from that by nontoxinogenic capsulated strains. Additionally, PA immunization counters bacterial growth and dissemination in vivo from the onset of infection. PMID:17635863

  19. Genomics of Clostridium tetani.

    Science.gov (United States)

    Brüggemann, Holger; Brzuszkiewicz, Elzbieta; Chapeton-Montes, Diana; Plourde, Lucile; Speck, Denis; Popoff, Michel R

    2015-05-01

    Genomic information about Clostridium tetani, the causative agent of the tetanus disease, is scarce. The genome of strain E88, a strain used in vaccine production, was sequenced about 10 years ago. One additional genome (strain 12124569) has recently been released. Here we report three new genomes of C. tetani and describe major differences among all five C. tetani genomes. They all harbor tetanus-toxin-encoding plasmids that contain highly conserved genes for TeNT (tetanus toxin), TetR (transcriptional regulator of TeNT) and ColT (collagenase), but substantially differ in other plasmid regions. The chromosomes share a large core genome that contains about 85% of all genes of a given chromosome. The non-core chromosome comprises mainly prophage-like genomic regions and genes encoding environmental interaction and defense functions (e.g. surface proteins, restriction-modification systems, toxin-antitoxin systems, CRISPR/Cas systems) and other fitness functions (e.g. transport systems, metabolic activities). This new genome information will help to assess the level of genome plasticity of the species C. tetani and provide the basis for detailed comparative studies. PMID:25638019

  20. Inhibition of aflatoxin biosynthesis in Aspergillus flavus by phenolic compounds extracted of Piper betle L.

    Directory of Open Access Journals (Sweden)

    Darab Yazdani

    2013-12-01

    Full Text Available Food contamination by aflatoxins is an important food safety concern for agricultural products. In order to identify and develop novel antifungal agents, several plant extracts and isolated compounds have been evaluated for their bioactivities. Anti-infectious activity of Piper betle used in traditional medicine of Malaysia has been reported previously.Crude methanol extract from P. betel powdered leaves was partitioned between chloroform and water. The fractions were tested against A. flavus UPMC 89, a strong aflatoxin producing strain. Inhibition of mycelial growth and aflatoxin biosynthesis were tested by disk diffusion and macrodillution techniques, respectively. The presence of aflatoxin was determined by thin-layer chromatography (TLC and fluorescence spectroscopy techniques using AFB1 standard. The chloroform soluble compounds were identified using HPLC-Tandem mass spectrometry technique.The results, evaluated by measuring the mycelial growth and quantification of aflatoxin B1(AFLB1 production in broth medium revealed that chloroform soluble compounds extract from P. betle dried leaves was able to block the aflatoxin biosynthesis pathway at concentration of 500μg/ml without a significant effect on mycelium growth. In analyzing of this effective fractions using HPLC-MS(2 with ESI ionization technique, 11 phenolic compounds were identified.The results showed that the certain phenolic compounds are able to decline the aflatoxin production in A. flavus with no significant effect on the fungus mycelia growth. The result also suggested P. betle could be used as potential antitoxin product.

  1. THE EFFECT OF A NEW SALICYLATE SYNTHESIS PRODUCT ON BLOOD GSH VALUES IN RATS

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    CORINA GRĂVILĂ

    2013-12-01

    Full Text Available GSH (γ-glutamylcysteinylglycine is a sulfhydril (-SH antioxidant, antitoxin and enzyme cofactor which is an important component of the cellular detoxification of reactive oxygen species (ROS. Being water soluble it is found mainly in the cytosol and other aqueous phases of the living system and thus constitute one of the most important intracellular antioxidants (10,7,9. GSH plays a role in removing various toxic chemicals and drugs from the body. As a result glutathione levels in the body are reduced by exposure to heavy metals and the chemicals used in chemotherapy (6. Sulfanilamide was the first sulfonamide discovered in this class of antimicrobial agents and its structure is considered to contain the minimum pharmacophore. They prevent or limit bacterial multiplication. Salicylic acid (2-hydroxybenzoic acid, is the basic substance of the salicylates which are non-steroidal anti-inflammatory drugs (NSAIDs. Salicylic acid and methyl salicylate (ester (methyl 2-hydroxybenzoate are the main therapeutically used substances of this group. This study was carried out to investigate the effect of a new synthesis product in comparison with the effect of sulfanilamide on GSH values in intraperitonally injected Wistar rats.

  2. Anti-snake venom: use and adverse reaction in a snake bite study clinic in Bangladesh

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    MR Amin

    2008-01-01

    Full Text Available Snakebites can present local or systemic envenomation, while neurotoxicity and respiratory paralysis are the main cause of death. The mainstay of management is anti-snake venom (ASV, which is highly effective, but liable to cause severe adverse reactions including anaphylaxis. The types of adverse reaction to polyvalent anti-snake venom have not been previously studied in Bangladesh. In this prospective observational study carried out between 1999 and 2001, in the Snake Bite Study Clinic of Chittagong Medical College Hospital, 35 neurotoxic-snake-bite patients who had received polyvalent anti-snake venom were included while the ones sensitized to different antitoxins and suffering from atopy were excluded. The common neurotoxic features were ptosis (100%, external ophthalmoplegia (94.2%, dysphagia (77.1%, dysphonia (68.5% and broken neck sign (80%. The percentage of anti-snake venom reaction cases was 88.57%; pyrogenic reaction was 80.64%; and anaphylaxis was 64.51%. The common features of anaphylaxis were urticaria (80%; vomiting and wheezing (40%; and angioedema (10%. The anti-snake venom reaction was treated mainly with adrenaline for anaphylaxis and paracetamol suppository in pyrogenic reactions. The average recovery time was 4.5 hours. Due to the danger of reactions the anti-snake venom should not be withheld from a snakebite victim when indicated and appropriate guidelines should be followed for its administration.

  3. Immunization Coverage Surveys and Linked Biomarker Serosurveys in Three Regions in Ethiopia.

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    Mark A Travassos

    Full Text Available Demographic and health surveys, immunization coverage surveys and administrative data often divergently estimate vaccination coverage, which hinders pinpointing districts where immunization services require strengthening. We assayed vaccination coverage in three regions in Ethiopia by coverage surveys and linked serosurveys.Households with children aged 12-23 (N = 300 or 6-8 months (N = 100 in each of three districts (woredas were randomly selected for immunization coverage surveys (inspection of vaccination cards and immunization clinic records and maternal recall and linked serosurveys. IgG-ELISA serologic biomarkers included tetanus antitoxin ≥ 0.15 IU/ml in toddlers (receipt of tetanus toxoid and Haemophilus influenzae type b (Hib anti-capsular titers ≥ 1.0 mcg/ml in infants (timely receipt of Hib vaccine.Coverage surveys enrolled 1,181 children across three woredas; 1,023 (87% also enrolled in linked serosurveys. Administrative data over-estimated coverage compared to surveys, while maternal recall was unreliable. Serologic biomarkers documented a hierarchy among the districts. Biomarker measurement in infants provided insight on timeliness of vaccination not deducible from toddler results.Neither administrative projections, vaccination card or EPI register inspections, nor parental recall, substitute for objective serological biomarker measurement. Including infants in serosurveys informs on vaccination timeliness.

  4. Structural Analysis of a Putative Aminoglycoside N-Acetyltransferase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Klimecka, Maria M.; Chruszcz, Maksymilian; Font, Jose; Skarina, Tatiana; Shumilin, Igor; Onopryienko, Olena; Porebski, Przemyslaw J.; Cymborowski, Marcin; Zimmerman, Matthew D.; Hasseman, Jeremy; Glomski, Ian J.; Lebioda, Lukasz; Savchenko, Alexei; Edwards, Aled; Minor, Wladek (SC); (Toronto); (UV)

    2012-02-15

    For the last decade, worldwide efforts for the treatment of anthrax infection have focused on developing effective vaccines. Patients that are already infected are still treated traditionally using different types of standard antimicrobial agents. The most popular are antibiotics such as tetracyclines and fluoroquinolones. While aminoglycosides appear to be less effective antimicrobial agents than other antibiotics, synthetic aminoglycosides have been shown to act as potent inhibitors of anthrax lethal factor and may have potential application as antitoxins. Here, we present a structural analysis of the BA2930 protein, a putative aminoglycoside acetyltransferase, which may be a component of the bacterium's aminoglycoside resistance mechanism. The determined structures revealed details of a fold characteristic only for one other protein structure in the Protein Data Bank, namely, YokD from Bacillus subtilis. Both BA2930 and YokD are members of the Antibiotic-NAT superfamily (PF02522). Sequential and structural analyses showed that residues conserved throughout the Antibiotic-NAT superfamily are responsible for the binding of the cofactor acetyl coenzyme A. The interaction of BA2930 with cofactors was characterized by both crystallographic and binding studies.

  5. Distribution of Tritiated Tetanus Toxin Following an Intraperitoneal Injection in Immunized and Non-Immunized Mice

    International Nuclear Information System (INIS)

    Tetanus toxin, purified by ultra-filtration and precipitation with ammonium sulphate, was lyophilized and exposed to 5 c of tritium gas (Wilzbach procedure) for 2½ d in a deep freeze cabinet at 0.38 atm of pressure. The toxin was then homogenized and the precipitated material removed by filtration through a HA millipore membrane. The filtrate was washed and concentrated in a membrane colloidal dialyzer. The resuspended material (particles estimated to be below 450 mpm in size) was highly toxic when injected into mice. Both the crude precipitate and the suspended toxin were injected into immunized mice and the animals autopsied at various times to determine the presence of radioactivity in the various inflammatory cells. These results were compared with those obtained when the toxins were neutralized and injected into non-immunized mice. In the inflammatory area produced by the injection of tritiated toxin, neutrophils containing radioactivity were found during the first 2 d, and macrophages containing radioactivity were found in the spleen as well as in the inflammatory area for as long as 15 d. Trace amounts of radioactivity were found in eosinophils. No radioactivity was found within mast cells in either the immunized or non-immunized animals. The significance of these results will be discussed in relation to initiation of antitoxin production. (author)

  6. Persistence and resistance as complementary bacterial adaptations to antibiotics.

    Science.gov (United States)

    Vogwill, T; Comfort, A C; Furió, V; MacLean, R C

    2016-06-01

    Bacterial persistence represents a simple of phenotypic heterogeneity, whereby a proportion of cells in an isogenic bacterial population can survive exposure to lethal stresses such as antibiotics. In contrast, genetically based antibiotic resistance allows for continued growth in the presence of antibiotics. It is unclear, however, whether resistance and persistence are complementary or alternative evolutionary adaptations to antibiotics. Here, we investigate the co-evolution of resistance and persistence across the genus Pseudomonas using comparative methods that correct for phylogenetic nonindependence. We find that strains of Pseudomonas vary extensively in both their intrinsic resistance to antibiotics (ciprofloxacin and rifampicin) and persistence following exposure to these antibiotics. Crucially, we find that persistence correlates positively to antibiotic resistance across strains. However, we find that different genes control resistance and persistence implying that they are independent traits. Specifically, we find that the number of type II toxin-antitoxin systems (TAs) in the genome of a strain is correlated to persistence, but not resistance. Our study shows that persistence and antibiotic resistance are complementary, but independent, evolutionary adaptations to stress and it highlights the key role played by TAs in the evolution of persistence. PMID:26999656

  7. YihE Kinase Is a Central Regulator of Programmed Cell Death in Bacteria

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    Angella Dorsey-Oresto

    2013-02-01

    Full Text Available Stress-mediated programmed cell death (PCD in bacteria has recently attracted attention, largely because it raises novel possibilities for controlling pathogens. How PCD in bacteria is regulated to avoid population extinction due to transient, moderate stress remains a central question. Here, we report that the YihE protein kinase is a key regulator that protects Escherichia coli from antimicrobial and environmental stressors by antagonizing the MazEF toxin-antitoxin module. YihE was linked to a reactive oxygen species (ROS cascade, and a deficiency of yihE stimulated stress-induced PCD even after stress dissipated. YihE was partially regulated by the Cpx envelope stress-response system, which, along with MazF toxin and superoxide, has both protective and destructive roles that help bacteria make a live-or-die decision in response to stress. YihE probably acts early in the stress response to limit self-sustaining ROS production and PCD. Inhibition of YihE may provide a way of enhancing antimicrobial lethality and attenuating virulence.

  8. Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

    Science.gov (United States)

    Baghban, Roghayyeh; Gargari, Seyed Latif Mousavi; Rajabibazl, Masoumeh; Nazarian, Shahram; Bakherad, Hamid

    2016-01-01

    Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property. PMID:24673401

  9. Structural and Thermodynamic Characterization of Vibrio fischeri CcdB*

    Science.gov (United States)

    De Jonge, Natalie; Hohlweg, Walter; Garcia-Pino, Abel; Respondek, Michal; Buts, Lieven; Haesaerts, Sarah; Lah, Jurij; Zangger, Klaus; Loris, Remy

    2010-01-01

    CcdBVfi from Vibrio fischeri is a member of the CcdB family of toxins that poison covalent gyrase-DNA complexes. In solution CcdBVfi is a dimer that unfolds to the corresponding monomeric components in a two-state fashion. In the unfolded state, the monomer retains a partial secondary structure. This observation correlates well with the crystal and NMR structures of the protein, which show a dimer with a hydrophobic core crossing the dimer interface. In contrast to its F plasmid homologue, CcdBVfi possesses a rigid dimer interface, and the apparent relative rotations of the two subunits are due to structural plasticity of the monomer. CcdBVfi shows a number of non-conservative substitutions compared with the F plasmid protein in both the CcdA and the gyrase binding sites. Although variation in the CcdA interaction site likely determines toxin-antitoxin specificity, substitutions in the gyrase-interacting region may have more profound functional implications. PMID:19959472

  10. Structural and thermodynamic characterization of Vibrio fischeri CcdB.

    Science.gov (United States)

    De Jonge, Natalie; Hohlweg, Walter; Garcia-Pino, Abel; Respondek, Michal; Buts, Lieven; Haesaerts, Sarah; Lah, Jurij; Zangger, Klaus; Loris, Remy

    2010-02-19

    CcdB(Vfi) from Vibrio fischeri is a member of the CcdB family of toxins that poison covalent gyrase-DNA complexes. In solution CcdB(Vfi) is a dimer that unfolds to the corresponding monomeric components in a two-state fashion. In the unfolded state, the monomer retains a partial secondary structure. This observation correlates well with the crystal and NMR structures of the protein, which show a dimer with a hydrophobic core crossing the dimer interface. In contrast to its F plasmid homologue, CcdB(Vfi) possesses a rigid dimer interface, and the apparent relative rotations of the two subunits are due to structural plasticity of the monomer. CcdB(Vfi) shows a number of non-conservative substitutions compared with the F plasmid protein in both the CcdA and the gyrase binding sites. Although variation in the CcdA interaction site likely determines toxin-antitoxin specificity, substitutions in the gyrase-interacting region may have more profound functional implications. PMID:19959472

  11. Molecular Structure of Endotoxins from Gram-negative Marine Bacteria: An Update

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    Antonio Molinaro

    2007-09-01

    Full Text Available Marine bacteria are microrganisms that have adapted, through millions of years, to survival in environments often characterized by one or more extreme physical or chemical parameters, namely pressure, temperature and salinity. The main interest in the research on marine bacteria is due to their ability to produce several biologically active molecules, such as antibiotics, toxins and antitoxins, antitumor and antimicrobial agents. Nonetheless, lipopolysaccharides (LPSs, or their portions, from Gram-negative marine bacteria, have often shown low virulence, and represent potential candidates in the development of drugs to prevent septic shock. Besides, the molecular architecture of such molecules is related to the possibility of thriving in marine habitats, shielding the cell from the disrupting action of natural stress factors. Over the last few years, the depiction of a variety of structures of lipids A, core oligosaccharides and O-specific polysaccharides from LPSs of marine microrganisms has been given. In particular, here we will examine the most recently encountered structures for bacteria belonging to the genera Shewanella, Pseudoalteromonas and Alteromonas, of the γ-Proteobacteria phylum, and to the genera Flavobacterium, Cellulophaga, Arenibacter and Chryseobacterium, of the Cytophaga- Flavobacterium-Bacteroides phylum. Particular attention will be paid to the chemical features expressed by these structures (characteristic monosaccharides, non-glycidic appendages, phosphate groups, to the typifying traits of LPSs from marine bacteria and to the possible correlation existing between such features and the adaptation, over years, of bacteria to marine environments.

  12. Botulinum neurotoxin: Progress in negating its neurotoxicity; and in extending its therapeutic utility via molecular engineering. MiniReview.

    Science.gov (United States)

    Kostrzewa, Richard M; Kostrzewa, Rose Anna; Kostrzewa, John P

    2015-10-01

    While the poisonous effects of botulinum neurotoxin (BoNT) have been recognized since antiquity, the overall actions and mechanisms of effects of BoNT have been elucidated primarily over the past several decades. The general utility of BoNT is described in the paper, but the focus is mainly on the approaches towards negating the toxic effects of BoNT, and on the projection of an engineered BoNT molecule serving as a Trojan Horse to deliver a therapeutic load for treatment of a host of medical disorders. The BoNT molecule is configured with a binding domain, a zinc-dependent protease with specificity primarily for vesicular proteins, and a translocation domain for delivery of the metalloprotease into the cytoplasm. The anti-toxin approaches for BoNT include the use of vaccines, antibodies, block of BoNT binding or translocation, inhibition of metalloprotease activity, impeded translocation of the protease/catalytic domain, and inhibition of the downstream Src signaling pathway. Projections of BoNT as a therapeutic include its targeting to non-cholinergic nerves, also targeting to non-neuronal cells for treatment of hypersecretory disorders (e.g., cystic fibrosis), and treatment of hormonal disorders (e.g., acromegaly). Still in the exploratory phase, there is the expectation of major advances in BoNT neuroprotective strategies and burgeoning utility of engineered BoNTs as therapeutics. PMID:26192475

  13. Locating therapeutic vaccines in nineteenth-century history.

    Science.gov (United States)

    Gradmann, Christoph

    2008-06-01

    This essay places some therapeutic vaccines, including particularly the diphtheria antitoxin, into their larger historical context of the late nineteenth century. As industrially produced drugs, these vaccines ought to be seen in connection with the structural changes in medicine and pharmacology at the time. Given the spread of industrial culture and technology into the field of medicine and pharmacology, therapeutic vaccines can be understood as boundary objects that required and facilitated communication between industrialists, medical researchers, public health officials, and clinicians. It was in particular in relation to evaluation and testing for efficacy in animal models that these medicines became a model for twentieth-century medicine. In addition, these medicines came into being as a parallel invention in two very distinct local cultures of research: the Institut Pasteur in Paris and the Institut für Infektionskrankheiten in Berlin. While their local cultural origins were plainly visible, the medicines played an important role in the alignment of the methods and objects that took place in bacteriology research in France and Germany in the 1890s. This article assesses the two locally specific regimes for control in France and in Imperial Germany. In France the Institut Pasteur, building on earlier successful vaccines, enjoyed freedom from scrutinizing control. The tight and elaborate system of control that evolved in Imperial Germany is portrayed as being reliant on experiences that were drawn from the dramatic events that surrounded the launching of a first example of so-called "bacteriological medicine," tuberculin, in 1890. PMID:18831134

  14. Fatal course of foodborne botulism in an eigth-month old Infant

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    Davide Lonati

    2011-12-01

    Full Text Available An 8-month old girl, weighing 9 kg, was brought by her parents at 8.15 am to the Emergency Department (ED for a progressive worsening of weakness and acute respiratory failure. On admission, the baby presented with poor oral intake, a weak cry and extremely weak muscular body control. Poor gag and suck, unreactive mydriasis, hypotonia, lethargy and absence of peristalsis were noted. Laboratory data showed severe respiratory acidosis. Chest X-ray, electroencephalography, encephalic CT scan and MRI were all normal, as were cerebrospinal fluid analysis and viral tests. Orotracheal intubation and continuous mechanical ventilation were applied. The patient received fluids, corticosteroids, aerosol therapy, large-spectrum antibiotics and enteral- nutrition. Further investigation revealed ingestion of an improperly prepared homecanned homogenized turkey meal. Type A botulinum neurotoxin was identified. Trivalent botulinum antitoxin, prostigmine and oral activated charcoal were administered. Generalized flaccid paralysis, areflexic bilateral mydriasis, gastric stasis and deep coma persisted for the duration of the hospital stay, and the patient died of severe respiratory failure and cardiac arrest 12 days after ED admission. Botulism poisoning should be suspected in any infant presenting with feeding difficulties, constipation, descendent paralysis or acute respiratory failure. Supportive treatment and antidotal therapy should be performed as soon as a clinical diagnosis is made. We describe a case of foodborne botulism in an 8-month old infant caused by ingestion of an improperly prepared home-canned homogenized turkey meal, representing the youngest fatal case reported in medical literature.

  15. Structural analysis of the active site architecture of the VapC toxin from Shigella flexneri.

    Science.gov (United States)

    Xu, Kehan; Dedic, Emil; Brodersen, Ditlev E

    2016-07-01

    The VapC toxin from the Shigella flexneri 2a virulence plasmid pMYSH6000 belongs to the PIN domain protein family, which is characterized by a conserved fold with low amino acid sequence conservation. The toxin is a bona fide Mg(2+) -dependent ribonuclease and has been shown to target initiator tRNA(fMet) in vivo. Here, we present crystal structures of active site catalytic triad mutants D7A, D7N, and D98N of the VapC toxin in absence of antitoxin. In all structures, as well as in solution, VapC forms a dimer. In the D98N structure, a Hepes molecule occupies both active sites of the dimer and comparison with the structure of RNase H bound to a DNA/RNA hybrid suggests that the Hepes molecule mimics the position of an RNA nucleotide in the VapC active site. Proteins 2016; 84:892-899. © 2016 Wiley Periodicals, Inc. PMID:26833558

  16. In vitro Transcriptome Analysis of Two Chinese Isolates of Streptococcus suis Serotype 2

    Institute of Scientific and Technical Information of China (English)

    Dake Zhang; Nan Du; Sufang Ma; Qingtao Hu; Guangwen Lu; Wei Chen; Changqing Zeng

    2014-01-01

    The Streptococcus suis serotype 2 (S. suis 2) isolates 05ZYH33 and 98HAH33 have caused severe human infections in China. Using a strand-specific RNA-seq analysis, we compared the in vitro transcriptomes of these two Chinese isolates with that of a reference strain (P1/7). In the 89K genomic island that is specific to these Chinese isolates, a toxin–antitoxin system showed relatively high levels of transcription among the S. suis. The known virulence factors with high transcriptional activity in these two highly-pathogenic strains are mainly involved in adhesion, biofilm formation, hemolysis and the synthesis and transport of the outer membrane protein. Furthermore, our analysis of novel transcripts identified over 50 protein-coding genes with one of them encoding a toxin protein. We also predicted over 30 small RNAs (sRNAs) in each strain, and most of them are involved in riboswitches. We found that six sRNA candidates that are related to bacterial virulence, including cspA and rli38, are specific to Chinese isolates. These results provide insight into the factors responsible for the difference in virulence among the different S. suis 2 isolates.

  17. Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component

    International Nuclear Information System (INIS)

    The pharmacokinetic interaction of an affinity-purified 125I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 x 10-9 M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme could not dissociate the membrane-bound toxin formed at 4 or 370C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 40C but not at 370C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. It is proposed that, in addition to a ganglioside, interaction of tetanotoxin with synaptic membranes is facilitated by a protein and may also require an appropriate lipid environment. These latter membrane constituents may play a pivotal role in the sequestration of the toxin

  18. An unexpected tetanus case.

    Science.gov (United States)

    Ergonul, Onder; Egeli, Demet; Kahyaoglu, Bulent; Bahar, Mois; Etienne, Mill; Bleck, Thomas

    2016-06-01

    1 million cases of tetanus are estimated to occur worldwide each year, with more than 200 000 deaths. Tetanus is a life-threatening but preventable disease caused by a toxin produced by Clostridium tetani-a Gram-positive bacillus found in high concentrations in soil and animal excrement. Tetanus is almost completely preventable by active immunisation, but very rarely unexpected cases can occur in individuals who have been previously vaccinated. We report a case of generalised tetanus in a 22-year-old woman that arose despite the protective antitoxin antibody in her serum. The patient received all her vaccinations in the USA; her last vaccination was 6 years ago. The case was unusual because the patient had received all standard vaccinations, had no defined port of entry at disease onset, and had symptoms lasting for 6 months. Tetanus can present with unusual clinical forms; therefore, the diagnosis and management of this rare but difficult disease should be updated. In this Grand Round, we review the clinical features, epidemiology, treatment, and prognosis of C tetani infections. PMID:27301930

  19. A radioimmunoassay for tetanus-antibodies using protein A-containing Staphylococcus aureus

    International Nuclear Information System (INIS)

    To measure tetanus antibodies a trace amount of 125I-labeled tetanus toxin is mixed with appropriate dilutions of human serum or blood. The labeled antigen-antibody complexes are adsorbed to heat-killed staphylococci (Cowan I) via their surface protein A. The radioactivity of the washed solid phase is a function of the initial antibody concentration. The test allows the measurement of 6 x 10-0U of tetanus antitoxin in a volume of 0.03 ml. In order to avoid possible interferences, serum has to be diluted 20-fold before use. Taking that into account the real border limit of sensitivity is 4 x 10-3U/ml serum. Antibodies may be measured in serum, in plasma, and even in heparinized blood. As to its sensitivity, the test compares well with the toxin neutralization procedure. It is superior to the previous radioimmunologic, enzymoimmunologic, and hemagglutination techniques with respect to sensitivity and reproducibility. It reflects the values obtained in the toxin neutralization test better than the other in vitro procedures, as shown by parallel assays of 17 sera. (orig.)

  20. Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer.

    Science.gov (United States)

    Katsukawa, Chihiro; Komiya, Takako; Umeda, Kaoru; Goto, Minami; Yanai, Tokuma; Takahashi, Motohide; Yamamoto, Akihiko; Iwaki, Masaaki

    2016-03-01

    Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans. PMID:26853714

  1. Immunogenicity test of tetanus component in adsorbed vaccines by toxin binding inhibition test

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    Denise Cristina Souza Matos

    2002-09-01

    Full Text Available Samples from 20 lots of diphtheria-tetanus (adult use dT vaccine and from 20 lots of diphtheria-tetanus-pertussis (DTP vaccine were used to standardize and validate the in vitro toxin binding inhibition (ToBI test for the immunogenicity test of the tetanus component. The levels of tetanus antitoxin obtained by ToBI test were compared to those obtained using the toxin neutralization (TN test in mice routinely employed to perform the quality control of the tetanus component in adsorbed vaccines. The results ranged from 1.8 to 3.5 IU/ml for dT and 2 to 4 IU/ml for DTP by ToBI test and 1.4 to 3 IU/ml for dT and 1.8 to 3.5 IU/ml for DTP by TN in mice. These results were significantly correlated. From this study, it is concluded that the ToBI test is an alternative to the in vivo neutralization procedure in the immunogenicity test of the tetanus component in adsorbed vaccines. A substantial refinement and a reduction in use of animals can be achieved.

  2. Mutagenic Deimmunization of Diphtheria Toxin for Use in Biologic Drug Development

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    Joerg U. Schmohl

    2015-10-01

    Full Text Available Background: Targeted toxins require multiple treatments and therefore must be deimmunized. We report a method of protein deimmunization based on the point mutation of highly hydrophilic R, K, D, E, and Q amino acids on the molecular surface of truncated diphtheria-toxin (DT390. Methods: Based on their surface position derived from an X-ray-crystallographic model, residues were chosen for point mutation that were located in prominent positions on the molecular surface and away from the catalytic site. Mice were immunized with a targeted toxin containing either a mutated DT390 containing seven critical point mutations or the non-mutated parental toxin form. Results: Serum analysis revealed a significant 90% reduction in anti-toxin antibodies in mice immunized with the mutant, but not the parental drug form despite multiple immunizations. The experiment was repeated in a second strain of mice with a different MHC-haplotype to address whether point mutation removed T or B cell epitopes. Findings were identical indicating that B cell epitopes were eliminated from DT. The mutant drug form lost only minimal activity in vitro as well as in vivo. Conclusion: These findings indicate that this method may be effective for deimmunizing of other proteins and that discovery of a deimmunized form of DT may lead to the development of more effective targeted toxin.

  3. HASIL GUNA IMUNISASI DASAR DIFTERI DENGAN VAKSIN DPT 2 DOSIS

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    Dyah W. Isbagio

    2012-09-01

    Full Text Available A retrospective study on a basic diptheria immunization was done in Tulangan district of East Jawa, involving three hundred ninety two children with various immune status, that say 0,1 and 2 doses of DPT vaccine manufactured by Perum. Bio Farma, Bandung. The study was aimed to measure serological effectiviness 2 of doses of DPT vaccine for inducing immunity against diphtheria in real life situation. Diphtheria antitoxin titre was examined by Passive Haemagglutination Assay on 0,1 ml of capillary blood specimens. The result showed that 2 doses of DPT vaccine given at 1-3 months interval on children of 3—14 months old, were able to induce an adequate immune response on more than 80% of the children. Immune response with 0 and 1 doses of DPT vaccine has also been discussed. The potensial implication of this study result, supported by other study on a various of age, on the administration strategy of booster dose of Td ("adult type" vaccine among school children has also been suggested.

  4. High-Throughput Screening Uncovers Novel Botulinum Neurotoxin Inhibitor Chemotypes.

    Science.gov (United States)

    Bompiani, Kristin M; Caglič, Dejan; Krutein, Michelle C; Benoni, Galit; Hrones, Morgan; Lairson, Luke L; Bian, Haiyan; Smith, Garry R; Dickerson, Tobin J

    2016-08-01

    Botulism is caused by potent and specific bacterial neurotoxins that infect host neurons and block neurotransmitter release. Treatment for botulism is limited to administration of an antitoxin within a short time window, before the toxin enters neurons. Alternatively, current botulism drug development targets the toxin light chain, which is a zinc-dependent metalloprotease that is delivered into neurons and mediates long-term pathology. Several groups have identified inhibitory small molecules, peptides, or aptamers, although no molecule has advanced to the clinic due to a lack of efficacy in advanced models. Here we used a homogeneous high-throughput enzyme assay to screen three libraries of drug-like small molecules for new chemotypes that modulate recombinant botulinum neurotoxin light chain activity. High-throughput screening of 97088 compounds identified numerous small molecules that activate or inhibit metalloprotease activity. We describe four major classes of inhibitory compounds identified, detail their structure-activity relationships, and assess their relative inhibitory potency. A previously unreported chemotype in any context of enzyme inhibition is described with potent submicromolar inhibition (Ki = 200-300 nM). Additional detailed kinetic analyses and cellular cytotoxicity assays indicate the best compound from this series is a competitive inhibitor with cytotoxicity values around 4-5 μM. Given the potency and drug-like character of these lead compounds, further studies, including cellular activity assays and DMPK analysis, are justified. PMID:27314875

  5. Advanced medical countermeasures for radiological accidents and nuclear disasters: prevention, prophylaxis, treatment and pre- and post-exposure management.

    Science.gov (United States)

    Popov, Dmitri; Maliev, Slava; Jones, Jeffrey

    Countermeasures against nuclear terrorism to prevent or limit the number of irradiated human population or radiation intoxications include early identification of the nuclear terrorism event and all persons which exposed by radiation, decontamination program and procedures, radiation control, and medical countermeasures which include medical diagnosis,differential diagnosis of Acute Radiation Syndromes by Immune Enzyme Assay , pre-exposure vaccination with Human Antiradiation Vaccine, post-exposure specific treatment - de-intoxication with Radiation Antidote IgG (blocking Antiradiation Antibodies). Our Advanced Medical Technology elaborated as a part of effective countermeasure include Plan of Action.Countermeasures against nuclear terrorism to prevent or limit the number of high level of lethality and severe forms of radiation illness or intoxications include A.early identification of the nuclear terrorism event and persons exposed,b. appropriate decontamination, c. radiation control, and d.medical countermeasures and medical management of ARS. Medical countermeasures, which include medical interventions such as active immuneprophylaxis with Human Antiradiation Vaccine , passive immune-prophylaxis with Antiradiation Antitoxins immune-globulins IgG , and chemoprophylaxis - post-exposure antioxidants prophylaxis and antibioticprophylaxis. Medical countermeasures with Antiradiation Vaccine should be initiated before an exposure (if individuals are identified as being at high risk for exposure)but after a confirmed exposure event Antiradiation Vaccine not effective and Antiradiation Antidot IgG must be applyed for treatment of Acute Radiation Syndromes.

  6. A Family Outbreak of Foodborne Botulism Following Consumption of Home-Canned Doogh in Hamadan, West of Iran

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    Hashemi

    2015-02-01

    Full Text Available Background Food-borne botulism is one of the potentially fatal forms of food poisoning, usually caused by ingestion of home-canned vegetables, fruits and fish products. Objectives The aim of this study was to report an outbreak of botulism due to homemade doogh in Hamadan, Iran. Patients and Methods During an outbreak, 10 members of a family referred to the hospital because of food poisoning. All patients had a history of consumption of doogh, a traditional drink. After careful physical examination, all of them were hospitalized. Botulism was suspected in all patients except for the first patient. Results The first patient was a 76-year-old man who died after 12 hours of admission due to respiratory distress. Nine subsequent patients were diagnosed as botulism with the following symptoms: diplopia (90%, dizziness (70%, nausea and vomiting (80%, ptosis (60%, symmetric weakness of extremities (60%, dysarthria (30%, chest discomfort (30%, mydriasis (20%, dysphasia (20% and dry mouth (20%. All of the nine patients received botulinum antitoxin and improved during 5-15 days of hospitalization. Conclusions Immediate diagnosis based on careful history and physical examination are essential for management of botulism. People should be notified about proper food handling and preparation of traditional homemade foods.

  7. Polyclonal Antibody Therapies for Clostridium difficile Infection

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    Michael R. Simon

    2014-10-01

    Full Text Available Clostridium difficile infection has emerged as a growing worldwide health problem. The colitis of Clostridium difficile infection results from the synergistic action of C. difficile secreted toxins A and B upon the colon mucosa. A human monoclonal IgG anti-toxin has demonstrated the ability in combination therapy to reduce mortality in C. difficile challenged hamsters. This antibody is currently in a clinical trial for the treatment of human Clostridium difficile infection. More than one group of investigators has considered using polyclonal bovine colostral antibodies to toxins A and B as an oral passive immunization. A significant proportion of the healthy human population possesses polyclonal antibodies to the Clostridium difficile toxins. We have demonstrated that polyclonal IgA derived from the pooled plasma of healthy donors possesses specificity to toxins A and B and can neutralize these toxins in a cell-based assay. This suggests that secretory IgA prepared from such pooled plasma IgA may be able to be used as an oral treatment for Clostridium difficile infection.

  8. Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion.

    Science.gov (United States)

    Krämer, Christina E M; Wiechert, Wolfgang; Kohlheyer, Dietrich

    2016-01-01

    Conventional propidium iodide (PI) staining requires the execution of multiple steps prior to analysis, potentially affecting assay results as well as cell vitality. In this study, this multistep analysis method has been transformed into a single-step, non-toxic, real-time method via live-cell imaging during perfusion with 0.1 μM PI inside a microfluidic cultivation device. Dynamic PI staining was an effective live/dead analytical tool and demonstrated consistent results for single-cell death initiated by direct or indirect triggers. Application of this method for the first time revealed the apparent antibiotic tolerance of wild-type Corynebacterium glutamicum cells, as indicated by the conversion of violet fluorogenic calcein acetoxymethyl ester (CvAM). Additional implementation of this method provided insight into the induced cell lysis of Escherichia coli cells expressing a lytic toxin-antitoxin module, providing evidence for non-lytic cell death and cell resistance to toxin production. Finally, our dynamic PI staining method distinguished necrotic-like and apoptotic-like cell death phenotypes in Saccharomyces cerevisiae among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The combination of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally resolved observations that deliver new insights into the dynamics of cellular behaviour. PMID:27580964

  9. An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli.

    Science.gov (United States)

    Yamaguchi, Yoshihiro; Inouye, Masayori

    2015-12-01

    Almost all free-living bacteria contain toxin-antitoxin (TA) systems on their genomes and the targets of toxins are highly diverse. Here, we found a novel, previously unidentified TA system in Escherichia coli named yjhX-yjhQ. Induction of YjhX (85 amino acid residues) causes cell-growth arrest resulting in cell death, while YjhQ (181 residues) co-induction resumes cell growth. The primary cellular target of YjhX was found to be topoisomerase I (TopA), inhibiting both DNA replication and RNA synthesis. Notably, YjhX has no homology to any other toxins of the TA systems. YjhX was expressed well with an N-terminal protein S (PrS) tag in soluble forms. PrS-YjhX specifically interacts with the N-terminal region of TopA (TopA67) but not full-TopA in the absence of plasmid DNA, while PrS-YjhX binds to full-TopA in the presence of DNA. Notably, YjhX does not directly interact with DNA and RNA. YjhX inhibits only topoisomerase I but not topoisomerase III and IV in vitro. Hence, yjhX is renamed as the gene for the TopA inhibitor (the topAI gene). TopAI is the first endogenous protein inhibitor specific for topoisomerase I. PMID:26553797

  10. Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain.

    Science.gov (United States)

    Pellett, Sabine; Tepp, William H; Bradshaw, Marite; Kalb, Suzanne R; Dykes, Janet K; Lin, Guangyun; Nawrocki, Erin M; Pier, Christina L; Barr, John R; Maslanka, Susan E; Johnson, Eric A

    2016-01-01

    to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a "treatment-resistant" and highly potent toxin. However, the toxin's chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons. PMID:27303710

  11. Revisiting the Concept of Targeting Only Bacillus anthracis Toxins as a Treatment for Anthrax.

    Science.gov (United States)

    Glinert, Itai; Bar-David, Elad; Sittner, Assa; Weiss, Shay; Schlomovitz, Josef; Ben-Shmuel, Amir; Mechaly, Adva; Altboum, Zeev; Kobiler, David; Levy, Haim

    2016-08-01

    Protective antigen (PA)-based vaccines are effective in preventing the development of fatal anthrax disease both in humans and in relevant animal models. The Bacillus anthracis toxins lethal toxin (lethal factor [LF] plus PA) and edema toxin (edema factor [EF] plus PA) are essential for the establishment of the infection, as inactivation of these toxins results in attenuation of the pathogen. Since the toxins reach high toxemia levels at the bacteremic stages of the disease, the CDC's recommendations include combining antibiotic treatment with antitoxin (anti-PA) immunotherapy. We demonstrate here that while treatment with a highly potent neutralizing monoclonal antibody was highly efficient as postexposure prophylaxis treatment, it failed to protect rabbits with any detectable bacteremia (≥10 CFU/ml). In addition, we show that while PA vaccination was effective against a subcutaneous spore challenge, it failed to protect rabbits against systemic challenges (intravenous injection of vegetative bacteria) with the wild-type Vollum strain or a toxin-deficient mutant. To test the possibility that additional proteins, which are secreted by the bacteria under pathogenicity-stimulating conditions in vitro, may contribute to the vaccine's potency, we immunized rabbits with a secreted protein fraction from a toxin-null mutant. The antiserum raised against the secreted fraction reacts with the bacteria in an immunofluorescence assay. Immunization with the secreted protein fraction did not protect the rabbits against a systemic challenge with the fully pathogenic bacteria. Full protection was obtained only by a combined vaccination with PA and the secreted protein fraction. Therefore, these results indicate that an effective antiserum treatment in advanced stages of anthrax must include toxin-neutralizing antibodies in combination with antibodies against bacterial cell targets. PMID:27270276

  12. THE EFFECT OF AYURVEDIC DRUGS WHEN USED AS DISEASE MODIFYING ANTIREUMATIC DRUGS (DMARD’S IN AMAVATA (RHEUMATOID ARTHRITIS

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    Panthulu Raghupathi Goud

    2012-02-01

    Full Text Available The Disease Modifying Anti-Rheumatic Drugs (DMARD’S are therapeutic agents which rapidly reduce the intensity of inflammation and facilitate induction of remission. The sages of Ayurveda invented many remedies to combat this disease. Here an effort is made to evaluate once again the efficacy of some of the remedies. Ama and Vata are the two chief pathognomonic factors in causing Amavata. Ama has the qualities of heaviness (guru, unctuousness (Snigdha, immobility (Sthira, bulkiness (Sthula, and sliminess or stickiness (Pichhila. Vata has the properties of lightness (Laghu, dryness (Ruksha, movement (Chala, subtleness (Sukshma, and clearness (Vishada. Ama is the undigested food which results due to Mandagni (sluggish digestive fire which is caused due to various reasons. All types of metabolic fires (Agnis become sluggish in this disease. The stagnant Ama is called Ama visha. Ama is the substance which is the resultant of improper digestion of the food due to hypo-functioning of the gastric juices (Jatharagni. The drugs having the qualities of Tiktam (astringent, Deepana (appetizer and Katu (pungent modify the disease due to their qualities. The purgation property (Virechana guna modifies the process of disease. Castor oil (Eranda Tailam cures Vata diseases. It has been observed that after administration of Castor oil, the fluid from the inflamed joints and tissues has been drained away. Castor oil relieves pain, reduces inflammation and swelling, increases lymphatic circulation, reduces flatulence, stimulates the liver and the gall bladder, and reduces toxins. A scientific study on the effect of castor oil on humans found castor oil to be an antitoxin, and as having an impact on the lymphatic system enhancing the immune functioning of the body. Panchakola churnam is anti-inflammatory; it is an anti-oxidant, an immunomodulator, and a rejuvenator too. Hingu Triguna Tailam is digestive, carminative, analgesic and anti-rheumatic.

  13. YgfX (CptA) is a multimeric membrane protein that interacts with the succinate dehydrogenase assembly factor SdhE (YgfY).

    Science.gov (United States)

    McNeil, Matthew B; Iglesias-Cans, Marina C; Clulow, James S; Fineran, Peter C

    2013-07-01

    Serratia sp. strain ATCC 39006 produces the red-pigmented antibiotic prodigiosin. Prodigiosin biosynthesis is regulated by a complex hierarchy that includes the uncharacterized protein YgfX (DUF1434). The ygfX gene is co-transcribed with sdhE, an FAD assembly factor essential for the flavinylation and activation of the SdhA subunit of succinate dehydrogenase (SDH), a central enzyme in the tricarboxylic acid cycle and electron transport chain. The sdhEygfX operon is highly conserved within the Enterobacteriaceae, suggesting that SdhE and YgfX function together. We performed an extensive mutagenesis to gain molecular insights into the uncharacterized protein YgfX, and have investigated the relationship between YgfX and SdhE. YgfX localized to the membrane, interacted with itself, forming dimers or larger multimers, and interacted with SdhE. The transmembrane helices of YgfX were critical for protein function and the formation of YgfX multimers. Site-directed mutagenesis of residues conserved in DUF1434 proteins revealed a periplasmic tryptophan and a cytoplasmic aspartate that were crucial for YgfX activity. Both of these amino acids were required for the formation of YgfX multimers and interactions with SdhE but not membrane localization. Multiple cell division proteins were identified as putative interaction partners of YgfX and overexpression of YgfX had effects on cell morphology. These findings represent an important step in understanding the function of DUF1434 proteins. In contrast to a recent report, we found no evidence that YgfX and SdhE form a toxin-antitoxin system. In summary, YgfX functions as a multimeric membrane-bound protein that interacts with SdhE, an important FAD assembly factor that controls SDH activity. PMID:23657679

  14. Clostridium botulinum type D/C intoxication in a dairy cow stock in Saxony-Anhalt (Germany)--report on an innovative diagnostic approach.

    Science.gov (United States)

    Dlabola, Janine; Hashish, Emad; Pauly, Birgit; Kubisiak, Bernd; Behm, Ingrid; Heseler, Rüdiger; Schliephake, Annette; Wieler, Lothar H; Neubauer, Heinrich; Seyboldt, Christian

    2016-01-01

    Botulism in cattle is a rare but serious disease. In Germany there is no obligation to report botulism in animals and therefore a precise morbidity rate is not available. In this manuscript we describe an outbreak of Clostridium (C.) botulinum neurotoxin (BoNT) intoxication in a Saxony-Anhalt dairy cow stock of 286 Holstein-Friesian cows and offspring in spring/summer 2009 and its diagnostic approach. 122 animals showed clinical signs of BoNT intoxication. 115 of the affected animals (40.2% of the herd) independent of age died or had to be euthanized. Therapeutic attempts failed in almost all diseased cows, only four calves and three heifers recovered. Diagnostic samples of several animals (n = 4) (liver, ruminal and intestinal contents) and feed (n = 6) were tested for BoNT genes by polymerase chain reaction (PCR). BoNT gene type D was found in several (n = 8) organ samples. The PCR results allowed a preselection of samples for BoNT that were then tested by the mouse bioassay. Thus, the number of mice being inoculated in the mouse bioassay could be reduced. The mouse bioassay turned out positive (wasp-waist) in three preselected organ samples and the neutralization test of one sample with type-specific antitoxin confirmed the presence of BoNT type D. We succeeded in isolating a C. botulinum strain from a liver sample which was typed as a D/C mosaic strain by sequence analysis of the toxin gene. However, the source of the BoNT intoxication could not be traced back. PMID:27169148

  15. How to Make a Non-Antigenic Protein (Auto) Antigenic: Molecular Complementarity Alters Antigen Processing and Activates Adaptive-Innate Immunity Synergy.

    Science.gov (United States)

    Root-Bernstein, Robert

    2015-01-01

    Evidence is reviewed that complementary proteins and peptides form complexes with increased antigenicity and/or autoimmunogenicity. Five case studies are highlighted: 1) diphtheria toxin-antitoxin (antibody), which induces immunity to the normally non-antigenic toxin, and autoimmune neuritis; 2) tryptophan peptide of myelin basic protein and muramyl dipeptide ("adjuvant peptide"), which form a complex that induces experimental allergic encephalomyelitis; 3) an insulin and glucagon complex that is far more antigenic than either component individually; 4) various causes of experimental autoimmune myocarditis such as C protein in combination with its antibody, or coxsackie B virus in combination with the coxsackie and adenovirus receptor; 5) influenza A virus haemagglutinin with the outer membrane protein of the Haemophilus influenzae, which increases antigenicity. Several mechanisms cooperate to alter immunogenicity. Complexation alters antigen processing, protecting the components against proteolysis, altering fragmentation and presenting novel antigens to the immune system. Complementary antigens induce complementary adaptive immune responses (complementary antibodies and/or T cell receptors) that produce circulating immune complexes (CIC). CIC stimulate innate immunity. Concurrently, complementary antigens stimulate multiple Toll-like receptors that synergize to over-produce cytokines, which further stimulate adaptive immunity. Thus innate and adaptive immunity form a positive feedback loop. If components of the complex mimic a host protein, then autoimmunity may result. Enhanced antigenicity for production of improved vaccines and/or therapeutic autoimmunity (e.g., against cancer cells) might be achieved by using information from antibody or TCR recognition sites to complement an antigen; by panning for complements in randomized peptide libraries; or using antisense peptide strategies to design complements. PMID:26179268

  16. Molecular basis for the cross-reactivity of antibodies elicited by a natural anatoxin with alpha- and beta-toxins from the venom of Tityus serrulatus scorpion.

    Science.gov (United States)

    Chavez-Olortegui, Carlos; Molina, Franck; Granier, Claude

    2002-03-01

    A non-toxic protein (TsNTxP) isolated from the venom of the noxious scorpion Tityus serrulatus (Ts) induces polyclonal antibodies cross-reactive with several toxins from the venom, in sharp contrast to anti-toxin antibodies which are toxin specific. To try to uncover the molecular basis for these unusual properties, peptide scanning experiments were performed and indicated that the N- and C-terminal parts of TsNTxP enclose continuous epitopes (residues 1-15 and 47-61). Antibodies raised against peptides corresponding to these two regions were found to have neutralizing properties against a mixture of all toxic proteins from the T. serrulatus venom, indicating that residues 1-15 and 47-61 correspond to neutralizing epitopes. The identification of key antigenic residues within these two epitopes revealed that several of them are well conserved in the amino-acid sequences of the three main toxins (Ts II, Ts IV and Ts VII) from the venom: Glu 3, Tyr 5, Asp 8, Asp 50, Trp 55 and Lys 61. A single key-residue (Glu 58) is unique to TsNTxP. By using homology modeling, a model of the three-dimensional structure of TsNTxP was obtained. The antigenically important residues from TsNTxP were found to be surface exposed, with five of them clustered on the facet of the protein reported to enclose the active site of toxins. Residues equivalent to the seven key-residues of the anatoxin were also found to be exposed in the active toxins from T. serrulatus venom. These results show that antibodies elicited by the non-toxic protein TsNTxP recognized, within the N- and C-terminal parts of toxins of T. serrulatus, conserved and surface exposed residues which might also be involved in the toxic action of the proteins. PMID:11922945

  17. Status of vaccine research and development for enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Bourgeois, A Louis; Wierzba, Thomas F; Walker, Richard I

    2016-06-01

    Enterotoxigenic Escherichia coli (ETEC) is one of the most common bacterial causes of diarrhea-associated morbidity and mortality, particularly among infants and young children in developing countries. Still, the true impact on child and traveler health is likely underestimated. There are currently no licensed vaccines for ETEC, but studies indicate high public health impact, cost-effectiveness, and feasibility of immune protection through vaccination. ETEC vaccine development remains a World Health Organization priority. Traditionally, ETEC vaccine development efforts have focused on inducing antitoxin and anticolonization antigen immunity, as studies indicate that antibodies against both antigen types can contribute to protection and thus have potential for vaccines. Leading cellular vaccine candidates are ETVAX (a mixture of four inactivated strains) and ACE527 (a mixture of three live attenuated strains), both of which have been found to be safe and immunogenic in Phase 1/2 trials. ETVAX is the furthest along in development with descending-age studies already underway in Bangladesh. Other ETEC vaccine candidates based on protein subunits, toxoids (both LT and ST), or novel, more broadly conserved ETEC antigens are also under development. Of these, a protein adhesin-based subunit approach is the most advanced. Impact and economic models suggest favorable vaccine cost-effectiveness, which may help expand market interest in ETEC vaccines. Combination vaccine formulations may help improve the economic case for development and use, and better point-of-care diagnostics will help to raise awareness of the true health burden of ETEC and highlight the potential public health benefit of ETEC vaccine introduction. Better diagnostics and vaccine demand forecasting will also improve vaccine development financing and support accelerated uptake once a licensed vaccine becomes available. PMID:26988259

  18. A stable luciferase reporter plasmid for in vivo imaging in murine models of Staphylococcus aureus infections.

    Science.gov (United States)

    Bacconi, Marta; Haag, Andreas F; Torre, Antonina; Castagnetti, Andrea; Chiarot, Emiliano; Delany, Isabel; Bensi, Giuliano

    2016-04-01

    In vivo imaging of bioluminescent bacteria permits their visualization in infected mice, allowing spatial and temporal evaluation of infection progression. Most available bioluminescent strains were obtained by integration of the luciferase genes into the bacterial chromosome, a challenging and time-consuming approach. Recently, episomal plasmids were used, which were introduced in bacteria and expressed all genes required for bioluminescence emission. However, the plasmid was progressively lost in vitro and in vivo, if bacteria were not maintained under antibiotic selective pressure. Increased stability could be obtained inserting into the plasmid backbone sequences that assured plasmid partition between daughter bacterial cells, or caused death of bacteria that had lost the plasmid. So far, no detailed analysis was performed of either plasmid stability in vivo or contribution of different stabilizing sequence types. Here we report the construction of a plasmid, which includes the Photorhabdus luminescens lux cassette expressed under the control of a Staphylococcus aureus specific gene promoter, and toxin/antitoxin (T/A) and partition sequences (Par) conferring stability and transmissibility of the plasmid. Following infection of mice with S. aureus carrying this plasmid, we demonstrated that the promoter-lux fusion was functional in vivo, that the plasmid was retained by 70-100% of bacterial cells 7 days post-infection, and that both stabilizing sequence types were required to maximize plasmid retention. These data suggest that the plasmid can be a valuable tool to study gene expression and bacterial spread in small laboratory animals infected with S. aureus or possibly other Gram-positive human pathogens. PMID:26685857

  19. Saccharomyces boulardii CNCM I-745 supports regeneration of the intestinal microbiota after diarrheic dysbiosis - a review.

    Science.gov (United States)

    Moré, Margret I; Swidsinski, Alexander

    2015-01-01

    The probiotic medicinal yeast Saccharomyces cerevisiae HANSEN CBS 5926 (Saccharomyces boulardii CNCM I-745) is used for the prevention and treatment of diarrhea. Its action is based on multiple mechanisms, including immunological effects, pathogen-binding and antitoxinic effects, as well as effects on digestive enzymes. Correlated with these effects, but also due to its inherent properties, S. boulardii is able to create a favorable growth environment for the beneficial intestinal microbiota, while constituting extra protection to the host mucus layer and mucosa. This review focuses on the positive influence of S. boulardii on the composition of the intestinal microbiota. In a dysbiosis, as during diarrhea, the main microbial population (especially Lachnospiraceae, Ruminococcaceae, Bacteroidaceae, and Prevotellaceae) is known to collapse by at least one order of magnitude. This gap generally leads to transient increases in pioneer-type bacteria (Enterobacteriaceae, Bifidobacteriaceae, and Clostridiaceae). Several human studies as well as animal models demonstrate that treatment with S. boulardii in dysbiosis leads to the faster reestablishment of a healthy microbiome. The most relevant effects of S. boulardii on the fecal composition include an increase of short chain fatty acid-producing bacteria (along with a rise in short chain fatty acids), especially of Lachnospiraceae and Ruminococcaceae, as well as an increase in Bacteroidaceae and Prevotellaceae. At the same time, there is a suppression of pioneer bacteria. The previously observed preventive action of S. boulardii, eg, during antibiotic therapy or regarding traveler's diarrhea, can be explained by several mechanisms, including a stabilizing effect on the healthy microbiota as well as possibly on the mucus layer. Several different dysbiotic situations could profit from the effects of S. boulardii CNCM I-745. Its additional potential lies in a general stabilization of the gut flora for at-risk populations

  20. Clinical and neurophysiological features of tick paralysis.

    Science.gov (United States)

    Grattan-Smith, P J; Morris, J G; Johnston, H M; Yiannikas, C; Malik, R; Russell, R; Ouvrier, R A

    1997-11-01

    The clinical and neurophysiological findings in six Australian children with generalized tick paralysis are described. Paralysis is usually caused by the mature female of the species Ixodes holocyclus. It most frequently occurs in the spring and summer months but can be seen at any time of year. Children aged 1-5 years are most commonly affected. The tick is usually found in the scalp, often behind the ear. The typical presentation is a prodrome followed by the development of an unsteady gait, and then ascending, symmetrical, flaccid paralysis. Early cranial nerve involvement is a feature, particularly the presence of both internal and external ophthalmoplegia. In contrast to the experience with North American ticks, worsening of paralysis in the 24-48 h following tick removal is common and the child must be carefully observed over this period. Death from respiratory failure was relatively common in the first half of the century and tick paralysis remains a potentially fatal condition. Respiratory support may be required for > 1 week but full recovery occurs. This is slow with several weeks passing before the child can walk unaided. Anti-toxin has a role in the treatment of seriously ill children but there is a high incidence of acute allergy and serum sickness. Neurophysiological studies reveal low-amplitude compound muscle action potentials with normal motor conduction velocities, normal sensory studies and normal response to repetitive stimulation. The biochemical structure of the toxin of I. holocyclus has not been fully characterized but there are many clinical, neurophysiological and experimental similarities to botulinum toxin. PMID:9397015

  1. Monitoring in real time the cytotoxic effect of Clostridium difficile upon the intestinal epithelial cell line HT29.

    Science.gov (United States)

    Valdés, Lorena; Gueimonde, Miguel; Ruas-Madiedo, Patricia

    2015-12-01

    The incidence and severity of Clostridium difficile infections (CDI) has been increased not only among hospitalized patients, but also in healthy individuals traditionally considered as low risk population. Current treatment of CDI involves the use of antibiotics to eliminate the pathogen, although recurrent relapses have also been reported. For this reason, the search of new antimicrobials is a very active area of research. The strategy to use inhibitors of toxin's activity has however been less explored in spite of being a promising option. In this regard, the lack of fast and reliable in vitro screening methods to search for novel anti-toxin drugs has hampered this approach. The aim of the current study was to develop a method to monitor in real time the cytotoxicity of C. difficile upon the human colonocyte-like HT29 line, since epithelial intestinal cells are the primary targets of the toxins. The label-free, impedance based RCTA (real time cell analyser) technology was used to follow overtime the behaviour of HT29 in response to C. difficile LMG21717 producing both A and B toxins. Results obtained showed that the selection of the medium to grow the pathogen had a great influence in obtaining toxigenic supernatants, given that some culture media avoided the release of the toxins. A cytotoxic dose- and time-dependent effect of the supernatant obtained from GAM medium upon HT29 and Caco2 cells was detected. The sigmoid-curve fit of data obtained with HT29 allowed the calculation of different toxicological parameters, such as EC50 and LOAEL values. Finally, the modification in the behaviour of HT29 reordered in the RTCA was correlated with the cell rounding effect, typically induced by these toxins, visualized by time-lapsed captures using an optical microscope. Therefore, this RTCA method developed to test cytotoxicity kinetics of C. difficile supernatants upon IEC could be a valuable in vitro model for the screening of new anti-CDI agents. PMID:26436983

  2. Sequencing and characterizing the genome of Estrella lausannensis as an undergraduate project: training students and biological insights.

    Science.gov (United States)

    Bertelli, Claire; Aeby, Sébastien; Chassot, Bérénice; Clulow, James; Hilfiker, Olivier; Rappo, Samuel; Ritzmann, Sébastien; Schumacher, Paolo; Terrettaz, Céline; Benaglio, Paola; Falquet, Laurent; Farinelli, Laurent; Gharib, Walid H; Goesmann, Alexander; Harshman, Keith; Linke, Burkhard; Miyazaki, Ryo; Rivolta, Carlo; Robinson-Rechavi, Marc; van der Meer, Jan Roelof; Greub, Gilbert

    2015-01-01

    With the widespread availability of high-throughput sequencing technologies, sequencing projects have become pervasive in the molecular life sciences. The huge bulk of data generated daily must be analyzed further by biologists with skills in bioinformatics and by "embedded bioinformaticians," i.e., bioinformaticians integrated in wet lab research groups. Thus, students interested in molecular life sciences must be trained in the main steps of genomics: sequencing, assembly, annotation and analysis. To reach that goal, a practical course has been set up for master students at the University of Lausanne: the "Sequence a genome" class. At the beginning of the academic year, a few bacterial species whose genome is unknown are provided to the students, who sequence and assemble the genome(s) and perform manual annotation. Here, we report the progress of the first class from September 2010 to June 2011 and the results obtained by seven master students who specifically assembled and annotated the genome of Estrella lausannensis, an obligate intracellular bacterium related to Chlamydia. The draft genome of Estrella is composed of 29 scaffolds encompassing 2,819,825 bp that encode for 2233 putative proteins. Estrella also possesses a 9136 bp plasmid that encodes for 14 genes, among which we found an integrase and a toxin/antitoxin module. Like all other members of the Chlamydiales order, Estrella possesses a highly conserved type III secretion system, considered as a key virulence factor. The annotation of the Estrella genome also allowed the characterization of the metabolic abilities of this strictly intracellular bacterium. Altogether, the students provided the scientific community with the Estrella genome sequence and a preliminary understanding of the biology of this recently-discovered bacterial genus, while learning to use cutting-edge technologies for sequencing and to perform bioinformatics analyses. PMID:25745418

  3. The Bacillus anthracis cholesterol-dependent cytolysin, Anthrolysin O, kills human neutrophils, monocytes and macrophages

    Directory of Open Access Journals (Sweden)

    Rest Richard F

    2006-06-01

    Full Text Available Abstract Background Bacillus anthracis is an animal and human pathogen whose virulence is characterized by lethal and edema toxin, as well as a poly-glutamic acid capsule. In addition to these well characterized toxins, B. anthracis secretes several proteases and phospholipases, and a newly described toxin of the cholesterol-dependent cytolysin (CDC family, Anthrolysin O (ALO. Results In the present studies we show that recombinant ALO (rALO or native ALO, secreted by viable B. anthracis, is lethal to human primary polymorphonuclear leukocytes (PMNs, monocytes, monocyte-derived macrophages (MDMs, lymphocytes, THP-1 monocytic human cell line and ME-180, Detroit 562, and A549 epithelial cells by trypan blue exclusion or lactate dehydrogenase (LDH release viability assays. ALO cytotoxicity is dose and time dependent and susceptibility to ALO-mediated lysis differs between cell types. In addition, the viability of monocytes and hMDMs was assayed in the presence of vegetative Sterne strains 7702 (ALO+, UT231 (ALO-, and a complemented strain expressing ALO, UT231 (pUTE544, and was dependent upon the expression of ALO. Cytotoxicity of rALO is seen as low as 0.070 nM in the absence of serum. All direct cytotoxic activity is inhibited by the addition of cholesterol or serum concentration as low as 10%. Conclusion The lethality of rALO and native ALO on human monocytes, neutrophils, macrophages and lymphocytes supports the idea that ALO may represent a previously unidentified virulence factor of B. anthracis. The study of other factors produced by B. anthracis, along with the major anthrax toxins, will lead to a better understanding of this bacterium's pathogenesis, as well as provide information for the development of antitoxin vaccines for treating and preventing anthrax.

  4. Bacterial persistence: some new insights into an old phenomenon

    Indian Academy of Sciences (India)

    R Jayaraman

    2008-12-01

    Bigger discovered more than 60 years ago, at the very beginning of the antibiotic era, that populations of antibiotic-sensitive bacteria contained a very small fraction (approximately 10–6) of antibiotic-tolerant cells (persisters). Persisters are different from antibiotic-resistant mutants in that their antibiotic tolerance is non-heritable and reversible. In spite of its importance as an interesting biological phenomenon and in the treatment of infectious diseases, persistence did not attract the attention of the scientific community for more than four decades since its discovery. The main reason for this lack of interest was the difficulty in isolating sufficient numbers of persister cells for experimentation, since the proportion of persisters in a population of wild-type cells is extremely small. However, with the discovery of high-persister (hip) mutants of Escherichia coli by Moyed and his group in the early 1980s, the phenomenon attracted the attention of many groups and significant progress has occurred since then. It is now believed that persistence is the end result of a stochastic switch in the expression of some toxin–antitoxin (TA) modules (of which the hipA and hipB genes could be examples), creating an imbalance in their intracellular levels. There are also models invoking the involvement of the alarmone (p) ppGpp in the generation of persisters. However, the precise mechanisms are still unknown. Bacterial persistence is part of a wider gamut of phenomena variously called as bistability, multistability, phenotypic heterogeneity, stochastic switching processes, etc. It has attracted the attention of not only microbiologists but also a diverse band of researchers such as biofilm researchers, evolutionary biologists, sociobiologists, etc. In this article, I attempt to present a broad overview of bacterial persistence to illustrate its significance and the need for further exploration.

  5. Botulism: Pathogenesis, Diagnosis and Prevention

    Directory of Open Access Journals (Sweden)

    lili natalia

    2012-09-01

    Full Text Available Botulism is a potential lethal disease in animals as well as in human, a neuroparalytic disease caused by Clostridium botulinum toxin. C. botulinum is widely distributed in the soil and vegetation, intestinal contents of mammals, birds and fish. Eight types of C. botulinum (A, B, C1, C2, D, E, F, G have been recognized, each elaborating an immunologically distinct form of toxin. Botulinum neurotoxins are the most powerful biological toxins known and in some countries they have been studied and developed as biological weapon. The medical aspects of the toxin were also developed for therapeutic uses in human diseases. The spores of C. botulinum are relatively heat resistant and in contrast to the spores, botulinum toxin is relatively heat labile. Botulinum toxins are inactivated by their antitoxins. Botulinum toxin produces clinical manifestations when either inhaled or ingested. After toxin is absorbed, it enters the bloodstream and travels to peripheral cholinergic synapses, primarily the neuromuscular junction. Once at these sites, botulinum toxin is internalized and enzymatically prevents the release of acteylcholine leads to paralysis. Laboratory diagnoses for botulism should include isolating C. botulinum and detecting of toxin in the patient. Rapid and sensitive detection of all types of botulinum toxin are needed. Cases of botulism in Indonesia were found primarily in poultry and many cases were suspected and remained undiagnosed. Cases of botulism were suspected affecting cattle in East Java and serologically results showed positive to C. botulinum type C. The botulismus prevention using vaccine induced a strong antibody response and could be remained protective for 12 months, while botulism treatment in animals is usually ineffective.

  6. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B

    Directory of Open Access Journals (Sweden)

    Yongfeng Fan

    2015-08-01

    Full Text Available Existing antibodies (Abs used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B contains a zinc endopeptidase light chain (LC domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS. The equilibrium dissociation constants (KD of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM. Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

  7. Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity.

    Directory of Open Access Journals (Sweden)

    Yongfeng Fan

    Full Text Available The paralytic disease botulism is caused by botulinum neurotoxins (BoNT, multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC of BoNT serotype A (BoNT/A was targeted for generation of monoclonal antibodies (mAbs that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS. Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10-11 M to 3.53×10-8 M (mean KD 5.38×10-9 M and median KD 1.53×10-9 M, as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10-9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors.

  8. ``Black Holes" and Bacterial Pathogenicity: A Large Genomic Deletion that Enhances the Virulence of Shigella spp. and Enteroinvasive Escherichia coli

    Science.gov (United States)

    Maurelli, Anthony T.; Fernandez, Reinaldo E.; Bloch, Craig A.; Rode, Christopher K.; Fasano, Alessio

    1998-03-01

    Plasmids, bacteriophages, and pathogenicity islands are genomic additions that contribute to the evolution of bacterial pathogens. For example, Shigella spp., the causative agents of bacillary dysentery, differ from the closely related commensal Escherichia coli in the presence of a plasmid in Shigella that encodes virulence functions. However, pathogenic bacteria also may lack properties that are characteristic of nonpathogens. Lysine decarboxylate (LDC) activity is present in ≈ 90% of E. coli strains but is uniformly absent in Shigella strains. When the gene for LDC, cadA, was introduced into Shigella flexneri 2a, virulence became attenuated, and enterotoxin activity was inhibited greatly. The enterotoxin inhibitor was identified as cadaverine, a product of the reaction catalyzed by LDC. Comparison of the S. flexneri 2a and laboratory E. coli K-12 genomes in the region of cadA revealed a large deletion in Shigella. Representative strains of Shigella spp. and enteroinvasive E. coli displayed similar deletions of cadA. Our results suggest that, as Shigella spp. evolved from E. coli to become pathogens, they not only acquired virulence genes on a plasmid but also shed genes via deletions. The formation of these ``black holes,'' deletions of genes that are detrimental to a pathogenic lifestyle, provides an evolutionary pathway that enables a pathogen to enhance virulence. Furthermore, the demonstration that cadaverine can inhibit enterotoxin activity may lead to more general models about toxin activity or entry into cells and suggests an avenue for antitoxin therapy. Thus, understanding the role of black holes in pathogen evolution may yield clues to new treatments of infectious diseases.

  9. A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins.

    Directory of Open Access Journals (Sweden)

    Preeti Sharma

    Full Text Available Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1 and streptococcal (SpeA and SpeC toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD50 values of 400 pg/ml (SEA, 3 pg/ml (SEB, 25 pg/ml (TSST-1, 6 ng/ml (SpeA, and 100 pg/ml (SpeC. These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.

  10. Construction of a Vibrio cholerae prototype vaccine strain O395-N1-E1 which accumulates cell-associated cholera toxin B subunit.

    Science.gov (United States)

    Rhie, Gi-eun; Jung, Hae-Mi; Kim, Bong Su; Mekalanos, John J

    2008-10-01

    Because of its production and use in Vietnam, the most widely used oral cholera vaccine consists of heat- or formalin-killed Vibrio cholerae whole cells (WC). An earlier version of this type of vaccine called whole cell-recombinant B subunit vaccine (BS-WC) produced in Sweden also contained the B subunit of cholera toxin (CTB). Both WC and BS-WC vaccines produced moderate levels of protection in field trials designed to evaluate their cholera efficacy. V. cholerae cells in these vaccines induce antibacterial immunity, and CTB contributes to the vaccine's efficacy presumably by stimulating production of anti-toxin neutralizing antibody. Although more effective than the WC vaccine, the BS-WC vaccine has not been adopted for manufacture by developing world countries primarily because the CTB component is difficult to manufacture and include in the vaccine in the doses needed to induce significant immune responses. We reasoned this was a technical problem that might be solved by engineering strains of V. cholerae that express cell-associated CTB that would co-purify with the bacterial cell fraction during the manufacture of WC vaccine. Here we report that construction of a V. cholerae O1 classical strain, O395-N1-E1, that has been engineered to accumulate CTB in the periplasmic fraction by disrupting the epsE gene of type II secretion pathway. O395-N1-E1 induces anti-CTB IgG and vibriocidal antibodies in mice immunized with two doses of formalin killed whole cells. Intraperitoneal immunization of mice with O395-N1-E1 induced a significantly higher anti-CTB antibody response compared to that of the parental strain, O395-N1. Our results suggest that this prototype cholera vaccine candidate strain may assist in preparing improved and inexpensive oral BS-WC cholera vaccine without the need to purify CTB separately. PMID:18582519

  11. Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation.

    Science.gov (United States)

    Lu, Canhua; Nakayasu, Ernesto S; Zhang, Li-Qun; Luo, Zhao-Qing

    2016-01-01

    The morphology of bacterial cells is important for virulence, evasion of the host immune system, and coping with environmental stresses. The widely distributed Fic proteins (filamentation induced by cAMP) are annotated as proteins involved in cell division because of the presence of the HPFx[D/E]GN[G/K]R motif. We showed that the presence of Fic-1 from Pseudomonas fluorescens significantly reduced the yield of plasmid DNA when expressed in Escherichia coli or P. fluorescens. Fic-1 interacted with GyrB, a subunit of DNA gyrase, which is essential for bacterial DNA replication. Fic-1 catalyzed the AMPylation of GyrB at Tyr(109), a residue critical for binding ATP, and exhibited auto-AMPylation activity. Mutation of the Fic-1 auto-AMPylated site greatly reduced AMPylation activity toward itself and toward GyrB. Fic-1-dependent AMPylation of GyrB triggered the SOS response, indicative of DNA replication stress or DNA damage. Fic-1 also promoted the formation of elongated cells when the SOS response was blocked. We identified an α-inhibitor protein that we named anti-Fic-1 (AntF), encoded by a gene immediately upstream of Fic-1. AntF interacted with Fic-1, inhibited the AMPylation activity of Fic-1 for GyrB in vitro, and blocked Fic-1-mediated inhibition of DNA replication in bacteria, suggesting that Fic-1 and AntF comprise a toxin-antitoxin module. Our work establishes Fic-1 as an AMPylating enzyme that targets GyrB to inhibit DNA replication and may target other proteins to regulate bacterial morphology. PMID:26814232

  12. "Isolation of Clostridum botiulinum (Types A, B, E in Sediments from Coastal Areas in the North of Iran"

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    HR Tavakoli

    2003-09-01

    Full Text Available Iranian J Publ Health, Vol. 32, No. 3, pp.37-40, 2003 Isolation of Clostridum botiulinum (Types A, B, E in Sediments from Coastal Areas in the North of Iran *HR Tavakoli 1,V Razavilar 2 1Dept. of Nutrition and Food Hygiene,School of public health, Baghyatollah University of Medical Sciences, Tehran, Iran 2Dept. of Food Hygiene, School of Veterinary Medicine, Tehran University , Iran Clostridium botulinum has long been recognized as an etiological agent of food borne botulism and has been reported as an important food safety hazard. The aim of this study was to obtain information about C. botulinum distribution in north areas sediments of Iran in order to ascertain the risks associated with consumption and processing of fish from these waters. Two hundred and seventy samples of sediments from coastal areas of "Gilan" and "Mazandaran" provinas of Iran were collected and analyzed. The supernatants were inoculated into cooked meat media and then , grown specimens were stained and cheked microscopically. After centrifuge ,the supernatants were divided into three (untreated ,heated , and tripsinised and toxicity of them were tested by mouse bioassay. All mice controlled for 4 days for symptoms of botulism. Monovalent standard antitoxins were used for detection of toxin type. The present study revealed that the prevalence of C. botulinum (types A, B and E in sediments from different areas of Gillan and Mazandaran was 3.6% and 4.6% respectively, Mean prevalence of C. botulinum in sediments from north regions of Iran was 4.1%. It is also demonstrated that C. botulinum type E is predominant type seen in aquatic environments of the coastal areas of Iran. This is the first report of C. botulinum distribution in the sediments coastal areas of Iran. Introduction *Corresponding author: +98 21 4811483

  13. Regulatory RNAs in the Less Studied Streptococcal Species: From Nomenclature to Identification

    Science.gov (United States)

    Zorgani, Mohamed A.; Quentin, Roland; Lartigue, Marie-Frédérique

    2016-01-01

    Streptococcal species are Gram-positive bacteria involved in severe and invasive diseases in humans and animals. Although, this group includes different pathogenic species involved in life-threatening infections for humans, it also includes beneficial species, such as Streptococcus thermophilus, which is used in yogurt production. In bacteria virulence factors are controlled by various regulatory networks including regulatory RNAs. For clearness and to develop logical thinking, we start this review with a revision of regulatory RNAs nomenclature. Previous reviews are mostly dealing with Streptococcus pyogenes and Streptococcus pneumoniae regulatory RNAs. We especially focused our analysis on regulatory RNAs in Streptococcus agalactiae, Streptococcus mutans, Streptococcus thermophilus and other less studied Streptococcus species. Although, S. agalactiae RNome remains largely unknown, sRNAs (small RNAs) are supposed to mediate regulation during environmental adaptation and host infection. In the case of S. mutans, sRNAs are suggested to be involved in competence regulation, carbohydrate metabolism, and Toxin–Antitoxin systems. A new category of miRNA-size small RNAs (msRNAs) was also identified for the first time in this species. The analysis of S. thermophilus sRNome shows that many sRNAs are associated to the bacterial immune system known as CRISPR-Cas system. Only few of the other different Streptococcus species have been the subject of studies pointed toward the characterization of regulatory RNAs. Finally, understanding bacterial sRNome can constitute one step forward to the elaboration of new strategies in therapy such as substitution of antibiotics in the management of S. agalactiae neonatal infections, prevention of S. mutans dental caries or use of S. thermophilus CRISPR-Cas system in genome editing applications. PMID:27507970

  14. A palindromic CpG-containing phosphodiester oligodeoxynucleotide as a mucosal adjuvant stimulates plasmacytoid dendritic cell-mediated T(H1 immunity.

    Directory of Open Access Journals (Sweden)

    Jun-ichi Maeyama

    Full Text Available BACKGROUND: CpG oligodeoxynucleotides (ODNs, resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC-mediated T(H1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA. METHODS: T(H1 and T(H2 immunity activation was evaluated by cytokine production pattern and T-bet/GATA-3 ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT through nasal vaccination. RESULTS: G9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased T-bet/GATA-3 ratio by enhancing T-bet expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of T(H1, but not T(H2-type Abs depended completely on pDCs, the first in vivo demonstration by CpG ODNs. CONCLUSIONS: G9.1 is a promising mucosal adjuvant for induction of pDC-mediated T(H1 immunity.

  15. History of the use of immunoglobulin%免疫球蛋白治疗发展简史

    Institute of Scientific and Technical Information of China (English)

    项宁; 王晶; 胡文立

    2011-01-01

    1890年,德国学者Von Behring发现了动物血清抗毒素.由于注射动物血清易发生过敏反应,Cenci在1907年改用恢复期病人的血清来替代动物血清.1937年,Tiselius运用电泳技术从血清中分离出免疫球蛋白.1940年,Cohn等人发表了免疫球蛋白的制备过程.第2次世界大战之后,免疫球蛋白开始用于民用,主要用于预防和治疗感染性疾病.1952年,Bruton首次发现原发性免疫缺陷病患者血清中缺少Υ-球蛋白,并应用免疫球蛋白进行治疗.此后,免疫球蛋白开始用于治疗免疫缺陷性疾病.1981年,Imbach应用免疫球蛋白成功治疗了特发性血小板减少性紫癜.自此,免疫球蛋白开始用于治疗自身免疫性疾病.%In 1890,the German scholar Von Behring found animal serum antitoxin.In 1940,Cenci advocated to use recovery-stage patients' serum instead of animal serum for its anaphylactic reaction.In 1973,Tiselius separated immunoglobulin from serum with electrophoresis techniques.In 1940,immunoglobulin' s preparation process was published by Cohn and his colleagues.After the Second World War,immunoglobulin was used by civilians for prevention and treatment of infectious diseases.Bruton found the lack ofΥ- globulins in primaryimmunodeficiency patients in 1952,and immunoglobulin was used to treat this disease.From then on,immunoglobulin began to be used for immunodeficiency disease.In 1981,Imbach achieved success in idiopathic thrombocytopenic purpura with immunoglobulin.Immunoglobulin came into use for autoimmune disease subsequent to that.

  16. Venom conjugated polylactide applied as biocompatible material for passive and active immunotherapy against scorpion envenomation.

    Science.gov (United States)

    Ayari-Riabi, Sana; Trimaille, Thomas; Mabrouk, Kamel; Bertin, Denis; Gigmes, Didier; Benlasfar, Zakaria; Zaghmi, Ahlem; Bouhaouala-Zahar, Balkiss; Elayeb, Mohamed

    2016-04-01

    Scorpion envenoming represents a public health issue in subtropical regions of the world. Treatment and prevention need to promote antitoxin immunity. Preserving antigenic presentation while removing toxin effect remains a major challenge in toxin vaccine development. Among particulate adjuvant, particles prepared with poly (d,l-lactide) polymer are the most extensively investigated due to their excellent biocompatibility and biodegradability. The aim of this study is to develop surfactant-free PLA nanoparticles that safely deliver venom toxic fraction to enhance specific immune response. PLA nanoparticles are coated with AahG50 (AahG50/PLA) and BotG50 (BotG50/PLA): a toxic fraction purified from Androctonus australis hector and Buthus occitanus tunetanus venoms, respectively. Residual toxicities are evaluated following injections of PLA-containing high doses of AahG50 (or BotG50). Immunization trials are performed with the detoxified fraction administered alone without adjuvant. A comparative study of the effect of Freund is also included. The neutralizing capacity of sera is determined in naive mice. Six months later, immunized mice are challenged subcutaneously with increased doses of AahG50. Subcutaneous lethal dose 50 (LD50) of AahG50 and BotG50 is of 575μg/kg and 1300μg/kg respectively. By comparison, BotG50/PLA is totally innocuous while 50% of tested mice survive 2875μg AahG50/kg. Alhydrogel and Freund are not able to detoxify such a high dose. Cross-antigenicity between particulate and soluble fraction is also, ensured. AahG50/PLA and BotG50/PLA induce high antibody levels in mice serum. The neutralizing capacity per mL of anti-venom was 258μg/mL and 186μg/mL calculated for anti-AahG50/PLA and anti-BotG50/PLA sera, respectively. Animals immunized with AahG50/PLA are protected against AahG50 injected dose of 3162μg/kg as opposed all non-immunized mice died at this dose. We find that the detoxification approach based PLA nanoparticles, benefit the

  17. Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A

    Science.gov (United States)

    Benoit, Roger M.; Frey, Daniel; Hilbert, Manuel; Kevenaar, Josta T.; Wieser, Mara M.; Stirnimann, Christian U.; McMillan, David; Ceska, Tom; Lebon, Florence; Jaussi, Rolf; Steinmetz, Michel O.; Schertler, Gebhard F. X.; Hoogenraad, Casper C.; Capitani, Guido; Kammerer, Richard A.

    2014-01-01

    Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral β-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open β-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.

  18. Encapsulated living choroid plexus cells: potential long-term treatments for central nervous system disease and trauma

    Science.gov (United States)

    Skinner, S. J. M.; Geaney, M. S.; Lin, H.; Muzina, M.; Anal, A. K.; Elliott, R. B.; Tan, P. L. J.

    2009-12-01

    In neurodegenerative disease and in acute brain injury, there is often local up-regulation of neurotrophin production close to the site of the lesion. Treatment by direct injection of neurotrophins and growth factors close to these lesion sites has repeatedly been demonstrated to improve recovery. It has therefore been proposed that transplanting viable neurotrophin-producing cells close to the trauma lesion, or site of degenerative disease, might provide a novel means for continuous delivery of these molecules directly to the site of injury or to a degenerative region. The aim of this paper is to summarize recent published information and present new experimental data that indicate that long-lasting therapeutic implants of choroid plexus (CP) neuroepithelium may be used to treat brain disease. CP produces and secretes numerous biologically active neurotrophic factors (NT). New gene microarray and proteomics data presented here indicate that many other anti-oxidant, anti-toxin and neuronal support proteins are also produced and secreted by CP cells. In the healthy brain, these circulate in the cerebrospinal fluid through the brain and spinal cord, maintaining neuronal networks and associated cells. Recent publications describe how transplanted CP cells and tissue, either free or in an immunoprotected encapsulated form, can effectively deliver therapeutic molecules when placed near the lesion or site of degenerative disease in animal models. Using simple techniques, CP neuroepithelial cell clusters in suspension culture were very durable, remaining viable for 6 months or more in vitro. The cell culture conditions had little effect on the wide range and activity of genes expressed and proteins secreted. Recently, completed experiments show that implanting CP within alginate-poly-ornithine capsules effectively protected these xenogeneic cells from the host immune system and allowed their survival for 6 months or more in the brains of rats, causing no adverse effects

  19. Screening of Bifidobacteria and Lactobacilli Able to Antagonize the Cytotoxic Effect of Clostridium difficile upon Intestinal Epithelial HT29 Monolayer

    Science.gov (United States)

    Valdés-Varela, Lorena; Alonso-Guervos, Marta; García-Suárez, Olivia; Gueimonde, Miguel; Ruas-Madiedo, Patricia

    2016-01-01

    showed that this strain prevents HT29 cell rounding; this was achieved by preserving the F-actin microstructure and tight-junctions between adjacent cells, thus keeping the typical epithelium-like morphology. Besides, preliminary evidence showed that the viability of B. longum IPLA20022 is needed to exert the protective effect and that secreted factors seems to have anti-toxin activity. PMID:27148250

  20. Saccharomyces boulardii CNCM I-745 supports regeneration of the intestinal microbiota after diarrheic dysbiosis – a review

    Directory of Open Access Journals (Sweden)

    Moré MI

    2015-08-01

    Full Text Available Margret I Moré,1 Alexander Swidsinski2 1analyze & realize GmbH, Berlin, Germany; 2Laboratory for Molecular Genetics, Polymicrobial Infections and Bacterial Biofilms, Department of Medicine, Gastroenterology, Charité Hospital, CCM, Universitätsmedizin Berlin, Berlin, Germany Abstract: The probiotic medicinal yeast Saccharomyces cerevisiae HANSEN CBS 5926 (Saccharomyces boulardii CNCM I-745 is used for the prevention and treatment of diarrhea. Its action is based on multiple mechanisms, including immunological effects, pathogen-binding and antitoxinic effects, as well as effects on digestive enzymes. Correlated with these effects, but also due to its inherent properties, S. boulardii is able to create a favorable growth environment for the beneficial intestinal microbiota, while constituting extra protection to the host mucus layer and mucosa. This review focuses on the positive influence of S. boulardii on the composition of the intestinal microbiota. In a dysbiosis, as during diarrhea, the main microbial population (especially Lachnospiraceae, Ruminococcaceae, Bacteroidaceae, and Prevotellaceae is known to collapse by at least one order of magnitude. This gap generally leads to transient increases in pioneer-type bacteria (Enterobacteriaceae, Bifidobacteriaceae, and Clostridiaceae. Several human studies as well as animal models demonstrate that treatment with S. boulardii in dysbiosis leads to the faster reestablishment of a healthy microbiome. The most relevant effects of S. boulardii on the fecal composition include an increase of short chain fatty acid-producing bacteria (along with a rise in short chain fatty acids, especially of Lachnospiraceae and Ruminococcaceae, as well as an increase in Bacteroidaceae and Prevotellaceae. At the same time, there is a suppression of pioneer bacteria. The previously observed preventive action of S. boulardii, eg, during antibiotic therapy or regarding traveler’s diarrhea, can be explained by several

  1. Replication and Active Partition of Integrative and Conjugative Elements (ICEs of the SXT/R391 Family: The Line between ICEs and Conjugative Plasmids Is Getting Thinner.

    Directory of Open Access Journals (Sweden)

    Nicolas Carraro

    2015-06-01

    Full Text Available Integrative and Conjugative Elements (ICEs of the SXT/R391 family disseminate multidrug resistance among pathogenic Gammaproteobacteria such as Vibrio cholerae. SXT/R391 ICEs are mobile genetic elements that reside in the chromosome of their host and eventually self-transfer to other bacteria by conjugation. Conjugative transfer of SXT/R391 ICEs involves a transient extrachromosomal circular plasmid-like form that is thought to be the substrate for single-stranded DNA translocation to the recipient cell through the mating pore. This plasmid-like form is thought to be non-replicative and is consequently expected to be highly unstable. We report here that the ICE R391 of Providencia rettgeri is impervious to loss upon cell division. We have investigated the genetic determinants contributing to R391 stability. First, we found that a hipAB-like toxin/antitoxin system improves R391 stability as its deletion resulted in a tenfold increase of R391 loss. Because hipAB is not a conserved feature of SXT/R391 ICEs, we sought for alternative and conserved stabilization mechanisms. We found that conjugation itself does not stabilize R391 as deletion of traG, which abolishes conjugative transfer, did not influence the frequency of loss. However, deletion of either the relaxase-encoding gene traI or the origin of transfer (oriT led to a dramatic increase of R391 loss correlated with a copy number decrease of its plasmid-like form. This observation suggests that replication initiated at oriT by TraI is essential not only for conjugative transfer but also for stabilization of SXT/R391 ICEs. Finally, we uncovered srpMRC, a conserved locus coding for two proteins distantly related to the type II (actin-type ATPase parMRC partitioning system of plasmid R1. R391 and plasmid stabilization assays demonstrate that srpMRC is active and contributes to reducing R391 loss. While partitioning systems usually stabilizes low-copy plasmids, srpMRC is the first to be reported

  2. Inhibition of botulinum toxin's association with rat brain synaptosomes by toosendanin,an antibotulismic triterpenoid

    Institute of Scientific and Technical Information of China (English)

    Jianying Zhou; Yuliang Shi

    2006-01-01

    BACKGROUND:Toosendanin(TSN),a triterpenoid derivative extracted from the bark of Melia toosendanin Sieb et Zucc,has been demonstrated to be an effective drug for treatment of experimental botulism.OBJECTIVE:To explore its antibotulismic mechanism by observing the effect of toosendanin on association of botulinum toxin(BoTx)with rat brain synaptosomes under different conditions.DESIGN:A randomized controlled experiment.SETTING:Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences.MATERIALS:Sprague-Dawley rats,weighing(220±12)g,were involved.TSN was a sample recrystallized in ethanol with a purity>98%.BoTx/A and BoTx/C of 500 ku(1.8x107mouse LD50/mg)were purchased from Wako(Japan),Horse antitoxins to BoTx/A and/C(pdmary antibodies)were purchased from Lanzhou Institute of Biological Products(China).METHODS:Major experiments were finished between March 2005 and October 2005 in key laboratory of neurobiology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences.①Preparation of synaptosome of rats:one aliquot of synaptosome was pre-incubated with TSN(17μmol/L)for 20 minutes at 4℃or 37℃(another aliquot of synaptosome which was untouched was used as control).Then,BoTx was respectively added for another 20-minute incubation.②A homochronous experiment was still Derformed at 37℃.Differently,high level of K+ was used to stimulate synaptosome for 25 minutee.Two aliquots of synaptosomes with or without TSN(17 μmol/L)were preincubated for 15 minutes.Then,30 mmol/L KCl was separately added in two aliquots,5 minutes later,13 nmol/L BoTx/C was separately added followed by 20-minute incubation.[3]The effect of TSN on BoTx binding was observed by Western blot and synchronization method.③ttest was used for comparing the difference of measurement data.MAIN OUTCOME MEASURES:The gray value of BoTx bands from westem blot was used to estimate the bound amount of BoTx.RESULTS:①Preincubation of synaptosomes with TSN(17 μmol/L)at 4

  3. Doctor’s identity in modern Western society

    Directory of Open Access Journals (Sweden)

    KIM Ock-Joo

    2005-06-01

    Full Text Available Two centuries ago doctors perceived themselves quite differently as they do today Doctor’s identity in modern Western society shaped from the modernization of medicine starting in the nineteenth century Modern medicine as practiced today was established from 1800 to the World War I In the eighteenth century three medical groups (physicians surgeons and apothecaries struggled to elevate their position and to organize their education Surgery and surgical education in hospitals developed greatly while physicians tried to theorize their own medical system in the eighteenth century In the early nineteenth century hospital medicine emerged hospitals moved from the place for the poor and the social inadequate to the center of medical education and research Especially French hospitals became the birth places of clinico-pathology new diagnosis with stethoscope careful observation and the numerical method The influence of the hospital medicine spread from France to England America and other parts of Europe After the birth of clinic in France laboratory medicine emerged in Germany France Britain and the United States Surpassing other nations Germany developed university-centered laboratory research system Most of all the reward and status of the laboratory researchers were established so that they could concentrate on their research Although other countries were influenced by German system and knowledge they did not develop research system at the same degree as Germany Rise of scientific medicine transformed self-perception of doctors Science made a great impact not only on the doctors’ practice of medicine but also on the public’s perception of medicine and doctors In the late nineteenth century new discoveries and new armament of scientific medicine marched through antiseptic surgery tropical medicine new laboratories antitoxin therapies from immunology the rise of pharmaceutical industry and the discovery of X-ray Payment system also was changed

  4. 生理盐水联合酒精应用的不同方式对皮试消毒的效果分析%An Analysis on Effect of Different Application Ways of Normal Saline plus Alcohol on Skin Test

    Institute of Scientific and Technical Information of China (English)

    罗艳; 孔碧华; 李冬芬

    2015-01-01

    Objective:To study the sterilizing effect of different application ways of sodium chloride(0 .9% ) plus alcohol(75% ) in the area of skin test .Methods:Select a total of 600 cases of patients who were infused in clinic and has to have a allergy skin test of tetanus antitoxin in some hospital during the period from January 2013 to December , randomly divide them into wiping group and spraying group .The wiping group were wiped with sodium chloride (0 .9% ) firstly ,and then were sterilized with alcohol(75% ) in the same way ,finally took the skin test .The spraying group were sterilized with alcohol(75% ) in the same way above first ,and then were sprayed with the spray bottle containing sodium chloride(0 .9% ) towards the area wiped with alcohol ,dry the sterilized area with sterile swabs and take the skin test .20 minutes later ,Compare and observe the false positive rate after skin test ,infection rate ,the prev‐alence of anaphylactic reaction after medication in the two groups ,sterilizing quantity in sterilized area .Results:False positive rate of the wiping group and spraying group was 0 .7% and 3 .3 respectively (P< 0 .05) .None of them showed any skin infection and allergy in both group .As for the quantity of sterilized bacteria in the two groups after medicating ,the quantity of the wiping group was less than that of spraying group ,the difference was comparatively obvious (P<0 .05) .Conclusion:The safety application of the wipe and spray of normal saline plus and alcohol in the sterilization of skin test lower the false positive rate resulted from skin sanitizer ,alleviate patients'suffering and it is more effective ,saves health resources ,reduces the workload and improves clinical working efficiency .%目的:探讨运用0.9%氯化钠(即生理盐水)联合75%乙醇应用的不同方式对皮试区皮肤消毒效果的研究。方法:选取某院2013年1~12月的门诊输液需进行过伤风抗病毒素过敏皮试的患者共600