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Sample records for antitoxins

  1. 9 CFR 113.451 - Tetanus Antitoxin.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Tetanus Antitoxin. 113.451 Section 113... Products § 113.451 Tetanus Antitoxin. Tetanus Antitoxin is a specific antibody product containing... Tetanus Antitoxin shall yield not less than the labeled unitage of antitoxin throughout the dating...

  2. 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, ...

  3. Toxin-Antitoxin Systems of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Christopher F. Schuster

    2016-05-01

    Full Text Available Toxin-antitoxin (TA systems are small genetic elements found in the majority of prokaryotes. They encode toxin proteins that interfere with vital cellular functions and are counteracted by antitoxins. Dependent on the chemical nature of the antitoxins (protein or RNA and how they control the activity of the toxin, TA systems are currently divided into six different types. Genes comprising the TA types I, II and III have been identified in Staphylococcus aureus. MazF, the toxin of the mazEF locus is a sequence-specific RNase that cleaves a number of transcripts, including those encoding pathogenicity factors. Two yefM-yoeB paralogs represent two independent, but auto-regulated TA systems that give rise to ribosome-dependent RNases. In addition, omega/epsilon/zeta constitutes a tripartite TA system that supposedly plays a role in the stabilization of resistance factors. The SprA1/SprA1AS and SprF1/SprG1 systems are post-transcriptionally regulated by RNA antitoxins and encode small membrane damaging proteins. TA systems controlled by interaction between toxin protein and antitoxin RNA have been identified in S. aureus in silico, but not yet experimentally proven. A closer inspection of possible links between TA systems and S. aureus pathophysiology will reveal, if these genetic loci may represent druggable targets. The modification of a staphylococcal TA toxin to a cyclopeptide antibiotic highlights the potential of TA systems as rather untapped sources of drug discovery.

  4. Toxin-Antitoxin Systems of Staphylococcus aureus.

    Science.gov (United States)

    Schuster, Christopher F; Bertram, Ralph

    2016-05-05

    Toxin-antitoxin (TA) systems are small genetic elements found in the majority of prokaryotes. They encode toxin proteins that interfere with vital cellular functions and are counteracted by antitoxins. Dependent on the chemical nature of the antitoxins (protein or RNA) and how they control the activity of the toxin, TA systems are currently divided into six different types. Genes comprising the TA types I, II and III have been identified in Staphylococcus aureus. MazF, the toxin of the mazEF locus is a sequence-specific RNase that cleaves a number of transcripts, including those encoding pathogenicity factors. Two yefM-yoeB paralogs represent two independent, but auto-regulated TA systems that give rise to ribosome-dependent RNases. In addition, omega/epsilon/zeta constitutes a tripartite TA system that supposedly plays a role in the stabilization of resistance factors. The SprA1/SprA1AS and SprF1/SprG1 systems are post-transcriptionally regulated by RNA antitoxins and encode small membrane damaging proteins. TA systems controlled by interaction between toxin protein and antitoxin RNA have been identified in S. aureus in silico, but not yet experimentally proven. A closer inspection of possible links between TA systems and S. aureus pathophysiology will reveal, if these genetic loci may represent druggable targets. The modification of a staphylococcal TA toxin to a cyclopeptide antibiotic highlights the potential of TA systems as rather untapped sources of drug discovery.

  5. Antitoxin Treatment of Inhalation Anthrax: A Systematic Review.

    Science.gov (United States)

    Huang, Eileen; Pillai, Satish K; Bower, William A; Hendricks, Katherine A; Guarnizo, Julie T; Hoyle, Jamechia D; Gorman, Susan E; Boyer, Anne E; Quinn, Conrad P; Meaney-Delman, Dana

    2015-01-01

    Concern about use of anthrax as a bioweapon prompted development of novel anthrax antitoxins for treatment. Clinical guidelines for the treatment of anthrax recommend antitoxin therapy in combination with intravenous antimicrobials; however, a large-scale or mass anthrax incident may exceed antitoxin availability and create a need for judicious antitoxin use. We conducted a systematic review of antitoxin treatment of inhalation anthrax in humans and experimental animals to inform antitoxin recommendations during a large-scale or mass anthrax incident. A comprehensive search of 11 databases and the FDA website was conducted to identify relevant animal studies and human reports: 28 animal studies and 3 human cases were identified. Antitoxin monotherapy at or shortly after symptom onset demonstrates increased survival compared to no treatment in animals. With early treatment, survival did not differ between antimicrobial monotherapy and antimicrobial-antitoxin therapy in nonhuman primates and rabbits. With delayed treatment, antitoxin-antimicrobial treatment increased rabbit survival. Among human cases, addition of antitoxin to combination antimicrobial treatment was associated with survival in 2 of the 3 cases treated. Despite the paucity of human data, limited animal data suggest that adjunctive antitoxin therapy may improve survival. Delayed treatment studies suggest improved survival with combined antitoxin-antimicrobial therapy, although a survival difference compared with antimicrobial therapy alone was not demonstrated statistically. In a mass anthrax incident with limited antitoxin supplies, antitoxin treatment of individuals who have not demonstrated a clinical benefit from antimicrobials, or those who present with more severe illness, may be warranted. Additional pathophysiology studies are needed, and a point-of-care assay correlating toxin levels with clinical status may provide important information to guide antitoxin use during a large-scale anthrax

  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. Toxins-antitoxins: diversity, evolution and function.

    Science.gov (United States)

    Hayes, Finbarr; Van Melderen, Laurence

    2011-10-01

    Genes for toxin-antitoxin (TA) complexes are widespread in prokaryote genomes, and species frequently possess tens of plasmid and chromosomal TA loci. The complexes are categorized into three types based on genetic organization and mode of action. The toxins universally are proteins directed against specific intracellular targets, whereas the antitoxins are either proteins or small RNAs that neutralize the toxin or inhibit toxin synthesis. Within the three types of complex, there has been extensive evolutionary shuffling of toxin and antitoxin genes leading to considerable diversity in TA combinations. The intracellular targets of the protein toxins similarly are varied. Numerous toxins, many of which are sequence-specific endoribonucleases, dampen protein synthesis levels in response to a range of stress and nutritional stimuli. Key resources are conserved as a result ensuring the survival of individual cells and therefore the bacterial population. The toxin effects generally are transient and reversible permitting a set of dynamic, tunable responses that reflect environmental conditions. Moreover, by harboring multiple toxins that intercede in protein synthesis in response to different physiological cues, bacteria potentially sense an assortment of metabolic perturbations that are channeled through different TA complexes. Other toxins interfere with the action of topoisomersases, cell wall assembly, or cytoskeletal structures. TAs also play important roles in bacterial persistence, biofilm formation and multidrug tolerance, and have considerable potential both as new components of the genetic toolbox and as targets for novel antibacterial drugs.

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

    Directory of Open Access Journals (Sweden)

    Marek Gabriško

    2016-06-01

    Full Text Available 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.

  9. 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

  10. Prokaryotic toxin-antitoxin systems: novel regulations of the toxins.

    Science.gov (United States)

    Otsuka, Yuichi

    2016-05-01

    Toxin-antitoxin (TA) systems are widely conserved in prokaryotic plasmids and chromosomes and are linked to many roles in cell physiology, including plasmid maintenance, stress response, persistence and protection from phage infection. A TA system is composed of a stable toxin and a labile antitoxin that inhibits a harmful effect of the cognate toxin. When gene expression from the TA loci is repressed under certain conditions such as nutrient starvation, the toxin is freed from the rapidly degrading antitoxin and obstructs an essential cellular process, such as DNA replication, translation and peptidoglycan synthesis, which subsequently causes growth arrest. TA systems are classified into five types according to the nature and the function of antitoxins, and the activity of toxins is tightly regulated in a variety of ways. This short-review highlights several novel regulatory mechanisms for Escherichia coli toxins that we recently discovered.

  11. Toxin-Antitoxin Systems in Clinical Pathogens

    Directory of Open Access Journals (Sweden)

    Laura Fernández-García

    2016-07-01

    Full Text Available 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.

  12. Equine Botulinum Antitoxin for the Treatment of Infant Botulism ▿

    OpenAIRE

    Vanella de Cuetos, Elida E.; Fernandez, Rafael A.; Bianco, María I.; Sartori, Omar J.; Piovano, María L.; Lúquez, Carolina; de Jong, Laura I. T.

    2011-01-01

    Infant botulism is the most common form of human botulism in Argentina and the United States. BabyBIG (botulism immune globulin intravenous [human]) is the antitoxin of choice for specific treatment of infant botulism in the United States. However, its high cost limits its use in many countries. We report here the effectiveness and safety of equine botulinum antitoxin (EqBA) as an alternative treatment. We conducted an analytical, observational, retrospective, and longitudinal study on cases ...

  13. A regulatory role for Staphylococcus aureus toxin-antitoxin system PemIKSa.

    Science.gov (United States)

    Bukowski, Michal; Lyzen, Robert; Helbin, Weronika M; Bonar, Emilia; Szalewska-Palasz, Agnieszka; Wegrzyn, Grzegorz; Dubin, Grzegorz; Dubin, Adam; Wladyka, Benedykt

    2013-01-01

    Toxin-antitoxin systems were shown to be involved in plasmid maintenance when they were initially discovered, but other roles have been demonstrated since. Here we identify and characterize a novel toxin-antitoxin system (pemIKSa) located on Staphylococcus aureus plasmid pCH91. The toxin (PemKSa) is a sequence-specific endoribonuclease recognizing the tetrad sequence U↓AUU, and the antitoxin (PemISa) inhibits toxin activity by physical interaction. Although the toxin-antitoxin system is responsible for stable plasmid maintenance our data suggest the participation of pemIKSa in global regulation of staphylococcal virulence by alteration of the translation of large pools of genes. We propose a common mechanism of reversible activation of toxin-antitoxin systems based on antitoxin transcript resistance to toxin cleavage. Elucidation of this mechanism is particularly interesting because reversible activation is a prerequisite for the proposed general regulatory role of toxin-antitoxin systems.

  14. 9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

    Science.gov (United States)

    2010-01-01

    ... Toxin. (v) Neutralize all toxin-antitoxin mixtures at room temperature for 1 hour, and hold in ice water... is acceptable to Animal and Plant Health Inspection Service. (vi) Diluent. The solution used to make... 0.25 gram of sodium chloride in each 100 ml of distilled water; adjusting the pH to 7.2;...

  15. 9 CFR 113.454 - Clostridium Perfringens Type C Antitoxin.

    Science.gov (United States)

    2010-01-01

    ...) Neutralize all toxin-antitoxin mixtures at room temperature for 1 hour and hold in ice water until injections... Animal and Plant Health Inspection Service. (vi) Diluent. The solution used to make proper dilutions... chloride in each 100 ml of distilled water; adjusting the pH to 7.2; autoclaving at 250 °F. for 25...

  16. THE NATURE OF THE TOXIN-ANTITOXIN FLOCCULATION PHENOMENON.

    Science.gov (United States)

    Bronfenbrenner, J J; Reichert, P

    1926-09-30

    1. Animals immunized with the formalinized filtrates of young toxic cultures of B. botulinus produce an antitoxic serum poor in precipitins. 2. Animals immunized with the formalinized filtrates of old and partly autolyzed toxic cultures produce an antitoxic serum containing precipitins. 3. Animals immunized with toxin-free autolyzed bacteria produce a serum free from antitoxin but rich in specific precipitins. 4. Animals immunized with the filtrates of an atoxic variant produce a serum free from antitoxin but rich in precipitins for the homologous toxin. 5. Animals immunized with the washed bacteria of the atoxic variant produce a serum that contains no antitoxin, but is rich in precipitins for the homologous toxin. 6. Removal of the precipitins by flocculation with a non-toxic antigen does not materially reduce the antitoxic value of a serum. 7. Removal of the proteins of the antigen by add coagulation removes the specific precipitable substance. 8. All the sera that contain precipitins produce the specific flocculus when combined with homologous toxins, anatoxins, or with the filtrates of the atoxic variant. The flocculation is restricted within the type. The amount of the precipitate and the width of the zone vary approximately with the estimated amount of bacterial protein in the antigen that is used for the immunization of animals. We conclude, therefore, that the toxin-antitoxin flocculation is a specific bacterial precipitation phenomenon.

  17. 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...

  18. 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...

  19. The Antitoxin Protein of a Toxin-Antitoxin System from Xylella fastidiosa Is Secreted via Outer Membrane Vesicles

    Science.gov (United States)

    Santiago, André da Silva; Mendes, Juliano S.; dos Santos, Clelton A.; de Toledo, Marcelo A. S.; Beloti, Lilian L.; Crucello, Aline; Horta, Maria A. C.; Favaro, Marianna T. de Pinho; Munar, Duber M. M.; de Souza, Alessandra A.; Cotta, Mônica A.; de Souza, Anete P.

    2016-01-01

    The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA) operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp. pauca strain 9a5c. These proteins display a high similarity to their homologs in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC), size exclusion chromatography, isothermal titration calorimetry, and Western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by Western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss the possible

  20. Emerging Roles of Toxin-Antitoxin Modules in Bacterial Pathogenesis

    Directory of Open Access Journals (Sweden)

    Barbara Kędzierska

    2016-06-01

    Full Text Available Toxin-antitoxin (TA cassettes are encoded widely by bacteria. The modules typically comprise a protein toxin and protein or RNA antitoxin that sequesters the toxin factor. Toxin activation in response to environmental cues or other stresses promotes a dampening of metabolism, most notably protein translation, which permits survival until conditions improve. Emerging evidence also implicates TAs in bacterial pathogenicity. Bacterial persistence involves entry into a transient semi-dormant state in which cells survive unfavorable conditions including killing by antibiotics, which is a significant clinical problem. TA complexes play a fundamental role in inducing persistence by downregulating cellular metabolism. Bacterial biofilms are important in numerous chronic inflammatory and infectious diseases and cause serious therapeutic problems due to their multidrug tolerance and resistance to host immune system actions. Multiple TAs influence biofilm formation through a network of interactions with other factors that mediate biofilm production and maintenance. Moreover, in view of their emerging contributions to bacterial virulence, TAs are potential targets for novel prophylactic and therapeutic approaches that are required urgently in an era of expanding antibiotic resistance. This review summarizes the emerging evidence that implicates TAs in the virulence profiles of a diverse range of key bacterial pathogens that trigger serious human disease.

  1. 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.

  2. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.;

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... of the relB gene counteracted the effect of relE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae, Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial...

  3. 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.

  4. 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.

  5. 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.

  6. Protection Against Clostridium difficile Infection With Broadly Neutralizing Antitoxin Monoclonal Antibodies

    OpenAIRE

    2012-01-01

    The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI. Novel antitoxin mAbs were generated in mice and were humanized. The humanized antitoxin A mAb PA-50...

  7. Equine botulinum antitoxin for the treatment of infant botulism.

    Science.gov (United States)

    Vanella de Cuetos, Elida E; Fernandez, Rafael A; Bianco, María I; Sartori, Omar J; Piovano, María L; Lúquez, Carolina; de Jong, Laura I T

    2011-11-01

    Infant botulism is the most common form of human botulism in Argentina and the United States. BabyBIG (botulism immune globulin intravenous [human]) is the antitoxin of choice for specific treatment of infant botulism in the United States. However, its high cost limits its use in many countries. We report here the effectiveness and safety of equine botulinum antitoxin (EqBA) as an alternative treatment. We conducted an analytical, observational, retrospective, and longitudinal study on cases of infant botulism registered in Mendoza, Argentina, from 1993 to 2007. We analyzed 92 medical records of laboratory-confirmed cases and evaluated the safety and efficacy of treatment with EqBA. Forty-nine laboratory-confirmed cases of infant botulism demanding admission in intensive care units and mechanical ventilation included 31 treated with EqBA within the 5 days after the onset of signs and 18 untreated with EqBA. EqBA-treated patients had a reduction in the mean length of hospital stay of 23.9 days (P = 0.0007). For infants treated with EqBA, the intensive care unit stay was shortened by 11.2 days (P = 0.0036), mechanical ventilation was reduced by 11.1 days (P = 0.0155), and tube feeding was reduced by 24.4 days (P = 0.0001). The incidence of sepsis in EqBA-treated patients was 47.3% lower (P = 0.0017) than in the untreated ones. Neither sequelae nor adverse effects attributable to EqBA were noticed, except for one infant who developed a transient erythematous rash. These results suggest that prompt treatment of infant botulism with EqBA is safe and effective and that EqBA could be considered an alternative specific treatment for infant botulism when BabyBIG is not available.

  8. Equine Botulinum Antitoxin for the Treatment of Infant Botulism ▿

    Science.gov (United States)

    Vanella de Cuetos, Elida E.; Fernandez, Rafael A.; Bianco, María I.; Sartori, Omar J.; Piovano, María L.; Lúquez, Carolina; de Jong, Laura I. T.

    2011-01-01

    Infant botulism is the most common form of human botulism in Argentina and the United States. BabyBIG (botulism immune globulin intravenous [human]) is the antitoxin of choice for specific treatment of infant botulism in the United States. However, its high cost limits its use in many countries. We report here the effectiveness and safety of equine botulinum antitoxin (EqBA) as an alternative treatment. We conducted an analytical, observational, retrospective, and longitudinal study on cases of infant botulism registered in Mendoza, Argentina, from 1993 to 2007. We analyzed 92 medical records of laboratory-confirmed cases and evaluated the safety and efficacy of treatment with EqBA. Forty-nine laboratory-confirmed cases of infant botulism demanding admission in intensive care units and mechanical ventilation included 31 treated with EqBA within the 5 days after the onset of signs and 18 untreated with EqBA. EqBA-treated patients had a reduction in the mean length of hospital stay of 23.9 days (P = 0.0007). For infants treated with EqBA, the intensive care unit stay was shortened by 11.2 days (P = 0.0036), mechanical ventilation was reduced by 11.1 days (P = 0.0155), and tube feeding was reduced by 24.4 days (P = 0.0001). The incidence of sepsis in EqBA-treated patients was 47.3% lower (P = 0.0017) than in the untreated ones. Neither sequelae nor adverse effects attributable to EqBA were noticed, except for one infant who developed a transient erythematous rash. These results suggest that prompt treatment of infant botulism with EqBA is safe and effective and that EqBA could be considered an alternative specific treatment for infant botulism when BabyBIG is not available. PMID:21918119

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

    Science.gov (United States)

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

    2015-01-01

    We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VH H) 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 Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen-binding proteins and the heterodimer fusion protein containing two VH H domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin-producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism.

  10. 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.

  11. 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.

  12. Development of an Innovative in Vitro Potency Assay for Anti-Botulinum Antitoxins.

    Science.gov (United States)

    Rosen, Osnat; Ozeri, Eyal; Barnea, Ada; David, Alon Ben; Zichel, Ran

    2016-09-24

    Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming, labor-intensive, costly, and raises ethical issues related to the large numbers of laboratory animals needed. Until now, all efforts to develop an alternative in vitro assay have not provided a valid replacement to the mouse potency assay. In the present study, we report the development of an innovative in vitro assay for determining botulinum antitoxin potency, using botulinum type B as a model. The concept of the assay is to mimic two fundamental steps in botulinum intoxication: receptor binding and catalytic activity. By simulating these steps in vitro we were able to accurately determine the potency of antitoxin preparations. The reproducibility of the assay was high with a CV vitro assay highly correlated with that measured by the standard in vivo mouse assay (r = 0.9842, p vitro assay has the potential to be considered, after validation, as a replacement to the mouse assay for quantitating neutralizing antibody concentrations in pharmaceutical botulinum antitoxin preparations. Future adoption of this in vitro assay would minimize the use of laboratory animals, speed up the time, and reduce the cost of botulinum antitoxin approval.

  13. Analysis of the Bacillus cereus SpoIIS antitoxin-toxin system reveals its three-component nature.

    Science.gov (United States)

    Melničáková, Jana; Bečárová, Zuzana; Makroczyová, Jana; Barák, Imrich

    2015-01-01

    Programmed cell death in bacteria is generally associated with two-component toxin-antitoxin systems. The SpoIIS toxin-antitoxin system, consisting of a membrane-bound SpoIISA toxin and a small, cytosolic antitoxin SpoIISB, was originally identified in Bacillus subtilis. In this work we describe the Bacillus cereus SpoIIS system which is a three-component system, harboring an additional gene spoIISC. Its protein product serves as an antitoxin, and similarly as SpoIISB, is able to bind SpoIISA and abolish its toxic effect. Our results indicate that SpoIISC seems to be present not only in B. cereus but also in other Bacilli containing a SpoIIS toxin-antitoxin system. In addition, we show that B. cereus SpoIISA can form higher oligomers and we discuss the possible role of this multimerization for the protein's toxic function.

  14. An efficient method for the purification of proteins from four distinct toxin-antitoxin modules.

    Science.gov (United States)

    Sterckx, Yann G-J; De Gieter, Steven; Zorzini, Valentina; Hadži, San; Haesaerts, Sarah; Loris, Remy; Garcia-Pino, Abel

    2015-04-01

    Toxin-antitoxin (TA) modules are stress response elements that are ubiquitous in the genomes of bacteria and archaea. Production and subsequent purification of individual TA proteins is anything but straightforward as over-expression of the toxin gene is lethal to bacterial and eukaryotic cells and over-production of the antitoxin leads to its proteolytic degradation because of its inherently unstructured nature. Here we describe an effective production and purification strategy centered on an on-column denaturant-induced dissociation of the toxin-antitoxin complex. The success of the method is demonstrated by its application on four different TA families, encoding proteins with distinct activities and folds. A series of biophysical and in vitro activity tests show that the purified proteins are of high quality and suitable for structural studies.

  15. 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...... %. IMPORTANCE: The early stringent response elicited by amino-acid starvation is controlled by a sharp increase of the cellular (p)ppGpp level. Toxin-antitoxin encoded mRNases are activated by (p)ppGpp through enhanced degradation of antitoxins. The present work shows that this activation happens at a very...

  16. Conditional Cooperativity in Toxin-Antitoxin Regulation Prevents Random Toxin Activation and Promotes Fast Translational Recovery

    DEFF Research Database (Denmark)

    Cataudella, Ilaria; Trusina, Ala; Sneppen, Kim;

    2012-01-01

    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...... 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...

  17. RnlB Antitoxin of the Escherichia coli RnlA-RnlB Toxin-Antitoxin Module Requires RNase HI for Inhibition of RnlA Toxin Activity.

    Science.gov (United States)

    Naka, Kenta; Qi, Dan; Yonesaki, Tetsuro; Otsuka, Yuichi

    2017-01-11

    The Escherichia coli RnlA-RnlB toxin-antitoxin system is related to the anti-phage mechanism. Under normal growth conditions, an RnlA toxin with endoribonuclease activity is inhibited by binding of its cognate RnlB antitoxin. After bacteriophage T4 infection, RnlA is activated by the disappearance of RnlB, resulting in the rapid degradation of T4 mRNAs and consequently no T4 propagation when T4 dmd encoding a phage antitoxin against RnlA is defective. Intriguingly, E. coli RNase HI, which plays a key role in DNA replication, is required for the activation of RnlA and stimulates the RNA cleavage activity of RnlA. Here, we report an additional role of RNase HI in the regulation of RnlA-RnlB system. Both RNase HI and RnlB are associated with NRD (one of three domains of RnlA). The interaction between RnlB and NRD depends on RNase HI. Exogenous expression of RnlA in wild-type cells has no effect on cell growth because of endogenous RnlB and this inhibition of RnlA toxicity requires RNase HI and NRD. These results suggest that RNase HI recruits RnlB to RnlA through NRD for inhibiting RnlA toxicity and thus plays two contrary roles in the regulation of RnlA-RnlB system.

  18. Counterselection method based on conditional silencing of antitoxin genes in Escherichia coli.

    Science.gov (United States)

    Tsukuda, Miyuki; Nakashima, Nobutaka; Miyazaki, Kentaro

    2015-11-01

    Counterselection is a genetic engineering technique to eliminate specific genetic fragments containing selectable marker genes. Although the technique is widely used in bacterial genome engineering and plasmid curing experiments, the repertoire of the markers usable in Escherichia coli is limited. Here we developed a novel counterselection method in E. coli based on antisense RNA (asRNA) technology directed against toxin-antitoxin (TA) modules. Under normal conditions, excess antitoxin neutralizes its cognate toxin and thus the module is stably maintained in the genome. We hypothesised that repression of an antitoxin gene would perturb cell growth due to the toxin being released. We designed asRNAs corresponding to all 19 type II antitoxins encoded in the E. coli genome. asRNAs were then conditionally expressed; repression of MqsA in the MqsR/MqsA module had the greatest inhibitory effect, followed by RnlB in the RnlA/RnlB module. The utility of asRNA(MqsA) as a counterselection marker was demonstrated by efficient plasmid curing and strain improvement experiments.

  19. Discovery of Peptide-Based Antitoxins against Neurotoxins from Green and Black Mamba (Dendroaspis Family)

    DEFF Research Database (Denmark)

    Jappe, Emma Christine; Munk, Andreas; Laustsen, Andreas Hougaard

    dendrotoxins α-dendrotoxin (α-Dtx, UniProtKBP00980), isolated from Dendroaspisangusticeps (Green mamba), and dendrotoxin I (Dtx I, UniProtKBP00979) from Dendroaspis polylepis (Black mamba) by phage display [5,6].Cross-reactive antitoxins with theability to neutralise several toxins are of interest to antivenom...

  20. 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...

  1. A Conserved Mode of Protein Recognition and Binding in a ParD−ParE Toxin−Antitoxin Complex

    Energy Technology Data Exchange (ETDEWEB)

    Dalton, Kevin M.; Crosson, Sean (UC)

    2010-05-06

    Toxin-antitoxin (TA) systems form a ubiquitous class of prokaryotic proteins with functional roles in plasmid inheritance, environmental stress response, and cell development. ParDE family TA systems are broadly conserved on plasmids and bacterial chromosomes and have been well characterized as genetic elements that promote stable plasmid inheritance. We present a crystal structure of a chromosomally encoded ParD-ParE complex from Caulobacter crescentus at 2.6 {angstrom} resolution. This TA system forms an {alpha}{sub 2}{beta}{sub 2} heterotetramer in the crystal and in solution. The toxin-antitoxin binding interface reveals extensive polar and hydrophobic contacts of ParD antitoxin helices with a conserved recognition and binding groove on the ParE toxin. A cross-species comparison of this complex structure with related toxin structures identified an antitoxin recognition and binding subdomain that is conserved between distantly related members of the RelE/ParE toxin superfamily despite a low level of overall primary sequence identity. We further demonstrate that ParD antitoxin is dimeric, stably folded, and largely helical when not bound to ParE toxin. Thus, the paradigmatic model in which antitoxin undergoes a disorder-to-order transition upon toxin binding does not apply to this chromosomal ParD-ParE TA system.

  2. ACTIVE IMMUNITY PRODUCED BY SO CALLED BALANCED OR NEUTRAL MIXTURES OF DIPHTHERIA TOXIN AND ANTITOXIN.

    Science.gov (United States)

    Smith, T

    1909-03-01

    The foregoing and earlier data taken together demonstrate that an active immunity lasting several years can be produced in guinea-pigs, by the injection of toxin-antitoxin mixtures which have no recognizable harmful effect either immediate or remote. They also show, what might have been anticipated, that under the same conditions mixtures which produce local lesions and which, therefore, contain an excess of toxin produce a much higher degree of immunity than the neutral mixtures, and that an excess of antitoxin reduces the possibility of producing an active immunity, and may extinguish it altogether. There is, therefore, a certain definite relation between the components of the mixture and the degree of immunity producible. Furthermore, toxin-antitoxin mixtures do not change materially within five days at room temperature. They are apparently more efficacious at the end of forty-eight hours than immediately after preparation. The experiments finally prove that a relatively high degree of active immunity can be induced by a harmless procedure, whereas the use of toxin alone leading to very severe local lesions is incapable of producing more than an insignificant protection. The method, therefore, invites further tests in regard to its ultimate applicability to the human being. Unless the subcutis of the guinea-pig reacts to toxin-antitoxin mixtures in a manner peculiar to itself, a practical, easily controlled method for active immunization can be worked out which should afford a larger protection than the serum alone and avoid the complications associated with horse serum. That proportion of toxin and antitoxin which would produce the highest desirable immunity consistent with the least discomfort would have to be carefully worked out for the human subject. From the nature of the immunity induced it is obvious, however, that such a method of immunization cannot take the place of a large dose of antitoxin in exposed individuals who must be protected at once. It

  3. Horizontal gene transfer of chromosomal Type II toxin-antitoxin systems of Escherichia coli.

    Science.gov (United States)

    Ramisetty, Bhaskar Chandra Mohan; Santhosh, Ramachandran Sarojini

    2016-02-01

    Type II toxin-antitoxin systems (TAs) are small autoregulated bicistronic operons that encode a toxin protein with the potential to inhibit metabolic processes and an antitoxin protein to neutralize the toxin. Most of the bacterial genomes encode multiple TAs. However, the diversity and accumulation of TAs on bacterial genomes and its physiological implications are highly debated. Here we provide evidence that Escherichia coli chromosomal TAs (encoding RNase toxins) are 'acquired' DNA likely originated from heterologous DNA and are the smallest known autoregulated operons with the potential for horizontal propagation. Sequence analyses revealed that integration of TAs into the bacterial genome is unique and contributes to variations in the coding and/or regulatory regions of flanking host genome sequences. Plasmids and genomes encoding identical TAs of natural isolates are mutually exclusive. Chromosomal TAs might play significant roles in the evolution and ecology of bacteria by contributing to host genome variation and by moderation of plasmid maintenance.

  4. Prokaryotic toxin-antitoxin systems--the role in bacterial physiology and application in molecular biology.

    Science.gov (United States)

    Bukowski, Michal; Rojowska, Anna; Wladyka, Benedykt

    2011-01-01

    Bacteria have developed multiple complex mechanisms ensuring an adequate response to environmental changes. In this context, bacterial cell division and growth are subject to strict control to ensure metabolic balance and cell survival. A plethora of studies cast light on toxin-antitoxin (TA) systems as metabolism regulators acting in response to environmental stress conditions. Many of those studies suggest direct relations between the TA systems and the pathogenic potential or antibiotic resistance of relevant bacteria. Other studies point out that TA systems play a significant role in ensuring stability of mobile genetic material. The evolutionary origin and relations between various TA systems are still a subject of a debate. The impact of toxin-antitoxin systems on bacteria physiology prompted their application in molecular biology as tools allowing cloning of some hard-to-maintain genes, plasmid maintenance and production of recombinant proteins.

  5. 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 ...

  6. 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.

  7. 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.

  8. Protection against Clostridium difficile infection with broadly neutralizing antitoxin monoclonal antibodies.

    Science.gov (United States)

    Marozsan, Andre J; Ma, Dangshe; Nagashima, Kirsten A; Kennedy, Brian J; Kang, Yun Kenneth; Arrigale, Robert R; Donovan, Gerald P; Magargal, Wells W; Maddon, Paul J; Olson, William C

    2012-09-01

    The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI. Novel antitoxin mAbs were generated in mice and were humanized. The humanized antitoxin A mAb PA-50 and antitoxin B mAb PA-41 have picomolar potencies in vitro and bind to novel regions of the respective toxins. In a hamster model for CDI, 95% of animals treated with a combination of humanized PA-50 and PA-41 showed long-term survival relative to 0% survival of animals treated with standard antibiotics or comparator mAbs. These humanized mAbs provide insight into C. difficile intoxication and hold promise as potential nonantibiotic agents for improving clinical management of CDI.

  9. Regulation of the vapBC-1 toxin-antitoxin locus in nontypeable Haemophilus influenzae.

    Directory of Open Access Journals (Sweden)

    Susan D Cline

    Full Text Available Nontypeable Haemophilus influenzae (NTHi are human-adapted commensal bacteria that can cause a number of chronic mucosal infections, including otitis media and bronchitis. One way for these organisms to survive antibiotic therapy and cause recurrent disease is to stop replicating, as most antimicrobials target essential biosynthetic pathways. Toxin-antitoxin (TA gene pairs have been shown to facilitate entry into a reversible bacteriostatic state. Characteristically, these operons encode a protein toxin and an antitoxin that associate following translation to form a nontoxic complex, which then binds to and regulates the cognate TA promoter. Under stressful conditions, the labile antitoxin is degraded and the complex disintegrates, freeing the stable toxin to facilitate growth arrest. How these events affected the regulation of the TA locus, as well as how the transcription of the operon was subsequently returned to its normal state upon resumption of growth, was not fully understood. Here we show that expression of the NTHi vapBC-1 TA locus is repressed by a complex of VapB-1 and VapC-1 under conditions favorable for growth, and activated by the global transactivator Factor for Inversion Stimulation (Fis upon nutrient upshift from stationary phase. Further, we demonstrate for the first time that the VapC-1 toxin alone can bind to its cognate TA locus control region and that the presence of VapB-1 directs the binding of the VapBC-1 complex in the transcriptional regulation of vapBC-1.

  10. Calibration and commutability assessment of the 1st International Standard for Diphtheria Antitoxin Human.

    Science.gov (United States)

    Stickings, Paul; Rigsby, Peter; Coombes, Laura; von Hunolstein, Christina; Ralli, Luisa; Pinto, Antonella; Sesardic, Dorothea

    2013-11-01

    The 1st International Standard for Diphtheria Antitoxin Human (coded 10/262) was established by the World Health Organization Expert Committee on Biological Standardization in 2012. This paper describes the production, characterization and calibration of the new standard which is intended for use in the standardization of assays used to measure diphtheria antibody responses in human serum. The new standard was calibrated in terms of the International Standard for Diphtheria Antitoxin Equine in an international collaborative study. A total of 8 participants from 8 different countries performed in vivo and/or in vitro toxin neutralization tests and returned data that was used to assign units to the proposed new standard. The new standard has a diphtheria antitoxin potency of 2 IU/ampoule and is predicted to be stable. A follow up study was performed to assess commutability of the new standard. The follow up study was an existing external quality assessment, modified to include the new standard. Results obtained suggest that the new standard is commutable, showing comparable behaviour to native human serum samples in the majority of the assays compared, and is therefore suitable for use as a reference preparation in assays used to measure the level of anti-diphtheria antibodies in human serum.

  11. A widespread bacteriophage abortive infection system functions through a Type IV toxin-antitoxin mechanism.

    Science.gov (United States)

    Dy, Ron L; Przybilski, Rita; Semeijn, Koen; Salmond, George P C; Fineran, Peter C

    2014-04-01

    Bacterial abortive infection (Abi) systems are 'altruistic' cell death systems that are activated by phage infection and limit viral replication, thereby providing protection to the bacterial population. Here, we have used a novel approach of screening Abi systems as a tool to identify and characterize toxin-antitoxin (TA)-acting Abi systems. We show that AbiE systems are encoded by bicistronic operons and function via a non-interacting (Type IV) bacteriostatic TA mechanism. The abiE operon was negatively autoregulated by the antitoxin, AbiEi, a member of a widespread family of putative transcriptional regulators. AbiEi has an N-terminal winged-helix-turn-helix domain that is required for repression of abiE transcription, and an uncharacterized bi-functional C-terminal domain, which is necessary for transcriptional repression and sufficient for toxin neutralization. The cognate toxin, AbiEii, is a predicted nucleotidyltransferase (NTase) and member of the DNA polymerase β family. AbiEii specifically bound GTP, and mutations in conserved NTase motifs (I-III) and a newly identified motif (IV), abolished GTP binding and subsequent toxicity. The AbiE systems can provide phage resistance and enable stabilization of mobile genetic elements, such as plasmids. Our study reveals molecular insights into the regulation and function of the widespread bi-functional AbiE Abi-TA systems and the biochemical properties of both toxin and antitoxin proteins.

  12. 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

    2016-02-19

    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.

  13. An endogenous antitoxin to the lethal venom of the funnel web spider, Atrax robustus, in rabbit sera.

    Science.gov (United States)

    Sheumack, D D; Comis, A; Claassens, R; Mylecharane, E J; Spence, I; Howden, M E

    1991-01-01

    1. An endogenous antitoxin fraction was isolated from non-immune rabbit sera by affinity chromatography with robustoxin bound to the solid support. 2. Robustoxin is the sole lethal toxin in the venom of the male funnel web spider, Atrax robustus. 3. The fraction was found to contain IgG and IgM immunoglobulins. 4. This fraction prevented or reversed the lethal actions of the crude venom in newborn mice, in mouse phrenic nerve-hemidiaphragm preparations, and in anaesthetized monkeys. 5. The antitoxin fraction is of potential value in the therapy of human envenomation by A. robustus.

  14. Structure, Biology, and Therapeutic Application of Toxin–Antitoxin Systems in Pathogenic Bacteria

    Directory of Open Access Journals (Sweden)

    Ki-Young Lee

    2016-10-01

    Full Text Available Bacterial toxin–antitoxin (TA systems have received increasing attention for their diverse identities, structures, and functional implications in cell cycle arrest and survival against environmental stresses such as nutrient deficiency, antibiotic treatments, and immune system attacks. In this review, we describe the biological functions and the auto-regulatory mechanisms of six different types of TA systems, among which the type II TA system has been most extensively studied. The functions of type II toxins include mRNA/tRNA cleavage, gyrase/ribosome poison, and protein phosphorylation, which can be neutralized by their cognate antitoxins. We mainly explore the similar but divergent structures of type II TA proteins from 12 important pathogenic bacteria, including various aspects of protein–protein interactions. Accumulating knowledge about the structure–function correlation of TA systems from pathogenic bacteria has facilitated a novel strategy to develop antibiotic drugs that target specific pathogens. These molecules could increase the intrinsic activity of the toxin by artificially interfering with the intermolecular network of the TA systems.

  15. 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.

  16. (p)ppGpp controls bacterial persistence by stochastic induction of toxin-antitoxin activity.

    Science.gov (United States)

    Maisonneuve, Etienne; Castro-Camargo, Manuela; Gerdes, Kenn

    2013-08-29

    Persistence refers to the phenomenon in which isogenic populations of antibiotic-sensitive bacteria produce rare cells that transiently become multidrug tolerant. Whether slow growth in a rare subset of cells underlies the persistence phenotype has not be examined in wild-type bacteria. Here, we show that an exponentially growing population of wild-type Escherichia coli cells produces rare cells that stochastically switch into slow growth, that the slow-growing cells are multidrug tolerant, and that they are able to resuscitate. The persistence phenotype depends hierarchically on the signaling nucleotide (p)ppGpp, Lon protease, inorganic polyphosphate, and toxin-antitoxins. We show that the level of (p)ppGpp varies stochastically in a population of exponentially growing cells and that the high (p)ppGpp level in rare cells induces slow growth and persistence. (p)ppGpp triggers slow growth by activating toxin-antitoxin loci through a regulatory cascade depending on inorganic polyphosphate and Lon protease.

  17. Structural studies of the toxin-antitoxin proteins RelE and RelB from E. coli

    DEFF Research Database (Denmark)

    Andersen, Kasper Røjkjær; Overgaard, Martin; Gerdes, Kenn;

    the special tRNA-mRNA mimic, tmRNA [1]. Questions to be addressed Many questions remain to be answered in the bacterial toxin-antitoxin system. The crystal structure of RelBE from Pyrococcus horikoshii OT3 was previously solved at 2.3Å [2]. This structure shows the molecule in an inactive state, but OT3......The bacterial toxin-antitoxin system The relBE operon in E. coli encodes two small proteins: A toxin, RelE (12 kDa) and an antitoxin, RelB (9 kDa). RelE is activated under nutritional stress and is able to inhibit protein synthesis by cleaving the mRNA in the ribosomal A-site. This stress response...... serves to down-regulate metabolism in the cell when growth conditions are limited. RelB is expressed in excess over RelE during balanced growth, and inhibits the toxicity of RelE by forming an extremely stable toxin-antitoxin complex. The activation of RelE is induced when the labile RelB protein...

  18. What Is the Link between Stringent Response, Endoribonuclease Encoding Type II Toxin-Antitoxin Systems and Persistence?

    Science.gov (United States)

    Ramisetty, Bhaskar C M; Ghosh, Dimpy; Roy Chowdhury, Maoumita; Santhosh, Ramachandran S

    2016-01-01

    Persistence is a transient and non-inheritable tolerance to antibiotics by a small fraction of a bacterial population. One of the proposed determinants of bacterial persistence is toxin-antitoxin systems (TASs) which are also implicated in a wide range of stress-related phenomena. Maisonneuve E, Castro-Camargo M, Gerdes K. 2013. Cell 154:1140-1150 reported an interesting link between ppGpp mediated stringent response, TAS, and persistence. It is proposed that accumulation of ppGpp enhances the accumulation of inorganic polyphosphate which modulates Lon protease to degrade antitoxins. The decrease in the concentration of antitoxins supposedly activated the toxin to increase in the number of persisters during antibiotic treatment. In this study, we show that inorganic polyphosphate is not required for transcriptional activation of yefM/yoeB TAS, which is an indirect indication of Lon-dependent degradation of YefM antitoxin. The Δ10 strain, an Escherichia coli MG1655 derivative in which the 10 TAS are deleted, is more sensitive to ciprofloxacin compared to wild type MG1655. Furthermore, we show that the Δ10 strain has relatively lower fitness compared to the wild type and hence, we argue that the persistence related implications based on Δ10 strain are void. We conclude that the transcriptional regulation and endoribonuclease activity of YefM/YoeB TAS is independent of ppGpp and inorganic polyphosphate. Therefore, we urge for thorough inspection and debate on the link between chromosomal endoribonuclease TAS and persistence.

  19. Remarkable Functional Convergence: Alarmone ppGpp Mediates Persistence by Activating Type I and II Toxin-Antitoxins.

    Science.gov (United States)

    Gerdes, Kenn; Maisonneuve, Etienne

    2015-07-01

    In this issue of Molecular Cell, Verstraeten et al. (2015) demonstrate that the conserved GTPase Obg and the second messenger ppGpp mediate persistence by activation of a type I toxin-antitoxin module (hokB/sokB) in E. coli.

  20. Identification and characterization of chromosomal relBE toxin-antitoxin locus in Streptomyces cattleya DSM46488.

    Science.gov (United States)

    Li, Peng; Tai, Cui; Deng, Zixin; Gan, Jianhua; Oggioni, Marco R; Ou, Hong-Yu

    2016-08-18

    The relBE family of Type II toxin-antitoxin (TA) systems have been widely reported in bacteria but none in Streptomyces. With the conserved domain searches for TA pairs in the sequenced Streptomyces genomes, we identified two putative relBE loci, relBE1sca and relBE2sca, on the chromosome of Streptomyces cattleya DSM 46488. Overexpression of the S. cattleya toxin RelE2sca caused severe growth inhibition of E. coli and S. lividans, but RelE1sca had no toxic effect. The toxicity of RelE2sca could be abolished by the co-expression of its cognate RelB2sca antitoxin. Moreover, the RelBE2sca complex, or the antitoxin RelB2sca alone, specifically interacted with the relBE2sca operon and repressed its transcription. The relBE2sca operon transcription was induced under osmotic stress, along with the ClpP proteinase genes. The subsequent in vivo analysis showed that the antitoxin was degraded by ClpP. Interestingly, the E. coli antitoxin RelBeco was able to alleviate the toxicity of S. cattleya RelE2sca while the mutant RelB2sca(N61V&M68L) but not the wild type could alleviate the toxicity of E. coli RelEeco as well. The experimental demonstration of the relBEsca locus might be helpful to investigate the key roles of type II TA systems in Streptomyces physiology and environmental stress responses.

  1. Structure and Proposed Activity of a Member of the VapBC Family of Toxin-Antitoxin Systems: VapBC-5 from Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Miallau, L.; Faller, M.; Chiang, J.; Arbing, M.; Guo, F.; Cascio, D.; Eisenberg, D.; (UCLA)

    2009-03-02

    In prokaryotes, cognate toxin-antitoxin pairs have long been known, but no three-dimensional structure has been available for any given complex from Mycobacterium tuberculosis. Here we report the crystal structure and activity of a member of the VapBC family of complexes from M. tuberculosis. The toxin VapC-5 is a compact, 150 residues, two domain {alpha}/{beta} protein. Bent around the toxin is the VapB-5 antitoxin, a 33-residue {alpha}-helix. Assays suggest that the toxin is an Mg-enabled endoribonuclease, inhibited by the antitoxin. The lack of DNase activity is consistent with earlier suggestions that the complex represses its own operon. Furthermore, analysis of the interactions in the binding of the antitoxin to the toxin suggest that exquisite control is required to protect the bacteria cell from toxic VapC-5.

  2. Comprehensive comparative-genomic analysis of Type 2 toxin-antitoxin systems and related mobile stress response systems in prokaryotes

    Directory of Open Access Journals (Sweden)

    Makarova Kira S

    2009-06-01

    Full Text Available Abstract Background The prokaryotic toxin-antitoxin systems (TAS, also referred to as TA loci are widespread, mobile two-gene modules that can be viewed as selfish genetic elements because they evolved mechanisms to become addictive for replicons and cells in which they reside, but also possess "normal" cellular functions in various forms of stress response and management of prokaryotic population. Several distinct TAS of type 1, where the toxin is a protein and the antitoxin is an antisense RNA, and numerous, unrelated TAS of type 2, in which both the toxin and the antitoxin are proteins, have been experimentally characterized, and it is suspected that many more remain to be identified. Results We report a comprehensive comparative-genomic analysis of Type 2 toxin-antitoxin systems in prokaryotes. Using sensitive methods for distant sequence similarity search, genome context analysis and a new approach for the identification of mobile two-component systems, we identified numerous, previously unnoticed protein families that are homologous to toxins and antitoxins of known type 2 TAS. In addition, we predict 12 new families of toxins and 13 families of antitoxins, and also, predict a TAS or TAS-like activity for several gene modules that were not previously suspected to function in that capacity. In particular, we present indications that the two-gene module that encodes a minimal nucleotidyl transferase and the accompanying HEPN protein, and is extremely abundant in many archaea and bacteria, especially, thermophiles might comprise a novel TAS. We present a survey of previously known and newly predicted TAS in 750 complete genomes of archaea and bacteria, quantitatively demonstrate the exceptional mobility of the TAS, and explore the network of toxin-antitoxin pairings that combines plasticity with selectivity. Conclusion The defining properties of the TAS, namely, the typically small size of the toxin and antitoxin genes, fast evolution, and

  3. Proteomic profiling of Mycobacterium tuberculosis identifies nutrient-starvation-responsive toxin-antitoxin systems

    DEFF Research Database (Denmark)

    Albrethsen, Jakob; Agner, Jeppe; Piersma, Sander R

    2013-01-01

    In order to successfully enter the latent stage, Mycobacterium tuberculosis must adapt to conditions such as nutrient limitation and hypoxia. In vitro models that mimic latent infection are valuable tools for describing the changes in metabolism that occur when the bacterium exists in a non......-growing form. We used two complementary proteomic approaches, label-free LC-MS/MS analysis and two-dimensional difference gel electrophoresis, to determine the proteome profile of extracellular proteins from M. tuberculosis cultured under nutrient starvation. Through the label-free LC-MS/MS analysis......, significant differences in the overall metabolism during nutrient starvation were detected. Notably, members of the toxin-antitoxin systems were present in larger quantities in nutrient-starved cultures, supporting a role for these global modules as M. tuberculosis switches its metabolism into dormancy...

  4. 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.

  5. Comparison of four serological methods for the detection of diphtheria anti-toxin antibody.

    Science.gov (United States)

    Walory, J; Grzesiowski, P; Hryniewicz, W

    2000-11-01

    The aim of this study was to compare four serological methods for the detection of Corynebacterium diphtheriae IgG anti-toxin antibodies (IgG-DTAb) in human serum. One hundred serum samples were evaluated for C. diphtheriae IgG-DTAb by four different methods: passive haemagglutination (PHA), latex agglutination test (LA), toxoid enzyme-linked immunosorbent assay (Toxoid-ELISA), and toxin-binding inhibition enzyme-linked immunosorbent assay (ToBI-ELISA). As the external standardisation the neutralisation test for C. diphtheriae toxin in Vero cells (TN Vero) was used. For internal standardisation of IgG-DTAb titres, the WHO standard serum of human diphtheria antitoxin was used. The study revealed a poor correlation between the reference test and the PHA (r=0.34 Pearson's correlation coefficient), an acceptable correlation for the LA (r=0.74), a good correlation for the Toxoid-ELISA (r=0.81) and a very good correlation for ToBI-ELISA (r=0.93). The sensitivity measurements of PHA, LA, Toxoid-ELISA and ToBI-ELISA tests, were 14, 100, 94, 96% respectively and the corresponding specificity characteristics were 86, 76, 94, 90 respectively. Of the four evaluated methods, the ToBI-ELISA could be recommended for scientific and precise laboratory assays of diphtheria antibody levels in humans. For screening purposes the Toxoid-ELISA could be used, but the accuracy of antibody titres below 0.1 IU/ml, considered as the limits of protection, is questionable. Both tests offer very useful alternatives to the in vitro diphtheria toxin neutralisation test in Vero cells. Because of their unsatisfactory correlation and sensitivity as compared to the reference method, PHA and LA should be avoided and replaced by one of the two enzyme immunoassays.

  6. What Is the Link between Stringent Response, Endoribonuclease Encoding Type II Toxin–Antitoxin Systems and Persistence?

    Science.gov (United States)

    Ramisetty, Bhaskar C. M.; Ghosh, Dimpy; Roy Chowdhury, Maoumita; Santhosh, Ramachandran S.

    2016-01-01

    Persistence is a transient and non-inheritable tolerance to antibiotics by a small fraction of a bacterial population. One of the proposed determinants of bacterial persistence is toxin–antitoxin systems (TASs) which are also implicated in a wide range of stress-related phenomena. Maisonneuve E, Castro-Camargo M, Gerdes K. 2013. Cell 154:1140–1150 reported an interesting link between ppGpp mediated stringent response, TAS, and persistence. It is proposed that accumulation of ppGpp enhances the accumulation of inorganic polyphosphate which modulates Lon protease to degrade antitoxins. The decrease in the concentration of antitoxins supposedly activated the toxin to increase in the number of persisters during antibiotic treatment. In this study, we show that inorganic polyphosphate is not required for transcriptional activation of yefM/yoeB TAS, which is an indirect indication of Lon-dependent degradation of YefM antitoxin. The Δ10 strain, an Escherichia coli MG1655 derivative in which the 10 TAS are deleted, is more sensitive to ciprofloxacin compared to wild type MG1655. Furthermore, we show that the Δ10 strain has relatively lower fitness compared to the wild type and hence, we argue that the persistence related implications based on Δ10 strain are void. We conclude that the transcriptional regulation and endoribonuclease activity of YefM/YoeB TAS is independent of ppGpp and inorganic polyphosphate. Therefore, we urge for thorough inspection and debate on the link between chromosomal endoribonuclease TAS and persistence. PMID:27933045

  7. Crystallization of two operator complexes from the Vibrio cholerae HigBA2 toxin–antitoxin module

    Science.gov (United States)

    Hadži, San; Garcia-Pino, Abel; Gerdes, Kenn; Lah, Jurij; Loris, Remy

    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 bp DNA duplex belong to space group P3221, with unit-cell parameters a = b = 94.0, c = 123.7 Å, and diffract to 2.3 Å resolution. The second form corresponding to HigA2 in complex with a 19 bp duplex belong to space group P43212 and only diffract to 3.45 Å resolution. Crystals of the HigBA2 toxin–antitoxin were obtained in complex with a 31 bp duplex and belonged to space group P41212, with unit-cell parameters a = b = 113.6, c = 121.1 Å. They diffract to 3.3 Å resolution. PMID:25664801

  8. The MazEF Toxin-Antitoxin System Alters the β-Lactam Susceptibility of Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Christopher F Schuster

    Full Text Available Toxin-antitoxin (TA systems are genetic elements of prokaryotes which encode a stable toxin and an unstable antitoxin that can counteract toxicity. TA systems residing on plasmids are often involved in episomal maintenance whereas those on chromosomes can have multiple functions. The opportunistic pathogen Staphylococcus aureus possesses at least four different families of TA systems but their physiological roles are elusive. The chromosomal mazEF system encodes the RNase toxin MazF and the antitoxin MazE. In the light of ambiguity regarding the cleavage activity, we here verify that MazF specifically targets UACAU sequences in S. aureus in vivo. In a native strain background and under non-stress conditions, cleavage was observed in the absence or presence of mazE. Transcripts of spa (staphylococcal protein A and rsbW (anti-σB factor were cut, but translational reporter fusions indicated that protein levels of the encoded products were unaffected. Despite a comparable growth rate as the wild-type, an S. aureus mazEF deletion mutant was more susceptible to β-lactam antibiotics, which suggests that further genes, putatively involved in the antibiotic stress response or cell wall synthesis or turnover, are controlled by this TA system.

  9. The MazEF Toxin-Antitoxin System Alters the β-Lactam Susceptibility of Staphylococcus aureus.

    Science.gov (United States)

    Schuster, Christopher F; Mechler, Lukas; Nolle, Nicoletta; Krismer, Bernhard; Zelder, Marc-Eric; Götz, Friedrich; Bertram, Ralph

    2015-01-01

    Toxin-antitoxin (TA) systems are genetic elements of prokaryotes which encode a stable toxin and an unstable antitoxin that can counteract toxicity. TA systems residing on plasmids are often involved in episomal maintenance whereas those on chromosomes can have multiple functions. The opportunistic pathogen Staphylococcus aureus possesses at least four different families of TA systems but their physiological roles are elusive. The chromosomal mazEF system encodes the RNase toxin MazF and the antitoxin MazE. In the light of ambiguity regarding the cleavage activity, we here verify that MazF specifically targets UACAU sequences in S. aureus in vivo. In a native strain background and under non-stress conditions, cleavage was observed in the absence or presence of mazE. Transcripts of spa (staphylococcal protein A) and rsbW (anti-σB factor) were cut, but translational reporter fusions indicated that protein levels of the encoded products were unaffected. Despite a comparable growth rate as the wild-type, an S. aureus mazEF deletion mutant was more susceptible to β-lactam antibiotics, which suggests that further genes, putatively involved in the antibiotic stress response or cell wall synthesis or turnover, are controlled by this TA system.

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

    Directory of Open Access Journals (Sweden)

    Fauziah Abu Bakar

    2016-04-01

    Full Text Available 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.

  11. 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.

  12. The administrative stabilization of vaccines: Regulating the diphtheria antitoxin in France and Germany, 1894-1900.

    Science.gov (United States)

    Hess, Volker

    2008-06-01

    It is well known that the development of a diphtheria anti-toxin serum evolved in a competitive race between two groups of researchers, one affiliated with Emil Behring in Berlin and Marburg, and another affiliated with Emile Roux in Paris. Proceeding on the basis of different theoretical assumptions and experimental practices, the two groups developed a therapeutic serum almost simultaneously. But the standardized substance they developed took on very different forms in the two countries. In Germany the new serum was marketed in the private sphere and subjected to state regulations, becoming a kind ofprototype of industrial medications. In France, however, the same substance was marketed as a gift of science to humanity and distributed through the communal health care system. This article demonstrates how a new medication emerged from the efforts to produce, market, regulate, distribute, and apply it in the two respective countries. It attributes the difference to the negotiations between the respective actors (scientists, industrialists, politicians, officers, and the public) and institutions (firms, academies, private and public institutes, legislative bodies, professional corporations). I develop this argument on three different levels: First, I stress the importance of the institutional foundations of serum production; second, I illustrate the decisive role played by existing "ways of regulating" in the rapid development of new legal statutes; and third, I describe the consequences that flowed from the respective administrative organization of marketing and dissemination. In sum, I explore how an experimental object was transformed into an object of the public health system and stabilized by administrative means.

  13. Type II toxin-antitoxin systems are unevenly distributed among Escherichia coli phylogroups.

    Science.gov (United States)

    Fiedoruk, Krzysztof; Daniluk, Tamara; Swiecicka, Izabela; Sciepuk, Malgorzata; Leszczynska, Katarzyna

    2015-01-01

    Type II toxin-antitoxin systems (TAs) are bicistronic operons ubiquitous in prokaryotic genomes, displaying multilevel association with cell physiology. Various possible functions have been assigned to TAs, ranging from beneficial for their hosts, such as a stress response, dormancy and protection against genomic parasites, to detrimental or useless functions, such as selfish alleles. As there is a link between several Escherichia coli features (e.g. virulence, lifestyle) and the phylogeny of this species, we hypothesized a similar association with TAs. Using PCR we studied the distribution of 15 chromosomal and plasmidic type II TA loci in 84 clinical E. coli isolates in relation to their main phylogenetic groups (A, B1, B2 and D). In addition, we performed in silico searching of these TA loci in 60 completely sequenced E. coli genomes deposited in GenBank. The highest number of TA loci per strain was observed in group A (mean 8.2, range 5-12) and the lowest in group B2 (mean 4.2, range 2-8). Moreover, significant differences in the prevalence of nine chromosomal TAs among E. coli phylogroups were noted. In conclusion, the presence of some chromosomal TAs in E. coli is phylogroup-related rather than a universal feature of the species. In addition, their limited collection in group B2 clearly distinguish it from the other E. coli phylogroups.

  14. Standardization and validation of Vero cell assay for potency estimation of diphtheria antitoxin serum.

    Science.gov (United States)

    Kumar, Sunil; Kanwar, Sarika; Bansal, Vivek; Sehgal, Rakesh

    2009-10-01

    Diphtheria toxin has the capacity to block protein synthesis in cultured mammalian cells, and thus causing cell death. This capacity of diphtheria toxin was utilized for in-vitro neutralization test to determine antibody titer, using Vero cells, which have been found to be susceptible to diphtheria toxin. In the present study, a Vero cell assay was standardized and validated for potency estimation of diphtheria antitoxin serum (DATS). The results obtained by Vero cell assay were compared with in-vivo biological assay. High degree of correlation (+0.98) was found between in-vivo biological assay and in-vitro Vero cell assay. The assay has also been found to be effective in determining the rising antibody titer in the equines inducted in DATS production. The present study indicated that although biological assays hold the key for final potency estimations till date but in the future scenario in-vitro Vero cell assay may be a good alternative to in-vivo biological assay.

  15. 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

    .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 Rel......The RelE and RelB proteins constitute the RNA interferase (toxin) and its cognate inhibitor (antitoxin) components of the Escherichia coli relBE toxin-antitoxin system. Despite the well-described functionality and physiological activity of this system in E. coli, no structural study was performed...... 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 infrared...

  16. 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.

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

    Science.gov (United States)

    Sonobe, M H; Trezena, A G; Guilhen, F B; Takano, V L; Fratelli, F; Sakauchi, D; Morais, J F; Prado, S M A; Higashi, H G

    2007-01-01

    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 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 (r(2) = 0.95, P diphtheria (r(2) = 0.93, P assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

  18. Identification of a human monoclonal antibody to replace equine diphtheria antitoxin for treatment of diphtheria intoxication.

    Science.gov (United States)

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

    2013-11-01

    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 alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 μg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.

  19. A toxin antitoxin system promotes the maintenance of the IncA/C-mobilizable Salmonella Genomic Island 1.

    Science.gov (United States)

    Huguet, Kevin T; Gonnet, Mathieu; Doublet, Benoît; Cloeckaert, Axel

    2016-08-31

    The multidrug resistance Salmonella Genomic Island 1 (SGI1) is an integrative mobilizable element identified in several enterobacterial pathogens. This chromosomal island requires a conjugative IncA/C plasmid to be excised as a circular extrachromosomal form and conjugally mobilized in trans. Preliminary observations suggest stable maintenance of SGI1 in the host chromosome but paradoxically also incompatibility between SGI1 and IncA/C plasmids. Here, using a Salmonella enterica serovar Agona clonal bacterial population as model, we demonstrate that a Toxin-Antitoxin (TA) system encoded by SGI1 plays a critical role in its stable host maintenance when an IncA/C plasmid is concomitantly present. This system, designated sgiAT for Salmonella genomic island 1 Antitoxin and Toxin respectively, thus seems to play a stabilizing role in a situation where SGI1 is susceptible to be lost through plasmid IncA/C-mediated excision. Moreover and for the first time, the incompatibility between SGI1 and IncA/C plasmids was experimentally confirmed.

  20. The ToxAvapA toxin-antitoxin locus contributes to the survival of nontypeable Haemophilus influenzae during infection.

    Science.gov (United States)

    Ren, Dabin; Kordis, Alexis A; Sonenshine, Daniel E; Daines, Dayle A

    2014-01-01

    Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that is a common cause of acute and recurrent mucosal infections. One uncharacterized NTHi toxin-antitoxin (TA) module, NTHI1912-1913, is a host inhibition of growth (higBA) homologue. We hypothesized that this locus, which we designated toxAvapA, contributed to NTHi survival during infection. We deleted toxAvapA and determined that growth of the mutant in defined media was not different from the parent strain. We tested the mutant for persistence during long-term in vitro co-culture with primary human respiratory tissues, which revealed that the ΔtoxAvapA mutant was attenuated for survival. We then performed challenge studies using the chinchilla model of otitis media and determined that mutant survival was also reduced in vivo. Following purification, the toxin exhibited ribonuclease activity on RNA in vitro, while the antitoxin did not. A microarray comparison of the transcriptome revealed that the tryptophan biosynthetic regulon was significantly repressed in the mutant compared to the parent strain. HPLC studies of conditioned medium confirmed that there was no significant difference in the concentration of tryptophan remaining in the supernatant, indicating that the uptake of tryptophan by the mutant was not affected. We conclude that the role of the NTHi toxAvapA TA module in persistence following stress is multifactorial and includes effects on essential metabolic pathways.

  1. A Type III protein-RNA toxin-antitoxin system from Bacillus thuringiensis promotes plasmid retention during spore development.

    Science.gov (United States)

    Short, Francesca L; Monson, Rita E; Salmond, George P C

    2015-01-01

    Members of the Bacillus cereus sensu lato group of bacteria often contain multiple large plasmids, including those encoding virulence factors in B. anthracis. Bacillus species can develop into spores in response to stress. During sporulation the genomic content of the cell is heavily compressed, which could result in counterselection of extrachromosomal genomic elements, unless they have robust stabilization and segregation systems. Toxin-antitoxin (TA) systems are near-ubiquitous in prokaryotes and have multiple biological roles, including plasmid stabilization during vegetative growth. Here, we have shown that a Type III TA system, based on an RNA antitoxin and endoribonuclease toxin, from plasmid pAW63 in Bacillus thuringiensis serovar kurstaki HD-73 can dramatically promote plasmid retention in populations undergoing sporulation and germination, and we provide evidence that this occurs through the post-segregational killing of plasmid-free forespores. Our findings show how an extremely common genetic module can be used to ensure plasmid maintenance during stress-induced developmental transitions, with implications for plasmid dynamics in B. cereus s.l. bacteria.

  2. The ToxAvapA toxin-antitoxin locus contributes to the survival of nontypeable Haemophilus influenzae during infection.

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    Dabin Ren

    Full Text Available Nontypeable Haemophilus influenzae (NTHi is an opportunistic pathogen that is a common cause of acute and recurrent mucosal infections. One uncharacterized NTHi toxin-antitoxin (TA module, NTHI1912-1913, is a host inhibition of growth (higBA homologue. We hypothesized that this locus, which we designated toxAvapA, contributed to NTHi survival during infection. We deleted toxAvapA and determined that growth of the mutant in defined media was not different from the parent strain. We tested the mutant for persistence during long-term in vitro co-culture with primary human respiratory tissues, which revealed that the ΔtoxAvapA mutant was attenuated for survival. We then performed challenge studies using the chinchilla model of otitis media and determined that mutant survival was also reduced in vivo. Following purification, the toxin exhibited ribonuclease activity on RNA in vitro, while the antitoxin did not. A microarray comparison of the transcriptome revealed that the tryptophan biosynthetic regulon was significantly repressed in the mutant compared to the parent strain. HPLC studies of conditioned medium confirmed that there was no significant difference in the concentration of tryptophan remaining in the supernatant, indicating that the uptake of tryptophan by the mutant was not affected. We conclude that the role of the NTHi toxAvapA TA module in persistence following stress is multifactorial and includes effects on essential metabolic pathways.

  3. 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.

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

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    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.

  5. 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

  6. Cell growth inhibition upon deletion of four toxin-antitoxin loci from the megaplasmids of Sinorhizobium meliloti.

    Science.gov (United States)

    Milunovic, Branislava; diCenzo, George C; Morton, Richard A; Finan, Turlough M

    2014-02-01

    Toxin and antitoxin (TA) gene pairs are addiction systems that are present in many microbial genomes. Sinorhizobium meliloti is an N2-fixing bacterial symbiont of alfalfa and other leguminous plants, and its genome consists of three large replicons, a circular chromosome (3.7 Mb) and the megaplasmids pSymA (1.4 Mb) and pSymB (1.7 Mb). S. meliloti carries 211 predicted type II TA genes, each encoding a toxin or an antitoxin. We constructed defined deletion strains that collectively removed the entire pSymA and pSymB megaplasmids except for their oriV regions. Of approximately 100 TA genes on pSymA and pSymB, we identified four whose loss was associated with cell death or stasis unless copies of the genes were supplied in trans. Orthologs of three of these loci have been characterized in other organisms (relB/E [sma0471/sma0473], Fic [DOC] [sma2105], and VapC [PIN] [orf2230/sma2231]), and this report contains the first experimental proof that RES/Xre (smb21127/smb21128) loci can function as a TA system. Transcriptome sequencing (RNA-seq) analysis did not reveal transcriptional differences between the TA systems to account for why deletion of the four "active" systems resulted in cell toxicity. These data suggest that severe cell growth phenotypes result from the loss of a few TA systems and that loss of most TA systems may result in more subtle phenotypes. These four TA systems do not appear to play a direct role in the S. meliloti-alfalfa symbiosis, as strains lacking these TA systems had a symbiotic N2 fixation phenotype that was indistinguishable from the wild type.

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

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    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.

  8. Crystal Structure of the Escherichia coli Fic Toxin-Like Protein in Complex with Its Cognate Antitoxin

    Science.gov (United States)

    Stanger, Frédéric V.; Harms, Alexander; Dehio, Christoph; Schirmer, Tilman

    2016-01-01

    FIC domain proteins mediate post-translational modifications of target proteins, which typically results in their inactivation. Depending on the conservation of crucial active site residues, the FIC fold serves as structural scaffold for various enzymatic activities, mostly target adenylylation. The founding member of the vast Fic protein family, EcFicT, was identified in Escherichia coli some time ago. The G55R point mutant of EcFicT displays the “filamentation induced by cAMP” (Fic) phenotype at high 3',5'-cyclic adenosine monophosphate (cAMP) concentrations and elevated temperature, but the underlying molecular mechanism and any putative biochemical activity of EcFicT have remained unknown. EcFicT belongs to class I Fic toxin proteins that are encoded together with a small inhibitory protein (antitoxin), named EcFicA in E. coli. Here, we report the crystal structures of two mutant EcFicT/EcFicA complexes (EcFicTG55RA and EcFicTAE28G) both showing close resemblance with the structure of the AMP-transferase VbhT from Bartonella schoenbuchensis in complex with its cognate antitoxin VbhA. However, crucial differences in the active site of EcFicT compared to VbhT and other AMP-transferases rationalize the lack of evidence for adenylylation activity. Comprehensive bioinformatic analysis suggests that EcFicT has evolved from canonical AMP-transferases and has acquired a conserved binding site for a yet to be discovered novel substrate. The G55R mutation has no effect on structure or thermal stability of EcFicT, such that the molecular basis for its associated Fic phenotype remains elusive. We anticipate that this structure will inspire further bioinformatic and experimental analyses in order to characterize the enzymatic activity of EcFicT and help revealing its physiological role. PMID:27657533

  9. Co-evolution of quaternary organization and novel RNA tertiary interactions revealed in the crystal structure of a bacterial protein–RNA toxin–antitoxin system

    Science.gov (United States)

    Rao, Feng; Short, Francesca L.; Voss, Jarrod E.; Blower, Tim R.; Orme, Anastasia L.; Whittaker, Tom E.; Luisi, Ben F.; Salmond, George P. C.

    2015-01-01

    Genes encoding toxin–antitoxin (TA) systems are near ubiquitous in bacterial genomes and they play key roles in important aspects of bacterial physiology, including genomic stability, formation of persister cells under antibiotic stress, and resistance to phage infection. The CptIN locus from Eubacterium rectale is a member of the recently-discovered Type III class of TA systems, defined by a protein toxin suppressed by direct interaction with a structured RNA antitoxin. Here, we present the crystal structure of the CptIN protein–RNA complex to 2.2 Å resolution. The structure reveals a new heterotetrameric quaternary organization for the Type III TA class, and the RNA antitoxin bears a novel structural feature of an extended A-twist motif within the pseudoknot fold. The retention of a conserved ribonuclease active site as well as traits normally associated with TA systems, such as plasmid maintenance, implicates a wider functional role for Type III TA systems. We present evidence for the co-variation of the Type III component pair, highlighting a distinctive evolutionary process in which an enzyme and its substrate co-evolve. PMID:26350213

  10. Co-evolution of quaternary organization and novel RNA tertiary interactions revealed in the crystal structure of a bacterial protein-RNA toxin-antitoxin system.

    Science.gov (United States)

    Rao, Feng; Short, Francesca L; Voss, Jarrod E; Blower, Tim R; Orme, Anastasia L; Whittaker, Tom E; Luisi, Ben F; Salmond, George P C

    2015-10-30

    Genes encoding toxin-antitoxin (TA) systems are near ubiquitous in bacterial genomes and they play key roles in important aspects of bacterial physiology, including genomic stability, formation of persister cells under antibiotic stress, and resistance to phage infection. The CptIN locus from Eubacterium rectale is a member of the recently-discovered Type III class of TA systems, defined by a protein toxin suppressed by direct interaction with a structured RNA antitoxin. Here, we present the crystal structure of the CptIN protein-RNA complex to 2.2 Å resolution. The structure reveals a new heterotetrameric quaternary organization for the Type III TA class, and the RNA antitoxin bears a novel structural feature of an extended A-twist motif within the pseudoknot fold. The retention of a conserved ribonuclease active site as well as traits normally associated with TA systems, such as plasmid maintenance, implicates a wider functional role for Type III TA systems. We present evidence for the co-variation of the Type III component pair, highlighting a distinctive evolutionary process in which an enzyme and its substrate co-evolve.

  11. A vapBC-type toxin-antitoxin module of Sinorhizobium meliloti influences symbiotic efficiency and nodule senescence of Medicago sativa.

    Science.gov (United States)

    Lipuma, Justine; Cinege, Gyöngyi; Bodogai, Monica; Oláh, Boglárka; Kiers, Aurélie; Endre, Gabriella; Dupont, Laurence; Dusha, Ilona

    2014-12-01

    The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti carries a large number of toxin-antitoxin (TA) modules both on the chromosome and megaplasmids. One of them, the vapBC-5 module that belongs to the type II systems was characterized here. It encodes an active toxin vapC-5, and was shown to be controlled negatively by the complex of its own proteins. Different mutants of the vapBC-5 genes exhibited diverse effects on symbiotic efficiency during interaction with the host plant Medicago sativa. The absence of the entire vapBC-5 region had no influence on nodule formation and nitrogen fixation properties. The strain carrying an insertion in the antitoxin gene showed a reduced nitrogen fixation capacity resulting in a lower plant yield. In contrast, when the toxin gene was mutated, the strain developed more efficient symbiosis with the host plant. The nitrogen fixing root nodules had a delayed senescent phenotype and contained elevated level of plant-derived molecules characteristic of later steps of nodule development. The longer bacteroid viability and abundance of active nitrogen fixing zone resulted in increased production of plant material. These data indicate that modification of the toxin/antitoxin production may influence bacteroid metabolism and may have an impact on the adaptation to changing environmental conditions.

  12. 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

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    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.

  13. TasA-tasB, a new putative toxin-antitoxin (TA system from Bacillus thuringiensis pGI1 plasmid is a widely distributed composite mazE-doc TA system

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    Mahillon Jacques

    2006-10-01

    Full Text Available Abstract Background Post-segregational killing systems are present in a large variety of microorganisms. When found on plasmids, they are described as addiction systems that act to maintain the plasmid during the partitioning of the cell. The plasmid to be maintained through the generations harbours a group of two genes, one coding for a stable toxin and the other coding for an unstable antitoxin that inhibits the effects of the toxin. If, during cell division, the plasmid is lost, the toxin and antitoxin proteins present in the cytosol cease to be newly expressed. The level of unstable antitoxin protein then rapidly decreases, leaving the toxin free to act on the cellular target, leading to cell death. Consequently, only cells harbouring the plasmid can survive. Results The pGI1 plasmid of Bacillus thuringiensis H1.1 harbours a group of two genes, one showing similarities with the Doc toxin of the phd-doc toxin-antitoxin system, potentially coding for a toxin-antitoxin system. Attempts were made to clone this putative system in the Gram-negative host Escherichia coli. The putative antitoxin tasA was easily cloned in E. coli. However, although several combinations of DNA fragment were used in the cloning strategy, only clones containing a mutation in the toxin gene could be recovered, suggesting a toxic activity of TasB. An exhaustive search was carried out in order to index genes homologous to those of the putative tasA-tasB system among microorganisms. This study revealed the presence of this system in great number and in a large variety of microorganisms, either as tasA-tasB homologues or in association with toxins (or antitoxins from other TA systems. Conclusion In this work, we showed that the pGI1 plasmid of B. thuringiensis H1.1 harbours genes resembling a toxin-antitoxin system, named tasA-tasB for thuringiensis addiction system. This system appeared to be functional but unregulated in E. coli. Bioinformatics studies showed that the tas

  14. Role of vapBC toxin-antitoxin loci in the thermal stress response of Sulfolobus solfataricus.

    Science.gov (United States)

    Cooper, Charlotte R; Daugherty, Amanda J; Tachdjian, Sabrina; Blum, Paul H; Kelly, Robert M

    2009-02-01

    TA (toxin-antitoxin) loci are ubiquitous in prokaryotic micro-organisms, including archaea, yet their physiological function is largely unknown. For example, preliminary reports have suggested that TA loci are microbial stress-response elements, although it was recently shown that knocking out all known chromosomally located TA loci in Escherichia coli did not have an impact on survival under certain types of stress. The hyperthermophilic crenarchaeon Sulfolobus solfataricus encodes at least 26 vapBC (where vap is virulence-associated protein) family TA loci in its genome. VapCs are PIN (PilT N-terminus) domain proteins with putative ribonuclease activity, while VapBs are proteolytically labile proteins, which purportedly function to silence VapCs when associated as a cognate pair. Global transcriptional analysis of S. solfataricus heat-shock-response dynamics (temperature shift from 80 to 90 degrees C) revealed that several vapBC genes were triggered by the thermal shift, suggesting a role in heat-shock-response. Indeed, knocking out a specific vapBC locus in S. solfataricus substantially changed the transcriptome and, in one case, rendered the crenarchaeon heat-shock-labile. These findings indicate that more work needs to be done to determine the role of VapBCs in S. solfataricus and other thermophilic archaea, especially with respect to post-transcriptional regulation.

  15. Role of vapBC toxin–antitoxin loci in the thermal stress response of Sulfolobus solfataricus

    Science.gov (United States)

    Cooper, Charlotte R.; Daugherty, Amanda J.; Tachdjian, Sabrina; Blum, Paul H.; Kelly, Robert M.

    2010-01-01

    TA (toxin–antitoxin) loci are ubiquitous in prokaryotic microorganisms, including archaea, yet their physiological function is largely unknown. For example, preliminary reports have suggested that TA loci are microbial stress-response elements, although it was recently shown that knocking out all known chromosomally located TA loci in Escherichia coli did not have an impact on survival under certain types of stress. The hyperthermophilic crenarchaeon Sulfolobus solfataricus encodes at least 26 vapBC (where vap is virulence-associated protein) family TA loci in its genome. VapCs are PIN (PilT N-terminus) domain proteins with putative ribonuclease activity, while VapBs are proteolytically labile proteins, which purportedly function to silence VapCs when associated as a cognate pair. Global transcriptional analysis of S. solfataricus heat-shock-response dynamics (temperature shift from 80 to 90°C) revealed that several vapBC genes were triggered by the thermal shift, suggesting a role in heat-shock-response. Indeed, knocking out a specific vapBC locus in S. solfataricus substantially changed the transcriptome and, in one case, rendered the crenarchaeon heat-shock-labile. These findings indicate that more work needs to be done to determine the role of VapBCs in S. solfataricus and other thermophilic archaea, especially with respect to post-transcriptional regulation. PMID:19143615

  16. The crystal structure of the Rv0301-Rv0300 VapBC-3 toxin-antitoxin complex from M. tuberculosis reveals a Mg2+ ion in the active site and a putative RNA-binding site

    Energy Technology Data Exchange (ETDEWEB)

    Min, Andrew B; Miallau, Linda; Sawaya, Michael R; Habel, Jeff; Cascio, Duilio; Eisenberg, David [UCLA; (UCB)

    2013-01-10

    VapBC pairs account for 45 out of 88 identified toxin-antitoxin (TA) pairs in the Mycobacterium tuberculosis (Mtb) H37Rv genome. A working model suggests that under times of stress, antitoxin molecules are degraded, releasing the toxins to slow the metabolism of the cell, which in the case of VapC toxins is via their RNase activity. Otherwise the TA pairs remain bound to their promoters, autoinhibiting transcription. The crystal structure of Rv0301-Rv0300, an Mtb VapBC TA complex determined at 1.49 Å resolution, suggests a mechanism for these three functions: RNase activity, its inhibition by antitoxin, and its ability to bind promoter DNA. The Rv0301 toxin consists of a core of five parallel beta strands flanked by alpha helices. Three proximal aspartates coordinate a Mg2+ ion forming the putative RNase active site. The Rv0300 antitoxin monomer is extended in structure, consisting of an N-terminal beta strand followed by four helices. The last two helices wrap around the toxin and terminate near the putative RNase active site, but with different conformations. In one conformation, the C-terminal arginine interferes with Mg2+ ion coordination, suggesting a mechanism by which the antitoxin can inhibit toxin activity. At the N-terminus of the antitoxin, two pairs of Ribbon-Helix-Helix (RHH) motifs are related by crystallographic twofold symmetry. The resulting hetero-octameric complex is similar to the FitAB system, but the two RHH motifs are about 30 Å closer together in the Rv0301-Rv0300 complex, suggesting either a different span of the DNA recognition sequence or a conformational change.

  17. Transcriptional profiling of type II toxin-antitoxin genes of Helicobacter pylori under different environmental conditions: identification of HP0967-HP0968 system

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    MIGUEL A DE LA CRUZ

    2016-11-01

    Full Text Available Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa and is responsible for causing peptic ulcers and gastric carcinoma. The expression of virulence factors allows the persistence of H. pylori in the stomach, which results in a chronic, sometimes uncontrolled inflammatory response. Type II toxin-antitoxin systems have emerged as important virulence factors in many pathogenic bacteria. Three type II toxin-antitoxin (TA systems have previously been identified in the genome of H. pylori 26695: HP0315-HP0316, HP0892-HP0893, and HP0894-HP0895. Here we characterized a heretofore undescribed type II TA system in H. pylori, HP0967-HP0968, which is encoded by the bicistronic operon hp0968-hp0967 and belongs to the Vap family. The predicted HP0967 protein is a toxin with ribonuclease activity whereas HP0968 is an antitoxin that binds to its own regulatory region. We found that all type II TA systems were expressed in H. pylori during early stationary growth phase, and differentially expressed in the presence of urea, nickel, and iron, although the hp0968-hp0967 pair was the most affected under these environmental conditions. Transcription of hp0968-hp0967 was strongly induced in a mature H. pylori biofilm and when the bacteria interacted with AGS epithelial cells. Kanamycin and chloramphenicol considerably boosted transcription levels of all the four type II TA systems. The hp0968-hp0967 TA system was the most frequent among 317 H. pylori strains isolated from all over the world. This study is the first report on the transcription of type II TA genes in H. pylori under different environmental conditions. Our data show that the HP0967 and HP0968 proteins constitute a bona fide type II TA system in H. pylori, whose expression is regulated by environmental cues, which are relevant in the context of infection of the human gastric mucosa.

  18. Mutagenesis and functional characterization of the RNA and protein components of the toxIN abortive infection and toxin-antitoxin locus of Erwinia.

    Science.gov (United States)

    Blower, T R; Fineran, P C; Johnson, M J; Toth, I K; Humphreys, D P; Salmond, G P C

    2009-10-01

    Bacteria are constantly challenged by bacteriophage (phage) infection and have developed multiple adaptive resistance mechanisms. These mechanisms include the abortive infection systems, which promote "altruistic suicide" of an infected cell, protecting the clonal population. A cryptic plasmid of Erwinia carotovora subsp. atroseptica, pECA1039, has been shown to encode an abortive infection system. This highly effective system is active across multiple genera of gram-negative bacteria and against a spectrum of phages. Designated ToxIN, this two-component abortive infection system acts as a toxin-antitoxin module. ToxIN is the first member of a new type III class of protein-RNA toxin-antitoxin modules, of which there are multiple homologues cross-genera. We characterized in more detail the abortive infection phenotype of ToxIN using a suite of Erwinia phages and performed mutagenesis of the ToxI and ToxN components. We determined the minimal ToxI RNA sequence in the native operon that is both necessary and sufficient for abortive infection and to counteract the toxicity of ToxN. Furthermore, site-directed mutagenesis of ToxN revealed key conserved amino acids in this defining member of the new group of toxic proteins. The mechanism of phage activation of the ToxIN system was investigated and was shown to have no effect on the levels of the ToxN protein. Finally, evidence of negative autoregulation of the toxIN operon, a common feature of toxin-antitoxin systems, is presented. This work on the components of the ToxIN system suggests that there is very tight toxin regulation prior to suicide activation by incoming phage.

  19. The mazEF toxin-antitoxin system as an attractive target in clinical isolates of Enterococcus faecium and Enterococcus faecalis.

    Science.gov (United States)

    Soheili, Sara; Ghafourian, Sobhan; Sekawi, Zamberi; Neela, Vasantha Kumari; Sadeghifard, Nourkhoda; Taherikalani, Morovat; Khosravi, Afra; Ramli, Ramliza; Hamat, Rukman Awang

    2015-01-01

    The toxin-antitoxin (TA) system is a regulatory system where two sets of genes encode the toxin and its corresponding antitoxin. In this study, the prevalence of TA systems in independently isolated clinical isolates of Enterococcus faecium and Enterococcus faecalis was determined, the dominant TA system was identified, different virulence genes in E. faecium and E. faecalis were surveyed, the level of expression of the virulence and TA genes in normal and stress conditions was determined, and finally their associations with the TA genes were defined. Remarkably, the analysis demonstrated higBA and mazEF in all clinical isolates, and their locations were on chromosomes and plasmids, respectively. On the other hand, a quantitative analysis of TA and virulence genes revealed that the expression level in both genes is different under normal and stress conditions. The results obtained by anti-mazF peptide nucleic acids demonstrated that the expression level of virulence genes had decreased. These findings demonstrate an association between TA systems and virulence factors. The mazEF on the plasmids and the higBA TA genes on the chromosomes of all E. faecium and E. faecalis strains were dominant. Additionally, there was a decrease in the expression of virulence genes in the presence of anti-mazF peptide nucleic acids. Therefore, it is suggested that mazEF TA systems are potent and sensitive targets in all E. faecium and E. faecalis strains.

  20. Construction of a novel, stable, food-grade expression system by engineering the endogenous toxin-antitoxin system in Bacillus subtilis.

    Science.gov (United States)

    Yang, Sen; Kang, Zhen; Cao, Wenlong; Du, Guocheng; Chen, Jian

    2016-02-10

    Bacillus subtilis as an important workhorse that has been widely used to produce enzymes and metabolites. To broaden its applications, especially in the food and feed industry, we constructed a novel, stable, food-grade expression system by engineering its type II toxin-antitoxin system. The expression of the toxin EndoA, encoded by the chromosomal ydcE gene, was regulated by an endogenous, xylose-inducible promoter, while the ydcD gene, which encodes the unstable antitoxin EndoB, was inserted into a food-grade vector backbone, where its expression was driven by the native, constitutive promoter PylxM. By maintaining the xylose concentration above 2.0 g L(-1), this auto-regulated expression system was absolutely stable after 100 generations. Compared with traditional antibiotic-dependent expression systems, this novel expression system resulted in greater biomass and higher titers of desired products (enzymes or metabolites). Our results demonstrate that this stable, food-grade expression system is suitable for enzyme production and pathway engineering, especially for the production of food-grade enzymes and metabolites.

  1. 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.

  2. The Monoclonal Antitoxin Antibodies (Actoxumab–Bezlotoxumab) Treatment Facilitates Normalization of the Gut Microbiota of Mice with Clostridium difficile Infection

    Science.gov (United States)

    Džunková, Mária; D'Auria, Giuseppe; Xu, Hua; Huang, Jun; Duan, Yinghua; Moya, Andrés; Kelly, Ciarán P.; Chen, Xinhua

    2016-01-01

    detected by the end of the experiments. In conclusion, MK-3415A (actoxumab–bezlotoxumab) treatment facilitates normalization of the gut microbiota in CDI mice. It remains to be examined whether or not the prevention of recurrent CDI by the antitoxin antibodies observed in clinical trials occurs through modulation of microbiota. PMID:27757389

  3. The monoclonal antitoxin antibodies (actoxumab-bezlotoxumab treatment facilitates normalization of the gut microbiota of mice with Clostridium difficile infection

    Directory of Open Access Journals (Sweden)

    Mária Džunková

    2016-10-01

    detected by the end of the experiments. In conclusion, MK-3415A (actoxumab-bezlotoxumab treatment facilitates normalization of the gut microbiota in CDI mice. It remains to be examined whether or not the prevention of recurrent CDI by the antitoxin antibodies observed in clinical trials occurs through modulation of microbiota.

  4. Progress of the Biological Roles of Toxin-Antitoxin System in the Stress Response%毒素-抗毒素系统在应激环境下的生物学作用的研究进展

    Institute of Scientific and Technical Information of China (English)

    熊鸣; 李明; 郑丹阳; 黄伟

    2013-01-01

    毒素-抗毒素系统(toxin-antitoxin system,TAS)广泛存在于细菌染色体及质粒上,是细菌中含量丰富的小型遗传元件.TAS通常由两个紧密相连的基因组成,分别编码毒素(toxin)和抗毒素(antitoxin),稳定的毒素能够损伤宿主细胞,不稳定的抗毒素能够保护宿主细胞免于毒素的损伤作用.依据其性质和作用方式,目前已经发现三种型别的TAS.TAS具有多种生物学作用,如诱导程序性细胞死亡(programmed cell death,PCD),应激条件下介导持留菌形成(persistence),稳定基因大片段等.本文就近几年TAS在应激条件下的生物学作用的研究进展做一综述.%Toxin-antitoxin (TA) systems are small genetic modules that are widespread and abundant in bacterial chromosomes and plasmids. TA systems are usually composed of two closely linked genes that encode a stable toxin and a labile antitoxin, respectively. The toxin can harm the host cell, while the cognate antitoxin protects the host cell from the toxin's damage. So far, three types of TA system have been described based on their nature and mode of action. Several biological functions of the TA systems have been proposed, such as inducing programmed cell death (PCD), mediating arrest under stress conditions (persistence), promoting stability of large genomic fragments, etc. This review summarizes recent findings on the biological roles of TA systems under stress conditions.

  5. Identification of mazEF Toxin – Antitoxin system and biofilm formation in clinical isolates of MRSA isolated from Eastern India

    Directory of Open Access Journals (Sweden)

    Sonia Jain

    2016-01-01

    Full Text Available Introduction: Interest on Toxin-Antitoxin (TA systems has increased dramatically over recent years. It is ubiquitously present in many bacterial genomes including pathogens like MRSA. Several cellular functions of TA systems are proposed like programmed cell death, persister cell formation, biofilm formation etc. However, their exact role in cell physiology remains unclear. Due to the biofilm development the rate of morbidity and mortality of chronic MRSA infections are increasing day by day. Objectives: The aim of the study is to find any association between biofilm formation and mazEF toxin-antitoxin systems in clinical isolates of MRSA, isolated from tertiary care hospital, Kolkata. Materials and Methods: MRSA isolates were detected for biofilm formation by tube method and simultaneously the presence of mazEF TA gene in these biofilm forming isolates were evaluated. Results: It was found that 52.47 % MRSA isolates were potent biofilm producers by Tube method. The results indicated that 55.26 % of the biofilm forming isolates possessed mazEF gene. And 44% of the biofilm forming isolates did not possessed mazEF gene. Conclusions: The analysis recommended that TA genes are prevalent in clinical isolates of MRSA strains but no significant difference is found in the presence of mazEF TA system in the biofilm producer and non-biofilm producer isolates. Further to come into any conclusion reverse transcription studies are needed to be done in order to find out the exact amount of mazEF expression in all MRSA isolates.

  6. Analysis of 1 Case of Recurrent Allergic Reactions Induced by Tetanus Antitoxin Injection%破伤风抗毒素致过敏反应反复发作1例分析

    Institute of Scientific and Technical Information of China (English)

    李勇; 刘立平

    2011-01-01

    OBJECTIVE: To alert clinicians pay attention to recurrent allergic reactions caused by Tetanus antitoxin injection.METHODS: 1 patient with left leg injury who suffered from recurrent allergic reactions induced by Tetanus antitoxin injection was reported and analyzed. RESULTS & CONCLUSION: There is no report about recurrent allergic reactions induced by Tetanus antitoxin injection. Clinical medical staff should be aware of the characteristics and pathogenesis of ADR induced by Tetanus antitoxin injection, master application pointer, adopt desensitization therapy for skin test-positive patients on the TAT and make observations and rescue preparation, or use human tetanus immune globulin (TIG) instead of TAT in order to avoid or reduce ADR.%目的:提示临床重视破伤风抗毒素(TAT)引起的过敏反应反复发作.方法:报道并分析1例因左腿外伤注射TAT致过敏反应反复发作.结果与结论:未见TAT注射液引起过敏反应反复发作的报道.临床医务人员应了解TAT注射液致不良反应(ADR)的特点及发病机制,掌握TAT应用指征,对皮肤试验阳性患者采取脱敏注射并做好观察及抢救准备,或使用人破伤风免疫球蛋白(TIG)替代TAT以避免或减少ADR的发生.

  7. Mapping Protein–Protein Interactions of the Resistance-Related Bacterial Zeta Toxin–Epsilon Antitoxin Complex (ε2ζ2 with High Affinity Peptide Ligands Using Fluorescence Polarization

    Directory of Open Access Journals (Sweden)

    María Isabel Fernández-Bachiller

    2016-07-01

    Full Text Available Toxin–antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta–Epsilon toxin–antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε2ζ2 complex. Three α helices of Zeta forming the protein–protein interaction (PPI site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε2ζ2 complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay.

  8. 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.

  9. Transcriptional Profiling of Type II Toxin–Antitoxin Genes of Helicobacter pylori under Different Environmental Conditions: Identification of HP0967–HP0968 System

    Science.gov (United States)

    Cárdenas-Mondragón, María G.; Ares, Miguel A.; Panunzi, Leonardo G.; Pacheco, Sabino; Camorlinga-Ponce, Margarita; Girón, Jorge A.; Torres, Javier; De la Cruz, Miguel A.

    2016-01-01

    Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa and is responsible for causing peptic ulcers and gastric carcinoma. The expression of virulence factors allows the persistence of H. pylori in the stomach, which results in a chronic, sometimes uncontrolled inflammatory response. Type II toxin–antitoxin (TA) systems have emerged as important virulence factors in many pathogenic bacteria. Three type II TA systems have previously been identified in the genome of H. pylori 26695: HP0315–HP0316, HP0892–HP0893, and HP0894–HP0895. Here we characterized a heretofore undescribed type II TA system in H. pylori, HP0967–HP0968, which is encoded by the bicistronic operon hp0968–hp0967 and belongs to the Vap family. The predicted HP0967 protein is a toxin with ribonuclease activity whereas HP0968 is an antitoxin that binds to its own regulatory region. We found that all type II TA systems were expressed in H. pylori during early stationary growth phase, and differentially expressed in the presence of urea, nickel, and iron, although, the hp0968–hp0967 pair was the most affected under these environmental conditions. Transcription of hp0968–hp0967 was strongly induced in a mature H. pylori biofilm and when the bacteria interacted with AGS epithelial cells. Kanamycin and chloramphenicol considerably boosted transcription levels of all the four type II TA systems. The hp0968–hp0967 TA system was the most frequent among 317 H. pylori strains isolated from all over the world. This study is the first report on the transcription of type II TA genes in H. pylori under different environmental conditions. Our data show that the HP0967 and HP0968 proteins constitute a bona fide type II TA system in H. pylori, whose expression is regulated by environmental cues, which are relevant in the context of infection of the human gastric mucosa. PMID:27920769

  10. Progress in The Study of Bacterial Toxin-antitoxin System%细菌毒素-抗毒素系统的研究进展

    Institute of Scientific and Technical Information of China (English)

    王晓蕾; 赵龙旋; 张俊杰

    2008-01-01

    毒素-抗毒素系统(toxin-antitoxin system,TA)由两个共表达的基因组成,其中一个基因编码不稳定的抗毒素蛋白(antitoxin),另一个基因编码稳定的毒素蛋白(toxin).毒素-抗毒素系统最早发现于一些低拷贝的质粒,用来维持低拷贝质粒在菌群中的稳定存在.随后的研究表明,毒素-抗毒素系统广泛存在于细菌,包括一些致病菌的染色体上.在营养缺乏等不良生长条件下,由于基因表达的抑制和蛋白酶的降解作用,不稳定的抗毒素蛋白减少,从而产生游离的毒素蛋白,导致细菌的生长抑制和死亡.毒素-抗毒素系统的生理功能目前还存在争议,有学者认为细茼染色体上的毒素-抗毒素系统可以在不良生长状况下介导细菌的死亡,即细茼程序性细胞死亡(baeterial programmedcell death).但也有证据显示,毒素-抗毒素系统的功能更偏向于应激状态下的生理调节方面,即只起应激状态下的抑菌作用而不是杀菌作用.对细菌生长调控中毒素-抗毒素系统的作用机理进行综述,并探讨毒素-抗毒素系统研究的理论和应用价值.

  11. Progress of Clinical Application and Safety of Tetanus Antitoxin%破伤风抗毒素临床应用及安全性研究进展

    Institute of Scientific and Technical Information of China (English)

    余超; 徐玉茗; 徐瑾; 万凯化; 袁兴东; 李穗; 周鹃

    2016-01-01

    目的了解破伤风抗毒素(tetanus antitoxin, TAT)的临床应用现状及其安全性研究进展,为开展上市后安全性评估提供参考。方法检索国内外研究TAT临床应用安全性相关的文献,对其进行归类分析研究。结果目前TAT在临床上主要应用于破伤风被动免疫和破伤风治疗;大量安全性研究显示,其皮试阳性率较高,主要与患者体质、药物因素、操作因素及其他原因造成假阳性过高有关。其不良反应主要有过敏性休克、血清病及其他类型不良反应。结论 TAT仍是国内为外伤患者预防破伤风的首选注射药物,建议在临床应用中应重视过敏,注意观察,并尽快制定适合我国的破伤风预防指南和规范,促进合理用药。%Objective To understand the progress of the clinical utilization status and safety of tetanus antitoxin (TAT), and to provide references for post-marketing safety evaluation.Methods Domestic and foreign literatures on the clinical application and safety of TAT were reviewed for further analysis and comparison.Results Now TAT has been mainly used for passive immunity and tetanus therapeutics in clinic. Intensive safety studies showed that higher false-positive rate of its skin test mainly related to patients’ physique, medicine factor, infusion operation factor, and so on. Its main adverse effects included anaphylactic shock, erum sickness, and other kinds of adverse reactions.Conclusion TAT is still the ifrst choice against tetanus of trauma patients in China, the paper suggests that medical workers must put emphasis on anaphylaxis and close observation, along with our country should expeditiously enact a speciifc against tetanus guidelines and standards of Chinese style, and promoting rational drug use.

  12. Effect of different solvents on skin test results of tetanus antitoxin prepared%不同溶媒配制破伤风抗毒素皮试液对皮试结果的影响

    Institute of Scientific and Technical Information of China (English)

    施雪雁; 范爱飞; 彭丽萍; 龚海琴

    2011-01-01

    Objective To observe skin test results of tetanus antitoxin prepared by different solvents.Methods Patients who needed injecting tetanus antitoxin as required by trauma were randomly divided into two groups, Group A and Group B, respectively. Group A was administered with tetanus antitoxin prepared by water for injection, while Group B with tetanus antitoxin prepared by saline. Skin test results were observed, the incidence of positive skin test results and self-inductance of pain intensity were compared.Results The incidence of positive skin test results in Group A was significantly higher than that in Group B (34.19% vs 17.50%,χ2=8.631,P<0.01), and the self-inductance of pain intensity were also greater than that in Group B. Patients with positive skin test results (40 patients) in Group A, were later injected tetanus antitoxin prepared by saline, 42.50% of them (17 patients) shown negative skin test results.Conclusions There might be a high false positive rate by using tetanus antitoxin prepared by water for injection; therefore tetanus antitoxin prepared by saline is recommended, for it could decrease the false positive rate, reduce unnecessary desensitization injections, alleviate patients' pain and improve nurses' work efficiency.%目的 观察2种不同溶媒配制破伤风抗毒素(TAT)皮试液对皮试结果的影响.方法 将因外伤需要注射TAT的患者237例随机分为甲组117例和乙组120例,分别使用注射用水配制的TAT皮试液和生理盐水配制的TAT皮试液做皮肤过敏试验,观察两组患者的皮试结果,对比两组皮试结果阳性率以及患者自感疼痛度的高低.结果 甲组患者皮试结果阳性率明显高于乙组,差异具有统计学意义(34.19%比17.50%,χ2=8.631,P<0.01),且患者自感疼痛度也高于乙组.甲组皮试结果阳性的患者(40例)在改用生理盐水配制的TAT皮试液做皮试后,17例(42.50%)患者皮试结果转为阴性.结论 用注射用水配制的TAT皮试液做皮肤

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

    Directory of Open Access Journals (Sweden)

    Ocean R Cohen

    Full Text Available 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.

  14. Dominant negative mutants of Bacillus thuringiensis Cry1Ab toxin function as anti-toxins: demonstration of the role of oligomerization in toxicity.

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    Claudia Rodríguez-Almazán

    Full Text Available BACKGROUND: Bacillus thuringiensis Cry toxins, that are used worldwide in insect control, kill insects by a mechanism that depends on their ability to form oligomeric pores that insert into the insect-midgut cells. These toxins are being used worldwide in transgenic plants or spray to control insect pests in agriculture. However, a major concern has been the possible effects of these insecticidal proteins on non-target organisms mainly in ecosystems adjacent to agricultural fields. METHODOLOGY/PRINCIPAL FINDINGS: We isolated and characterized 11 non-toxic mutants of Cry1Ab toxin affected in different steps of the mechanism of action namely binding to receptors, oligomerization and pore-formation. These mutant toxins were analyzed for their capacity to block wild type toxin activity, presenting a dominant negative phenotype. The dominant negative phenotype was analyzed at two levels, in vivo by toxicity bioassays against susceptible Manduca sexta larvae and in vitro by pore formation activity in black lipid bilayers. We demonstrate that some mutations located in helix alpha-4 completely block the wild type toxin activity at sub-stoichiometric level confirming a dominant negative phenotype, thereby functioning as potent antitoxins. CONCLUSIONS/SIGNIFICANCE: This is the first reported case of a Cry toxin dominant inhibitor. These data demonstrate that oligomerization is a fundamental step in Cry toxin action and represent a potential mechanism to protect special ecosystems from the possible effect of Cry toxins on non-target organisms.

  15. The phage growth limitation system in Streptomyces coelicolor A(3)2 is a toxin/antitoxin system, comprising enzymes with DNA methyltransferase, protein kinase and ATPase activity.

    Science.gov (United States)

    Hoskisson, Paul A; Sumby, Paul; Smith, Margaret C M

    2015-03-01

    The phage growth limitation system of Streptomyces coelicolor A3(2) is an unusual bacteriophage defence mechanism. Progeny ϕC31 phage from an initial infection are thought to be modified such that subsequent infections are attenuated in a Pgl(+) host but normal in a Pgl(-) strain. Earlier work identified four genes required for phage resistance by Pgl. Here we demonstrate that Pgl is an elaborate and novel phage restriction system that, in part, comprises a toxin/antitoxin system where PglX, a DNA methyltransferase is toxic in the absence of a functional PglZ. In addition, the ATPase activity of PglY and a protein kinase activity in PglW are shown to be essential for phage resistance by Pgl. We conclude that on infection of a Pgl(+) cell by bacteriophage ϕC31, PglW transduces a signal, probably via phosphorylation, to other Pgl proteins resulting in the activation of the DNA methyltransferase, PglX and this leads to phage restriction.

  16. Advances in Biological Function of Toxin-Antitoxin Molecular Systems%毒性分子-抗毒性分子系统的生物学作用研究进展

    Institute of Scientific and Technical Information of China (English)

    李国利; 胡福泉

    2015-01-01

    毒性分子-抗毒性分子系统( toxin-antitoxin systems,TA systems)被发现广泛存在于细菌染色体、质粒以及古细菌基因组中。TA系统是由2个基因组成的操纵子,这2个基因分别编码稳定的毒性分子和不稳定的抗毒性分子。毒性分子总是蛋白质,抗毒性分子可能是蛋白质或RNA。因此,根据抗毒性分子的性质和作用方式的不同可将TA系统家族分为5种类型。Ⅰ型和Ⅲ型的抗毒性分子是RNA,能抑制毒性分子的合成或者与其隔离;II、IV和V型的抗毒性分子是蛋白质,能隔离、平衡毒性分子作用或抑制其合成。TA系统具有多种生物学功能。目前研究表明,TA系统可能在细菌应激应答、程序化细胞死亡、多重耐药的形成、防止DNA入侵、稳定大基因组片段等方面有重要的作用。%Toxin-antitoxin systems( TA systems)extensively exist in bacterial chromosomes and plasmids as well as in archaic genomes. TA systems is an operon consisting of two genes,they are respectively toxin molecule of stably encoded and anti-toxin molecule of unstably encoded. Toxin molecule is always protein,while antitoxin molecule may be protein or RNA. Therefore,currently TA systems family can be divided into five different types. Antitoxin molecule of type I and type III is RNA,it can inhibit the synthesis of toxin molecule or isolate toxin molecule. Antitoxin mole-cule of type II,IV,and V is protein,it can isolate,balance the role of toxin molecule or inhibit its synthesis. TA systems possess multiple biological functions. Recent studies suggested that TA systems might play important role and have involved in the cell stress response,programmed cell death,the form of multidrug resistance,prevention from DNA invasion,as well as stabilization of large genomic fragments and other aspects.

  17. Structure of the Escherichia coli Antitoxin MqsA (YgiT/b3021) Bound to Its Gene Promoter Reveals Extensive Domain Rearrangements and the Specificity of Transcriptional Regulation

    Energy Technology Data Exchange (ETDEWEB)

    B Brown; T Wood; W Peti; R Page

    2011-12-31

    Bacterial cultures, especially biofilms, produce a small number of persister cells, a genetically identical subpopulation of wild type cells that are metabolically dormant, exhibit multidrug tolerance, and are highly enriched in bacterial toxins. The gene most highly up-regulated in Escherichia coli persisters is mqsR, a ribonuclease toxin that, along with mqsA, forms a novel toxin-antitoxin (TA) system. Like all known TA systems, both the MqsR-MqsA complex and MqsA alone regulate their own transcription. Despite the importance of TA systems in persistence and biofilms, very little is known about how TA modules, and antitoxins in particular, bind and recognize DNA at a molecular level. Here, we report the crystal structure of MqsA bound to a 26-bp fragment from the mqsRA promoter. We show that MqsA binds DNA predominantly via its C-terminal helix-turn-helix domain, with direct binding of recognition helix residues Asn{sup 97} and Arg{sup 010} to the DNA major groove. Unexpectedly, the structure also revealed that the MqsA N-terminal domain interacts with the DNA phosphate backbone. This results in a more than 105{sup o} rotation of the N-terminal domains between the free and complexed states, an unprecedented rearrangement for an antitoxin. The structure also shows that MqsA bends the DNA by more than 55{sup o} in order to achieve symmetrical binding. Finally, using a combination of biochemical and NMR studies, we show that the DNA sequence specificity of MqsA is mediated by direct readout.

  18. Clinical features of 42 patients with drug eruption caused by tetanus antitoxin injection%破伤风抗毒素所致药疹42例临床特点分析

    Institute of Scientific and Technical Information of China (English)

    邵成明; 陈婷婷; 葛永兴; 张敏杰

    2016-01-01

    Objective:To determine the clinical characteristics of drug eruption caused by tetanus antitoxin injection . Methods:The clinical data of 42 cases with drug eruption caused by tetanus antitoxin were analyzed respectively. Results:The skin test was negative in 40 patients and positive in 2 patients. Incubation was 6 to 12 days and the average incubation was 6.5 days. The lesions were presented as erythema in injection area in 2 patients and as generalized rash in 40 patients ( exanthematous type in 6 patients, urticaria type in 32 and erythema multiforme type in 2) . Five of 42 patients accompanied with fever, 5 with voimt and 12 with joint pain. Glucocorticoid was effective in the treatment. Conclusion:The common type of drug eruption caused by tetanus antitoxin injection is urticaria type and some patients have fever, voimt and joint pain. There is no cor-relation between the drug eruption caused by tetanus antitoxin injection and the result of skin test.%目的::明确破伤风抗毒素所致药疹的临床特点。方法:对42例破伤风抗毒素所致药疹的临床资料进行回顾性分析。结果:42例患者中破伤风抗毒素皮试阴性40例,阳性2例。发疹潜伏期6~12天,平均为6.5天,皮疹表现为注射局部红斑2例,全身泛发性皮疹40例(发疹型6例,荨麻疹型32例,多型红斑型2例)。42例患者中伴发热5例,呕吐5例,关节痛12例。糖皮质激素治疗有效。结论:破伤风抗毒素所致药疹最常见类型为荨麻疹型,可伴发热、消化道症状及关节痛。药疹的发生与皮试结果无关。

  19. Bacteriophage ΦM1 of Pectobacterium evolves to escape two bifunctional Type III toxin-antitoxin and abortive infection systems through mutations in a single viral gene.

    Science.gov (United States)

    Blower, Tim R; Chai, Ray; Przybilski, Rita; Chindhy, Shahzad; Fang, Xinzhe; Kidman, Samuel E; Tan, Hui; Luisi, Ben F; Fineran, Peter C; Salmond, George P C

    2017-02-03

    Some bacteria, when infected by their viral parasites (bacteriophages), undergo a suicidal response that also terminates productive viral replication (abortive infection; Abi). This response can be viewed as an altruistic act protecting the uninfected bacterial clonal population. Abortive infection can occur through the action of Type III protein-RNA toxin-antitoxin (TA) systems, such as ToxINPa from the phytopathogen, Pectobacterium atrosepticum Rare spontaneous mutants evolved in the generalized transducing phage, ΦM1, which escaped ToxINPa-mediated abortive infection in P. atrosepticum ΦM1 is a member of the Podoviridae and member of the "KMV-like viruses", a subset of the T7 supergroup. Genomic sequencing of ΦM1 escape mutants revealed single-base changes which clustered in a single open reading frame. The "escape" gene product, M1-23, was highly toxic to the host bacterium when over-expressed, but mutations in M1-23 that enabled an escape phenotype caused M1-23 to be less toxic. M1-23 is encoded within the DNA metabolism modular section of the phage genome, and when it was over-expressed, it co-purified with the host nucleotide excision repair protein, UvrA. While the M1-23 protein interacted with UvrA in co-immunoprecipitation assays, a UvrA mutant strain still aborted ΦM1, suggesting that the interaction is not critical for the Type III TA Abi activity. Additionally, ΦM1 escaped a heterologous Type III TA system (TenpINPl) from Photorhabdus luminescens (reconstituted in P. atrosepticum) through mutations in the same protein, M1-23. The mechanistic action of M1-23 is currently unknown but further analysis of this protein could provide insights into the mode of activation of both systems.

  20. The proteolytic activation of the relNEs (ssr1114/slr0664) toxin-antitoxin system by both proteases Lons and ClpP2s/Xs of Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Ning, Degang; Ye, Sen; Liu, Biao; Chang, Jianing

    2011-11-01

    The proteolytic regulation of toxin-antitoxin (TA) systems has been well studied in Escherichia coli but remains unclear in other bacteria. A chromosomal gene pair ssr1114/slr0664, named relNEs, of Synechocystis sp. PCC 6803 forms a TA system belonging to rel family. Here, we used E. coli strain BL21 (DE3) as a host to characterize the proteolytic regulation of relNEs. The proteases of this strain could not degrade the antitoxin RelN, and the ectopic production of the ATP-dependant protease Lons or ClpP2s/Xs of Synechocystis sp. PCC6803 did not affect E. coli growth. Either Lons or ClpP2s/Xs was able to degrade RelN resulting in growth arrest of E. coli due to the activation of RelEs's toxicity, and the presence of RelEs could protect RelN to a certain extent against Lons and ClpP2s/Xs. Our observations suggest that both Lons and ClpP2s/Xs are responsible for RelN proteolysis in the native host under certain conditions. RelN is the first protein substrate identified for cyanobacterial ATP-dependent proteases.

  1. 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.

  2. 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.

  3. Recombinant cholera toxin B subunit (rCTB) as a mucosal adjuvant enhances induction of diphtheria and tetanus antitoxin antibodies in mice by intranasal administration with diphtheria-pertussis-tetanus (DPT) combination vaccine.

    Science.gov (United States)

    Isaka, Masanori; Komiya, Takako; Takahashi, Motohide; Yasuda, Yoko; Taniguchi, Tooru; Zhao, Yanqiu; Matano, Keiko; Matsui, Hideyuki; Maeyama, Jun-Ichi; Morokuma, Kazunori; Ohkuma, Kunio; Goto, Norihisa; Tochikubo, Kunio

    2004-08-13

    Recombinant cholera toxin B subunit (rCTB) which is produced by Bacillus brevis carrying pNU212-CTB acts as a mucosal adjuvant capable of enhancing host immune responses specific to unrelated, mucosally co-administered vaccine antigens. When mice were administered intranasally with diphtheria-pertussis-tetanus (DPT) combination vaccine consisting of diphtheria toxoid (DTd), tetanus toxoid (TTd), pertussis toxoid (PTd), and formalin-treated filamentous hemagglutinin (fFHA), the presence of rCTB elevated constantly high values of DTd- and TTd-specific serum ELISA IgG antibody titres, and protective levels of diphtheria and tetanus toxin-neutralizing antibodies but the absence of rCTB did not. Moreover, the addition of rCTB protected all mice against tetanic symptoms and deaths. DPT combination vaccine raised high levels of serum anti-PT IgG antibody titres regardless of rCTB and protected mice from Bordetella pertussis challenge. These results suggest that co-administration of rCTB as an adjuvant is necessary for induction of diphtheria and tetanus antitoxin antibodies on the occasion of intranasal administration of DPT combination vaccine.

  4. Clinical observation on influence of time of drinking of patients on skin test of tetanus antitoxin%饮酒时间对破伤风抗毒素皮试影响的临床观察

    Institute of Scientific and Technical Information of China (English)

    冯莉

    2007-01-01

    @@ 破伤风是常和创伤相关联的一种特异性感染,是一种严重的疾病,但是可以预防的[1].破伤风抗毒素(Tetanus Antitoxin TAT)是破伤风类毒素免疫马的血浆经胃酶消化后用盐析法制得的液体或冻干抗毒素球蛋白制剂,用于治疗及预防破伤风.然而对人体而言,TAT是一种异体蛋白质,具有抗原性,人体注射后很容易发生过敏反应,轻者皮疹,重者休克.因此在临床使用前应做TAT皮试,准确判断TAT皮试结果至关重要.若皮试阳性需采用脱敏注射,注射后引起的过敏反应给护理人员及病人造成极大的威胁与烦恼,严重时导致病人死亡.因此,对324例外伤伴饮酒的病人做TAT皮试临床观察及分析.现报告如下.

  5. 饮酒对破伤风抗毒素皮内试验阳性率影响%The Influence of Drink for Positive Rate of Tetanus Antitoxin Skin Test

    Institute of Scientific and Technical Information of China (English)

    贺慧玲; 贺丽玲

    2011-01-01

    目的:通过对比研究,探讨饮酒对破伤风抗毒素(Tetanus Antitoxin,TAT)皮内试验结果的影响.方法:于2009年1月~2011年1月,选取我院急诊门诊593例需要做破伤风抗毒素皮试的外伤患者,其中饮酒后患者322例,按照饮酒后0~12 h,12~24 h分为两组,A组0~12 h 82例,B组12~24 h 240例,C组对照组,非饮酒患者271例,结果采用卡方检验分析.结果:A组38例阳性,B组57例阳性,C组39例阳性.A与B组差异有统计学意义,A与C组差异有统计学意义,B与C组差异有统计学意义(P<0.05).结论:患者饮酒后时间的长短对TAT皮内试验结果影响很大,皮内试验时间距饮酒时间越长,阳性率越低.

  6. 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

  7. TOXINS AND ANTITOXINS OF BACILLUS DYSENTERIAE SHIGA.

    Science.gov (United States)

    Olitsky, P K; Kligler, I J

    1920-01-01

    With the methods which have been described we have separated an exotoxin and an endotoxin from cultures of the Shiga dysenteric bacillus. The study of the nature and effect of the poison of this microorganism is thus simplified. The two toxins are physically and biologically distinct. The exotoxin is relatively heat-labile, arises in the early period of growth, and yields an antiexotoxic immune serum. The endotoxin, on the other hand, is heat-stable, is formed in the later period of growth, and is not neutralized by the antiexotoxic serum. The exotoxin exhibits a specific affinity for the central nervous organs in the rabbit, giving rise to a characteristic lesion-mainly, hemorrhages, necroses, and possibly a perivascular infiltration in the gray matter of the upper spinal cord and medulla. The endotoxin exerts a typical action on the intestinal tract, producing edema, hemorrhages, necroses, and ulcerations, especially in the large intestine. In dysentery in man the intestinal lesions predominate, but in severe epidemics paralysis and neuritis have been observed (Osler(17)). These facts become specially significant from the standpoint of the serum therapy of bacillary dysentery. A potent antidysenteric serum should contain antibodies against the exotoxin as well as the endotoxin. That such a serum can be produced in horses has been experimentally demonstrated.

  8. Genetic fusions of a CFA/I/II/IV MEFA (multiepitope fusion antigen and a toxoid fusion of heat-stable toxin (STa and heat-labile toxin (LT of enterotoxigenic Escherichia coli (ETEC retain broad anti-CFA and antitoxin antigenicity.

    Directory of Open Access Journals (Sweden)

    Xiaosai Ruan

    Full Text Available Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs and two distinct enterotoxins [heat-labile toxin (LT and heat-stable toxin type Ib (STa or hSTa]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2:243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3, CFA/IV (CS4, CS5, CS6] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5:1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in

  9. Genetic fusions of a CFA/I/II/IV MEFA (multiepitope fusion antigen) and a toxoid fusion of heat-stable toxin (STa) and heat-labile toxin (LT) of enterotoxigenic Escherichia coli (ETEC) retain broad anti-CFA and antitoxin antigenicity.

    Science.gov (United States)

    Ruan, Xiaosai; Sack, David A; Zhang, Weiping

    2015-01-01

    Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent

  10. 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

  11. 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

  12. A Simple Model for Assessment of Anti-Toxin Antibodies

    Directory of Open Access Journals (Sweden)

    Alex Skvortsov

    2013-01-01

    Full Text Available The toxins associated with infectious diseases are potential targets for inhibitors which have the potential for prophylactic or therapeutic use. Many antibodies have been generated for this purpose, and the objective of this study was to develop a simple mathematical model that may be used to evaluate the potential protective effect of antibodies. This model was used to evaluate the contributions of antibody affinity and concentration to reducing antibody-receptor complex formation and internalization. The model also enables prediction of the antibody kinetic constants and concentration required to provide a specified degree of protection. We hope that this model, once validated experimentally, will be a useful tool for in vitro selection of potentially protective antibodies for progression to in vivo evaluation.

  13. A Simple Model for Assessment of Anti-Toxin Antibodies

    Science.gov (United States)

    Skvortsov, Alex; Gray, Peter

    2013-01-01

    The toxins associated with infectious diseases are potential targets for inhibitors which have the potential for prophylactic or therapeutic use. Many antibodies have been generated for this purpose, and the objective of this study was to develop a simple mathematical model that may be used to evaluate the potential protective effect of antibodies. This model was used to evaluate the contributions of antibody affinity and concentration to reducing antibody-receptor complex formation and internalization. The model also enables prediction of the antibody kinetic constants and concentration required to provide a specified degree of protection. We hope that this model, once validated experimentally, will be a useful tool for in vitro selection of potentially protective antibodies for progression to in vivo evaluation. PMID:23862138

  14. 21 CFR 610.20 - Standard preparations.

    Science.gov (United States)

    2010-04-01

    .... Perfringens Antitoxin. Antipertussis Serum. Antirabies Serum. Sordellii Antitoxin. Staphylococcus Antitoxin..., Purified Protein Derivative. Typhoid Vaccine. Blood Derivative Thrombin. (b) Opacity standard. The...

  15. Antitoxin use and pediatric intensive care for viper bites in Rome, Italy.

    Science.gov (United States)

    Marano, M; Pisani, M; Stoppa, F; Di Nardo, M; Pirozzi, N; Luca, E; Pulitanò, S; Conti, G; Marzano, L; De Luca, D; Valentini, P; Pietrini, D; Piastra, M

    2014-01-01

    In Italy viper bites represent an uncommon event, though envenomation can cause severe complications, more in children than adults, because of dose/body size ratio. We present a case series within a selected population: 10 Italian cases (from Rome surroundings) of viperbites requiring PICU admission, over a 5-year interval. Five children showed a systemic involvement, whereas the remaining patients showed a damage. All were managed and closely monitored in an ICU setting. Relevant clinical findings and therapeutic approach, ICU course and complications have been recorded. Age range was 3-15 years with mean age of 6,9 (SD±4,58) years; 2 patients needed respiratory support beyond oxygen supplementation. Most patients underwent fluid loading, while hemodynamic support was given to4/10. Median PICU stay was 60 hours (IQR=24.0-75.5). No mortality was reported. Indications and precautions for administration of antivenom in the last years have been reviewed: early treatment seems to reduce mortality/morbidity, though representing a threat for children. Current recommendations for the treatment of viper envenomation have been described, based on a literature's review and the application of these knowledges to clinical reality of our PICUs. Therefore, paediatric patients with systemic or rapidly evolving symptoms should be monitored carefully for the development of bite-related complications in an ICU setting mostly in younger children.

  16. Antitoxin activity of plants used in Mexican traditional medicine against scorpion poisoning.

    Science.gov (United States)

    Jiménez-Ferrer, J E; Pérez-Terán, Y Y; Román-Ramos, R; Tortoriello, J

    2005-01-01

    Scorpions, especially in urban areas of tropical and subtropical regions, present a common risk of poisoning. In Mexico, scorpion envenomation is considered a public health problem. Despite the frequency of scorpion sting cases, there are to date no uniform criteria for their treatment. In Mexican traditional medicine, different plant species have been widely used as a remedy for treating scorpion poisoning. The aim of this work was to evaluate the effect of Bouvardia ternifolia, Aristolochia elegans and Vitex mollis extracts on Centruroides limpidus limpidus venom lethality in mice, and to determine their antagonist activity on guinea pig ileum. The hexane and methanol extract from B. ternifolia modified the LD50 of C. limpidus limpidus venom from 0.750 +/- 0.08 to 1.64 +/- 0.19 and 1.16 +/- 0.14 mg/kg, respectively. The extracts of A. elegans produced lower antitoxic activity, while extracts of V. mollis did not show any protection. On in vitro test, addition of B. ternifolia and A. elegans extracts strongly inhibited, in a concentration-dependent manner, the ileum contractions induced by venom. In general, the results demonstrated the effectiveness of these two plant species in modifying the lethality of C. limpidus limpidus venom in mice.

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

    Science.gov (United States)

    ... News & Events Home News & Events Newsroom Press Announcements FDA NEWS RELEASE For Immediate Release: March 22, 2013 ... Inquiries: Morgan Liscinsky, 301-796-0397, morgan.liscinsky@fda.hhs.gov Consumer Inquiries: 888-INFO-FDA, OCOD@ ...

  18. External quality assessment for the determination of diphtheria antitoxin in human serum.

    Science.gov (United States)

    Di Giovine, Paolo; Pinto, Antonella; Olander, Rose-Marie; Sesardic, Dorothea; Stickings, Paul; Berbers, Guy; Neal, Shona; Efstratiou, Androulla; Paberza, Ruta; Dauksiene, Snieguole; Bujko, Marina; Detcheva, Antoaneta; Joks, Unna; Levent, Belkis; von Hunolstein, Christina

    2010-08-01

    Accurate determination of diphtheria toxin antibodies is of value in determining the rates of immunity within broad populations or the immune status of individuals who may be at risk of infection, by assessing responses to vaccination and immunization schedule efficacy. Here we report the results of an external quality assessment (EQA) study for diphtheria serology, performed within the dedicated surveillance network DIPNET. Twelve national laboratories from 11 European countries participated by testing a standard panel of 150 sera using their current routine method: Vero cell neutralization test (NT), double-antigen enzyme-linked immunosorbent assay (ELISA; DAE), dual double-antigen time-resolved fluorescence immunoassay (dDA-DELFIA), passive hemagglutination assay (PHA), toxin binding inhibition assay (ToBI), and in-house or commercial ELISAs. The objective of the study was not to identify the best assay, as the advantages and drawbacks of methods used were known, but to verify if laboratories using their routine method would have categorized (as negative, equivocal, or positive) a serum sample in the same way. The performance of each laboratory was determined by comparing its results on a quantitative and qualitative basis to NT results from a single reference laboratory, as this test is considered the in vitro "gold standard." The performance of laboratories using NT was generally very good, while the laboratories' performance using other in vitro methods was variable. Laboratories using ELISA and PHA performed less well than those using DAE, dDA-DELFIA, or ToBI. EQA is important for both laboratories that use in vitro nonstandardized methods and those that use commercial ELISA kits.

  19. European sero-epidemiology network: standardisation of the results of diphtheria antitoxin assays.

    Science.gov (United States)

    von Hunolstein, C; Aggerbeck, H; Andrews, N; Berbers, G; Fievet-Groyne, F; Maple, P A; Olander, R M; Raux, M; Tischer, A

    2000-08-01

    A European Sero-Epidemiological Network (ESEN) was established with the aim to co-ordinate and harmonise serological surveillance of immunity to communicable diseases in Europe. In this study the inter-laboratory standardisation of diphtheria toxin antibody measurements is reported. A standard panel of 162 sera was tested by the participating laboratories using an in vitro assay of their choice: VERO cell toxin neutralisation assay (NT), double-antigen delayed time-resolved fluorescence immuno-assay (DA-DELFIA), double-antigen enzyme-linked immunosorbent assay (DAE), toxin binding inhibition test (ToBI) and an indirect enzyme-linked immunosorbent assay (ELISA). The results were standardised using regression against the NT. The variations due to inter-laboratory and inter-assay variation, which would otherwise make it difficult directly to compare the main serum bank results by the different laboratories and the various assays were successfully minimised by the standardisation. The regression equations obtained will be used to transform the respective local results of testing the main serum bank into the reference test unitages. This study also gave the opportunity to compare the various assays within and between laboratories. This demonstrated a very high correlation between DA-DELFIA, DAE, ToBI and the NT. The ELISA showed a good correlation, too, however sera below some 0.1 IU/ml were overestimated.

  20. Benzene inhalation effects upon tetanus antitoxin. Responses and leukemogenesis in mice

    Energy Technology Data Exchange (ETDEWEB)

    Stoner, R D; Drew, R T; Bernstein, D M

    1980-01-01

    The effects of inhaled benzene on primary and secondary antibody responses and the incidence of leukemia in mice are reported. Young adult mice were given 5, 12, or 22 exposures to 400 ppM benzene for 6 hrs/day 5 days/week. After the exposure periods, the mice were immunized with absorbed tetanus toxoid (APTT) and/or fluid tetanus toxid (FTT). Exposure to benzene increasingly suppressed primary antibody responses to both antigens. Secondary antibody responses to FTT were nearly normal in animals given 10, 15, or 20 exposures to 400 ppM benzene. Other groups of mice were exposed to either 200 ppM or 50 ppM benzene. Primary antibody responses elicited with FTT and/or APTT were nearly normal in all mice exposed to 50 ppM benzene and in mice exposed to 200 ppM benzene for 5 days. However, 10 and 20 exposures to 200 ppM benzene inhibited antibody production. The effects of chronically inhaled 300 ppM benzene on the time of onset and incidence of leukemia in 400 7-month-old female HRS/J mice were also studied. Two genotypes were used; the (hr/hr) hairless mice are leukemia-prone, whereas the (hr/+) haired mice are more resistant to leukemia. The exposure continued for a period of 6 months. Lymphoid, myeloid, and mixed (lymphoid and myeloid) leukemias were observed. Ninety percent of the (hr/hr) mice exposed to benzene died from leukemia as compared with 91% for the (hr/hr) air control group. Eighty-five percent of the (hr/+) mice exposed to benzene died from leukemia as compared with 81% for the (hr/+) air control group. Exposures to 300 ppM benzene did not alter the time of onset or the incidence of leukemia commonly expected in HRS/J mice.

  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. 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

    obtained after successive injections of DM43 in a concentration series were globally fitted to a simple bimolecular interaction, yielding the following kinetic rates for the DM43/jararhagin interaction: k(a) = 3.54 ± 0.03 × 10(4) M(-1) s(-1) and k(d) = 1.16 ± 0.07 × 10(-5) s(-1), resulting...

  3. Improved ELISA for determination of anti-diphtheria and/or anti-tetanus antitoxin antibodies in sera.

    Science.gov (United States)

    Kristiansen, M; Aggerbeck, H; Heron, I

    1997-11-01

    Double-antigen ELISAs for detection and quantification of anti-tetanus or anti-diphtheria antibodies in serum have been developed. The assays showed good correlations with established toxin neutralizing assays and were functionally specific for IgG antibodies. The double-antigen set-up allows specific antibodies to bind to antigen-coated microtitre wells with one arm and the free arm to bind to biotin-labelled antigen. The amount of antibodies able to bind labelled antigen was assessed by adding enzyme-conjugated streptavidin and colour substrate followed by measurement of the colour using an ELISA reader. The double-antigen principle makes it possible to compare samples of different species on the same plate, permitting the direct use of existing international references of animal or human origin. The double-antigen ELISAs showed a detection limit of 0.00002 IU/ml for both antibodies and were suitable for quantifying antibodies in blood samples collected on filter paper as well as in serum. The assays required no special equipment compared to traditional ELISA.

  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. 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.

    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 a total...

  6. The HigB/HigA Toxin/Antitoxin System of Pseudomonas aeruginosa Influences the Virulence Factors Pyochelin, Pyocyanin, and Biofilm Formation

    Science.gov (United States)

    2016-08-24

    metabolism to allow cells to withstand stress such as that from antibiotics (Cheng et al. 2014; Islam et al. 2015) and bile acid (Kwan et al. 2015...debris. The lysate was filtered through a 0.22- μm membrane and loaded on a HisTrap HP column (GE Healthcare, Pittsburgh, PA, USA ). HigB and...using HRP- conjugated anti- His antibody and HRP- conjugated anti- FLAG antibody (Thermo Scientific, Waltham, MA, USA ). Blotted proteins were

  7. 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

    . 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...

  8. Drug: D05187 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available gs in Japan [BR:br08301] 6 Agents against pathologic organisms and parasites 63 Biological preparations 633 Antitoxins, anti-leptospi...ral sera 6331 Antitoxins D05187 Gas gangrene antitoxin, equine (JP16) PubChem: 17398252 ...

  9. 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

  10. Drug: D06480 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D06480 Drug Diphtheria antitoxin Immunizing agent [passive] ATC code: J06AA01 Anato...RA AND IMMUNOGLOBULINS J06A IMMUNE SERA J06AA Immune sera J06AA01 Diphtheria antitoxin D06480 Diphtheria antitoxin PubChem: 47208136 ...

  11. Modeling sRNA-Regulated Plasmid Maintenance

    Science.gov (United States)

    Klumpp, Stefan

    2017-01-01

    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, a short half-life of the protein toxin is also beneficial to the function of the toxin-antitoxin system. In addition, we study a therapeutic scenario in which a competitor mRNA is introduced to sequester the sRNA antitoxin, causing the toxic protein to be expressed. PMID:28085919

  12. 结核分枝杆菌毒素-抗毒素伴侣系统基因多态性初步研究%Polymorphisms of toxin-antitoxin-chaperone system of Mycobacterium tuberculosis complex in China

    Institute of Scientific and Technical Information of China (English)

    肖彤洋; 赵丽丽; 刘海灿; 李马超; 赵秀芹; 万康林

    2016-01-01

    目的 研究结核分枝杆菌毒素-抗毒素伴侣(TAC)系统中higA、higB和Rv1957在结核分枝杆菌及其不同亚型菌株中的基因多态性,并探讨其生理意义.方法 选取183株结核分枝杆菌临床菌株,经间隔区寡核苷酸方法进行基因分型,同时分析TAC系统基因higA、higB和Rv1957的PCR扩增及序列,利用I-Mutant 2.0软件预测非同义突变对蛋白结构和功能的影响.结果 183株中138株(75.41%)属北京家族,45株(24.59%)属非北京家族.共149株菌(81.42%)的TAC系统发生突变,包括2种同义突变和6种非同义突变:同义突变发生于higA基因,仅见于北京家族菌株;3个基因均可见非同义突变,其中2种非同义突变仅见于非北京家族菌株,其余4种突变仅见于北京家族菌株.6种非同义突变中有4种突变可能影响蛋白的功能.位于higA基因的CAC121CAT突变位点在单耐链霉素和单耐利福平菌株中的突变频率高于敏感株,且差异有统计学意义(P<0.05).结论 结核分枝杆菌中的TAC系统具有一定的基因多态性,其中北京家族呈现更高的多态性水平,可能更有利于适应不同的宿主环境.%Objective To investigate the single nucleotide polymorphism (SNP) of toxinantitoxin-chaperone (TAC) system of Mycobacterium (M.) tuberculosis with different genotypes and its biological significance.Methods A total of 183 clinical M.tuberculosis isolates were collected for spoligotyping.The sequences of higA,higB and Rv1957 were obtained by using PCR and DNA sequencing.The sequences were compared for possible mutations.Functional consequences of nonsynonymous SNPs were predicted by using I-Mutant 2.0 servers.Results Among the 183 M.tuberculosis isolates,138(75.41%) belonged to the Beijing family,while 45(24.59%) belonged to the non-Beijing family.A total of 149(81.42%) isolates showed polymorphisms in the TAC system.We discovered 6 nonsynonymous SNPs and 2 synonymous SNPs.All the synonymous mutations occurred in higA gene,while nonsynonymous SNPs were found in the higA,higB and Rv1957 genes either.All the synonymous mutations and 4 nonsynonymous SNPs were restricted to the Beijing family strains and only 2 nonsynonymous SNPs were observed in the non-Beijing family strains.Of the 6 nonsynonymous SNPs studied,4 were predicted to have ability to affect the stability of respective protein.Conclusion The SNPs in the coding sequences of TAC system in clinical isolates can be relatively high and the Beijing family strains are with higher polymorphism,which might benefit to adapt to different host environment.

  13. The Molecular Evolution of Type Ⅱ Toxin-antitoxin(TA)System-MazEF in Bifidobacterial Genomes%双歧杆菌Ⅱ型毒素-抗毒素系统-MazEF的分子进化分析

    Institute of Scientific and Technical Information of China (English)

    韦艳霞; 刘佃滨; 郑纪伟; 尤红娟; 张鹏; 李阳; 汤仁仙

    2014-01-01

    Objective:To perform molecular evolution analysis of MazEF in genomes of Bifidobacterium. Method:The Protein sequences of MazF in bifidobacterial genomes were obtained by employing the PSI-BLAST search from NCBI. The Protein sequences of MazF encoded by other bacteria were obtained from non-redundant protein database of NCBI. All of the 16 S rRNA sequences used in this study were obtained from non-redundant nucleotide database of NCBI. Clustal χ² and MEGA4 were used to analyze molecular evolution of MazF. Result:The total number of MazF in bifidobacterial genomes were searched of 26 The molecular evolution analysis showed that MazF in bifidobacterial genomes had less conservation;the co-evolution of MazF with the 16 S RNA was only observed in individual species and most homologous analysis results of MazF were not in concordance with 16 S rRNA. Conclusion:MazEF was integrated in bifidobacterial chromosomes through horizontal transfer,which was a part of the non-core genome.%目的:对双歧杆菌属编码的Ⅱ类毒素-抗毒素(TA)系统MazEF进行分子进化分析。方法:利用PSI-BLAST在NCBI中搜索获得双歧杆菌编码的MazF分子序列,在NCBI蛋白数据库、核酸数据库分别下载其他菌属基因组编码的MazF蛋白序列和本研究所涉及菌属的16 S rRNA序列,应用Clustal X2和MEGA4软件对MazF做分子进化分析。结果:共搜索到双歧杆菌株染色体编码毒素蛋白MazF 26个。分子进化分析显示:整个双歧杆菌属的MazF保守性不好;MazF与16 S rRNA的共进化仅在个别菌种中发现,大部分MazF的聚类结果与相应菌种16 S rRNA的聚类结果不同。结论:双歧杆菌中的TA系统MazEF可能是通过基因的水平转移整合入基因组中,属于菌株非核心基因组部分。

  14. 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.

  15. Progress in research on toxins-antitoxins system in bacterial programmed cell death%细菌的细胞程序性死亡中毒素-抗毒素系统的研究进展

    Institute of Scientific and Technical Information of China (English)

    季建军; 邱景富; 杨瑞馥

    2006-01-01

    与哺乳动物细胞相同,细菌也存在着细胞程序性死亡.本文对细菌中存在的细胞程序性死亡现象、引发细菌造成细胞程序性死亡的毒素-抗毒素系统的组成、分类、意义以及影响细胞程序性死亡的因素进行了介绍.

  16. THE SYNERGY OF BACTERIAL TOXINS,

    Science.gov (United States)

    TOXINS AND ANTITOXINS, STRENGTH(PHYSIOLOGY), BACTERIA, CLOSTRIDIUM PERFRINGENS, CLOSTRIDIUM TETANI , CLOSTRIDIUM , STAPHYLOCOCCUS, ESCHERICHIA COLI, PROTEUS, ETIOLOGY, ANTIGENS, ANTIBODIES, AMINO ACIDS.

  17. 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 ...

  18. 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 ...

  19. Bacillus globigii cell size is influenced by variants of the quorum sensing peptide extracellular death factor

    NARCIS (Netherlands)

    Sijbrandij, T.; Kaman, W.E.; Ligtenberg, A.J.M.; Nazmi, K.; Veerman, E.C.I.; Bikker, F.J.

    2014-01-01

    Toxin-antitoxin modules are necessary for the mode of action of several antibiotics. One of the most studied toxin-antitoxin modules is the quorum sensing - dependent MazEF system in Escherichia coli. The quorum sensing factor in this system is called the extracellular death factor (EDF), a linear p

  20. Bacillus globigii cell size is influenced by variants of the quorum sensing peptide extracellular death factor

    NARCIS (Netherlands)

    T. Sijbrandij (T.); W.E. Kaman (Wendy); A.J.M. Ligtenberg (A. J M); K. Nazmi (Kamran); E.C.I. Veerman (Enno); F.J. Bikker (Floris)

    2014-01-01

    textabstractToxin-antitoxin modules are necessary for the mode of action of several antibiotics. One of the most studied toxin-antitoxin modules is the quorum sensing - dependent MazEF system in Escherichia coli. The quorum sensing factor in this system is called the extracellular death factor (EDF)

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

    NARCIS (Netherlands)

    Swaan, C.M.; Ouwerkerk, van M.; 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 outbr

  2. Identification of Enterotoxin E

    Science.gov (United States)

    Bergdoll, Merlin S.; Borja, Concordia R.; Robbins, Ruth N.; Weiss, Karl F.

    1971-01-01

    Identification of a new enterotoxin was accomplished by purification of the enterotoxin produced by staphylococcal strain FRI-326 and by preparation of specific antitoxin to the enterotoxin. Toxicity of the preparations was determined in rhesus monkeys, and specificity of the enterotoxin-antitoxin reaction was determined in gel diffusion plates. The enterotoxin was designated enterotoxin E. PMID:5005309

  3. 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, ...

  4. The Bacillus cereus spoIIS programmed cell death system

    Directory of Open Access Journals (Sweden)

    Jana eMelnicakova

    2015-08-01

    Full Text Available Programmed cell death in bacteria is generally associated with two¬ component toxin antitoxin systems. The SpoIIS toxin-antitoxin system, consisting of a membrane bound SpoIISA toxin and a small, cytosolic antitoxin SpoIISB, was originally identified in Bacillus subtilis. In this work we describe the Bacillus cereus SpoIIS system which is a three-component system, harbouring an additional gene spoIISC. Its protein product serves as an antitoxin, and similarly as SpoIISB, is able to bind SpoIISA and abolish its toxic effect. Our results indicate that SpoIISC seems to be present not only in B. cereus but also in other Bacilli containing a SpoIIS toxin antitoxin system. In addition, we show that B. cereus SpoIISA can form higher oligomers and we discuss the possible role of this multimerization for the protein’s toxic function.

  5. Medical Countermeasure Models. Volume 8. Botulinum Neurotoxin

    Science.gov (United States)

    2013-04-12

    were treated with antibiotics , 5,000 would be entered as identical treated individuals and 5,000 would be entered as identical untreated individuals...toxin as a biological weapon.” Journal of the American Medical Association. 285(1059). 2001. 24 Tacket CO et al. “ Equine Antitoxin Use and Other...antitoxin available for non-infant cases of botulism in the United States.69,70 HBAT, an equine antitoxin, targets BoNT serotypes A, B, C, D, E, F and G

  6. 21 CFR 610.21 - Limits of potency.

    Science.gov (United States)

    2010-04-01

    ... Globulin (Human), 250 units of tetanus antitoxin per container. Antigens Cholera Vaccine, 8 units each of Inaba and Ogawa serotype antigens per milliliter. Pertussis Vaccine, 12 units per total human immunizing dose. Typhoid Vaccine, 8 units per milliliter....

  7. Immune response

    Science.gov (United States)

    ... and tetanus antitoxin are examples of passive immunization. BLOOD COMPONENTS The immune system includes certain types of white ... lymphocytes develop, they normally learn to tell the difference between your own body tissues and substances that ...

  8. Expression and Immunogenicity of a Mutant Diphtheria Toxin Molecule, CRM197, and Its Fragments in Salmonella typhi Vaccine Strain CVD 908-htrA

    Science.gov (United States)

    Orr, Nadav; Galen, James E.; Levine, Myron M.

    1999-01-01

    Mutant diphtheria toxin molecule CRM197 and fragments thereof were expressed in attenuated Salmonella typhi CVD 908-htrA, and the constructs were tested for their ability to induce serum antitoxin. Initially, expressed proteins were insoluble, and the constructs failed to induce neutralizing antitoxin. Soluble CRM197 was expressed at low levels by utilizing the hemolysin A secretion system from Escherichia coli. PMID:10417208

  9. 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

    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......RUM replicons (74%) than non-CC17 strains (2.2% and 35%, respectively). A prevalent genetic linkage between the pRUM-replicon type and axe-txe was demonstrated by cohybridization analyses. The vanA resistance determinant was associated with all four replicon types, but we also confirmed the genetic linkage...... of vanA to unknown transferable replicons. PCR-based replicon typing, linked to the detection of other important plasmid-encoded traits, seems to be a feasible tool for tracing disseminating resistance plasmids stably maintained in various environments....

  10. Selectivity and self-assembly in the control of a bacterial toxin by an antitoxic noncoding RNA pseudoknot.

    Science.gov (United States)

    Short, Francesca L; Pei, Xue Y; Blower, Tim R; Ong, Shue-Li; Fineran, Peter C; Luisi, Ben F; Salmond, George P C

    2013-01-15

    Bacterial small RNAs perform numerous regulatory roles, including acting as antitoxic components in toxin-antitoxin systems. In type III toxin-antitoxin systems, small processed RNAs directly antagonize their toxin protein partners, and in the systems characterized the toxin and antitoxin components together form a trimeric assembly. In the present study, we sought to define how the RNA antitoxin, ToxI, inhibits its potentially lethal protein partner, ToxN. We show through cross-inhibition experiments with the ToxIN systems from Pectobacterium atrosepticum (ToxIN(Pa)) and Bacillus thuringiensis (ToxIN(Bt)) that ToxI RNAs are highly selective enzyme inhibitors. Both systems have an "addictive" plasmid maintenance phenotype. We demonstrate that ToxI(Pa) can inhibit ToxN(Pa) in vitro both in its processed form and as a repetitive precursor RNA, and this inhibition is linked to the self-assembly of the trimeric complex. Inhibition and self-assembly are both mediated entirely by the ToxI(Pa) RNA, with no requirement for cellular factors or exogenous energy. Finally, we explain the origins of ToxI antitoxin selectivity through our crystal structure of the ToxIN(Bt) complex. Our results show how a processed RNA pseudoknot can inhibit a deleterious protein with exquisite molecular specificity and how these self-contained and addictive RNA-protein pairs can confer different adaptive benefits in their bacterial hosts.

  11. Expression, Purification, and Functional Analysis of Novel RelE Operon from X. nematophila

    Directory of Open Access Journals (Sweden)

    Jitendra Singh Rathore

    2014-01-01

    Full Text Available Bacterial toxin-antitoxin (TA complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms. Escherichia coli RelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin from Xenorhabdus nematophila possessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter in E. coli Top 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

  12. Orphan Toxin OrtT (YdcX of Escherichia coli Reduces Growth during the Stringent Response

    Directory of Open Access Journals (Sweden)

    Sabina Islam

    2015-01-01

    Full Text Available Toxin/antitoxin (TA systems are nearly universal in prokaryotes; toxins are paired with antitoxins which inactivate them until the toxins are utilized. Here we explore whether toxins may function alone; i.e., whether a toxin which lacks a corresponding antitoxin (orphan toxin is physiologically relevant. By focusing on a homologous protein of the membrane-damaging toxin GhoT of the Escherichia coli GhoT/GhoS type V TA system, we found that YdcX (renamed OrtT for orphan toxin related to tetrahydrofolate is toxic but is not part of TA pair. OrtT is not inactivated by neighboring YdcY (which is demonstrated to be a protein, nor is it inactivated by antitoxin GhoS. Also, OrtT is not inactivated by small RNA upstream or downstream of ortT. Moreover, screening a genomic library did not identify an antitoxin partner for OrtT. OrtT is a protein and its toxicity stems from membrane damage as evidenced by transmission electron microscopy and cell lysis. Furthermore, OrtT reduces cell growth and metabolism in the presence of both antimicrobials trimethoprim and sulfamethoxazole; these antimicrobials induce the stringent response by inhibiting tetrahydrofolate synthesis. Therefore, we demonstrate that OrtT acts as an independent toxin to reduce growth during stress related to amino acid and DNA synthesis.

  13. Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines - extended study: correlation of serology with in vivo toxin neutralisation.

    Science.gov (United States)

    Sesardic, D; Winsnes, R; Rigsby, P; Behr-Gross, M-E

    2004-01-01

    Phase I of BSP034 collaborative study was extended in two laboratories to include correlation of serology with in vivo toxin neutralisation test (TNT) using 2 separate sets of 20 serum pools, produced in-house. The study investigated the extent to which the in vitro methods for diphtheria antibodies, Vero cell assay and diphtheria enzyme-linked immunosorbent assay for diphtheria antitoxin (D-ELISA), can detect neutralising antibodies by comparison with TNT in guinea pigs. The study was also performed to compare the antibody neutralising potency obtained in relation to guinea pig (GP) or equine (DI) antitoxin standard. In addition, the study provided an opportunity to compare ELISA for tetanus antitoxin (T-ELISA) and TNT assay for detection of anti-tetanus antibodies, from the same set of serum pools. The data obtained show that antitoxin potency obtained by Vero cell assay, D-ELISA and T-ELISA using the same GP standard, highly correlated with neutralising potency as determined in respective TNT assays. Vero cell assay with DI provided estimates that also correlated with neutralising potency, but were of significantly lower titre. Since reference to DI standard is widely used in serodiagnosis, as well as in clinical studies where diphtheria antitoxin titres obtained in the Vero cell method are taken as surrogate markers for vaccine efficacy, it should be investigated if a similar difference is also observed for human serology.

  14. Seroprevalence of tetanus toxoid antibody and booster vaccination efficacy in Japanese travelers.

    Science.gov (United States)

    Mizuno, Yasutaka; Yamamoto, Akihiko; Komiya, Takako; Takeshita, Nozomi; Takahashi, Motohide

    2014-01-01

    Tetanus can be prevented by vaccination, which is especially important for overseas travelers. However, despite booster vaccination every 10 years being recommended, most Japanese adults do not receive it in the absence of physical injury or overseas travel. We aimed to investigate the level of protective immunity against tetanus among Japanese travelers, which may provide valuable information for formulating booster vaccination recommendations. 113 Japanese travelers given tetanus toxoid were recruited. The collected samples included paired samples prior to and 3-5 weeks after receiving the booster vaccination. Travelers who did not return and those lacking sample collection at the second visit were excluded. Finally, 96 paired blood samples were collected. History of immunization against tetanus, including DPT and DT vaccines, was determined from interviews or immunization records. The pre-vaccination geometric mean titer for the 96 participants was 1.07 IU/mL; 76% had a protective antitoxin level (>0.1 IU/mL), and 50% had a long-term protective antitoxin level (>1.0 IU/mL). Most participants 50 years of age had protective immunity. Among the 23 participants without protective antitoxin levels (50 years of age. Although the tetanus antitoxin level decreases with age, booster vaccination helped to achieve an adequate protective antitoxin levels in Japanese travelers tetanus especially in those >50 years old need to obtain protective immunity against tetanus according to a basic immunization schedule to prevent tetanus in travelers and residents of Japan.

  15. 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.

  16. Identification of a New Enterotoxin as Enterotoxin C

    Science.gov (United States)

    Bergdoll, Merlin S.; Borja, Concordia R.; Avena, Remedios M.

    1965-01-01

    Bergdoll, Merlin S. (University of Chicago, Chicago, Ill.), Concordia R. Borja, and Remedios M. Avena. Identification of a new enterotoxin as enterotoxin C. J. Bacteriol. 90:1481–1485. 1965.—Identification of a new enterotoxin was accomplished by purification of the enterotoxins produced by staphylococcal strains 137 and 361 and by the preparation of specific antitoxin to the enterotoxin. Toxicity of the preparations was determined in rhesus monkeys, and specificity of the enterotoxin-antitoxin reaction was determined in gel-diffusion plates. The enterotoxin has been designated enterotoxin C, and staphylococcal strain 137 (ATCC 19095) was selected as the prototype strain. Images PMID:4954560

  17. Durability of immunity to diphtheria, tetanus and poliomyelitis after a three dose immunization schedule completed in the first eight months of life.

    Science.gov (United States)

    Jones, A E; Johns, A; Magrath, D I; Melville-Smith, M; Sheffield, F

    1989-08-01

    Blood samples were obtained from school entrants whose primary immunization schedule had consisted of three doses of DT or DTP vaccine and three doses of OPV all given before the age of 8 months. The sera were separated and assayed for diphtheria antitoxin, tetanus antitoxin and antibodies to the three serotypes of poliovirus. The results of the assays showed that the abbreviated three dose schedule induced satisfactory immunity to all five infections until school entry and that a reinforcing dose at 18 months was unnecessary.

  18. RNA decay by messenger RNA interferases

    DEFF Research Database (Denmark)

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

    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....

  19. Molecular biology. A Swiss army knife of immunity

    NARCIS (Netherlands)

    Brouns, S.J.J.

    2012-01-01

    Selfish genetic elements are more than a daily nuisance in the life of prokaryotes. Whereas viruses can multiply by reprogramming host cells, or integrate in the host genome as “stowaways,” conjugative plasmids (transferrable extrachromosomal DNA) make cells addicted to plasmid-encoded antitoxin fac

  20. Investigation of immunity level against diphtheria and reinforcement of immunity by booster vaccination for infection control staff in Okayama prefecture.

    Science.gov (United States)

    Nakajima, Hiroshi; Kariya, Hideaki; Ohata, Ritsuko; Ogura, Hajime

    2008-03-01

    The prevalence of immunity against diphtheria among Okayama local government staff members involved in diphtheria infection control was measured. Diphtheria booster vaccination was administered to staff members with low antitoxin levels ( or =0.1 IU/ml), and the remaining 157 (63.3%) showed levels of diphtheria-pertussis mixed vaccine (born in 1958-1967) and diphtheria-pertussis-tetanus mixed vaccine (born in 1968-) (58.3-61.0%) groups than in diphtheria vaccine (born in 1948-1957) and non-vaccinated (born until 1947) (7.4-18.9%) groups. Though antitoxin levels of 13 (68.4%) out of 19 staff members given booster vaccinations increased to 0.1 IU/ml, 50% of these individuals then showed levels of or =0.1 IU/ml in the non-booster vaccination group maintained their immunity levels for 2-4 years, independent of their history of vaccination. To ensure that staff members of the local government have fully protective antitoxin levels against diphtheria, periodical confirmation of antitoxin levels and booster vaccination should both be systematically carried out.

  1. Dynamic metabolic adjustments and genome plasticity are implicated in the heat shock response of the extremely thermoacidophilic archaeon Sulfolobus solfataricus.

    Science.gov (United States)

    Tachdjian, Sabrina; Kelly, Robert M

    2006-06-01

    Approximately one-third of the open reading frames encoded in the Sulfolobus solfataricus genome were differentially expressed within 5 min following an 80 to 90 degrees C temperature shift at pH 4.0. This included many toxin-antitoxin loci and insertion elements, implicating a connection between genome plasticity and metabolic regulation in the early stages of stress response.

  2. Stochastic induction of persister cells by HipA through (p)ppGpp-mediated activation of mRNA endonucleases

    DEFF Research Database (Denmark)

    Germain-Maisonneuve, Elsa; Roghanian, Mohammad; Gerdes, Kenn

    2015-01-01

    The model organism Escherichia coli codes for at least 11 type II toxin-antitoxin (TA) modules, all implicated in bacterial persistence (multidrug tolerance). Ten of these encode messenger RNA endonucleases (mRNases) inhibiting translation by catalytic degradation of mRNA, and the 11th module, hi...

  3. 9 CFR 113.108 - Clostridium Novyi Bacterin-Toxoid.

    Science.gov (United States)

    2010-01-01

    ...-antitoxin mixtures at room temperature for 1 hour and hold in ice water until injections of mice can be made... Animal and Plant Health Inspection Service. (vi) Diluent. The solution used to make proper dilutions... chloride in each 100 ml of distilled water; adjusting the pH to 7.2; autoclaving at 121 °C for 25...

  4. 9 CFR 113.111 - Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid.

    Science.gov (United States)

    2010-01-01

    ... toxin-antitoxin mixtures at room temperature for 1 hour and hold in ice water until injections of mice.... The solution used to make proper dilutions prescribed in this test. Such solutions shall be made by dissolving 1 gram of peptone and 0.25 grams of sodium chloride in each 100 ml of distilled water;...

  5. 9 CFR 113.109 - Clostridium Sordellii Bacterin-Toxoid.

    Science.gov (United States)

    2010-01-01

    ... diluent. (v) Neutralize all toxin-antitoxin mixtures at room temperature for 1 hour and hold in ice water... Inspection Service. (vi) Diluent. The solution used to make proper dilutions prescribed in this test. Such... distilled water; adjusting the pH to 7.2; autoclaving at 121 °C for 25 minutes; and storing at 4 °C...

  6. HicA of Escherichia coli defines a novel family of translation-independent mRNA interferases in bacteria and archaea

    DEFF Research Database (Denmark)

    Jørgensen, Mikkel G; Pandey, Deo P; Jaskolska, Milena

    2009-01-01

    Toxin-antitoxin (TA) loci are common in free-living bacteria and archaea. TA loci encode a stable toxin that is neutralized by a metabolically unstable antitoxin. The antitoxin can be either a protein or an antisense RNA. So far, six different TA gene families, in which the antitoxins are proteins......, have been identified. Recently, Makarova et al. (K. S. Makarova, N. V. Grishin, and E. V. Koonin, Bioinformatics 22:2581-2584, 2006) suggested that the hicAB loci constitute a novel TA gene family. Using the hicAB locus of Escherichia coli K-12 as a model system, we present evidence that supports...... this inference: expression of the small HicA protein (58 amino acids [aa]) induced cleavage in three model mRNAs and tmRNA. Concomitantly, the global rate of translation was severely reduced. Using tmRNA as a substrate, we show that HicA-induced cleavage does not require the target RNA to be translated...

  7. Botulisme hos spaedbørn

    DEFF Research Database (Denmark)

    Hoffmann, Thomas; Mølbak, Kåre; Paerregaard, Anders

    2010-01-01

    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....

  8. Persistence of Vancomycin Resistance in Multiple Clones of Enterococcus faecium Isolated from Danish Broilers 15 Years after the Ban of Avoparcin

    DEFF Research Database (Denmark)

    Bortolaia, Valeria; Mander, Manuela; Jensen, Lars Bogø

    2015-01-01

    associated with a transferable nontypeable plasmid lineage occurring in multiple E. faecium clones. Coselection of sequence type 842 by tetracycline use only partly explained the persistence of vancomycin resistance in the absence of detectable plasmid coresistance and toxin-antitoxin systems....

  9. From Fangs to Pharmacology: The Future of Snakebite Envenoming Therapy

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard; Engmark, Mikael; Milbo, Christina

    2016-01-01

    therapies for snakebite envenomings. In this review, current state-of-the-art in biopharmaceutical antitoxin development is presented together with an overview of available bioinformatics and structural data on snake venom toxins. This growing body of scientific and technological tools could define...

  10. Messenger RNA Interferase RelE Controls relBE Transcription by Conditional Cooperativity

    DEFF Research Database (Denmark)

    Overgaard, Martin; Borch, Jonas; Jørgensen, Mikkel G;

    2008-01-01

    Prokaryotic toxin-antitoxin (TA) loci consist of two genes in an operon that encodes a metabolically stable toxin and an unstable antitoxin. The antitoxin neutralises its cognate toxin by forming a tight complex with it. In all cases known, the antitoxin autoregulates TA operon transcription...... by binding to one or more operators in the promoter region while the toxin functions as a co-repressor of transcription. Interestingly, the toxin can also stimulate TA operon transcription. Here we analyse mechanistic aspects of how RelE of Escherichia coli can function both as a co-repressor and derepressor...... of relBE transcription. When RelB was in excess to RelE, two trimeric RelB(2)*RelE complexes bound cooperatively to two adjacent operator-sites in the relBE promoter region and repressed transcription. By contrast, RelE in excess stimulated relBE transcription and released the RelB(2)*RelE complex from...

  11. 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

  12. Centers for disease control and prevention expert panel meetings on prevention and treatment of anthrax in adults.

    Science.gov (United States)

    Hendricks, Katherine A; Wright, Mary E; Shadomy, Sean V; Bradley, John S; Morrow, Meredith G; Pavia, Andy T; Rubinstein, Ethan; Holty, Jon-Erik C; Messonnier, Nancy E; Smith, Theresa L; Pesik, Nicki; Treadwell, Tracee A; Bower, William A

    2014-02-01

    The Centers for Disease Control and Prevention convened panels of anthrax experts to review and update guidelines for anthrax postexposure prophylaxis and treatment. The panels included civilian and military anthrax experts and clinicians with experience treating anthrax patients. Specialties represented included internal medicine, pediatrics, obstetrics, infectious disease, emergency medicine, critical care, pulmonology, hematology, and nephrology. Panelists discussed recent patients with systemic anthrax; reviews of published, unpublished, and proprietary data regarding antimicrobial drugs and anthrax antitoxins; and critical care measures of potential benefit to patients with anthrax. This article updates antimicrobial postexposure prophylaxis and antimicrobial and antitoxin treatment options and describes potentially beneficial critical care measures for persons with anthrax, including clinical procedures for infected nonpregnant adults. Changes from previous guidelines include an expanded discussion of critical care and clinical procedures and additional antimicrobial choices, including preferred antimicrobial drug treatment for possible anthrax meningitis.

  13. Evaluation of Peru-15, a new live oral vaccine for cholera, in volunteers.

    Science.gov (United States)

    Sack, D A; Sack, R B; Shimko, J; Gomes, G; O'Sullivan, D; Metcalfe, K; Spriggs, D

    1997-07-01

    A new live oral cholera vaccine, Peru-15, was studied for safety, immunogenicity, and excretion in 2 groups of healthy volunteers. Twelve inpatient volunteers received freshly harvested vaccine in doses of either 10(7) or 10(9) cfu. Subsequently 50 outpatient volunteers received freeze-dried vaccine in doses of 10(8) or 10(9) cfu or placebo in a three-cell, double-masked, placebo-controlled trial. The strain was well tolerated at all dose levels, and it stimulated high levels of vibriocidal antibodies in most inpatient volunteers and in all outpatient volunteers. Although antitoxin responses were less frequent and of lower magnitude than the vibriocidal responses, antitoxin responses were seen in >60% of the outpatient volunteers. About 60% of the volunteers excreted the vaccine in their feces; however, fecal excretion did not correlate with serologic responses. It is concluded that Peru-15 is a safe and immunogenic oral vaccine for cholera.

  14. Isolation of Clostridium absonum and its cultural and biochemical properties.

    Science.gov (United States)

    Hayase, M; Mitsui, N; Tamai, K; Nakamura, S; Nishida, S

    1974-01-01

    A new procedure for isolation of Clostridium absonum was devised. Sixtyseven strains of C. absonum were isolated from 135 soil samples, but no strain of C. absonum could be found from human fecal samples. The lecithinase, hemolysin, and lethal toxin in the culture filtrates of this species exhibited low avidity for C. perfringens type A antitoxin. The three activities were inseparable by the present method of purification. A reinvestigation of biochemical properties revealed that incomplete suppression of lecithinase reaction by C. perfringens type A antitoxin and no fermentation of raffinose, melibiose, and starch are useful criteria to differentiate C. absonum from C. perfringens, and that positive, although weak, gelatin liquefaction and fermentation of trehalose are useful to differentiate it from C. paraperfringens.

  15. Clinically and electrophysiologically diagnosed botulinum intoxication.

    Science.gov (United States)

    Kotan, Dilcan; Aygul, Recep; Ceylan, Mustafa; Yilikoglu, Yalcin

    2013-01-03

    In this case report, clinical and electrophysiological findings of 43-year-old female patient who developed Clostridium botulinum intoxication after consumption of home-made canned food are presented. Following the sudden onset of severe nausea and vomiting, diplopia, blurred vision, bilateral ptosis, weakness, speech and swallowing difficulties have developed and the patient declared that she has just tasted the canned beans after she had rinsed them several times. The case, where serological tests cannot be performed, was diagnosed clinically and treated with antitoxin immediately. During follow-up, consecutive nerve stimulation was performed and significant incremental response was observed. There was an improvement in symptoms within 2 weeks, and in 5 or 6 weeks the symptoms had disappeared completely. Electrodiagnostic studies revealed that the findings turned to normal. The case showed that immediate antitoxin treatment is life-saving even the diagnosis of botulinum intoxication is based on clinical findings.

  16. Plastic surgical management of a cobra bite – a case study

    Directory of Open Access Journals (Sweden)

    Kuhbier, Jörn W.

    2017-02-01

    Full Text Available Cobra bites are quite rare in European countries as these snakes are not native there. Toxins are devastating for tissue resulting in massive necrosis, thus plastic surgery might play a role in reconstruction of the lost tissue. A case of a male patient bitten by a thai cobra in the left index finger is presented. Antitoxin administration was delayed due to secondary patient admission. Progressive tissue necrosis made radical debridement necessary, resulting in the need for plastic surgical defect coverage with a flap. While a radical debridement to prevent toxic necrosis due to lytic enzymes in cobra venom has been favoured beforehand, large case studies led to a more restrained initial surgical intervention. However, antitoxin administration should be first line therapy in management of these cases. If severe necrosis is present as it might occur in delayed admission, a plastic surgical management of the patient might be advantageous.

  17. 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.

  18. Antibody-based biological toxin detection

    Energy Technology Data Exchange (ETDEWEB)

    Menking, D.E.; Goode, M.T. [Army Edgewood Research, Development and Engineering Center, Aberdeen Proving Ground, MD (United States)

    1995-12-01

    Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a direct competition assay, antibodies against Cholera toxin, Staphylococcus Enterotoxin B or ricin were noncovalently immobilized on quartz fibers and probed with fluorescein isothiocyanate (FITC) - labeled toxins. In the indirect competition assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified anti-toxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or anti-toxin IgG in a dose dependent manner and the detection of the toxins was in the nanomolar range.

  19. Botulinum Neurotoxins

    Science.gov (United States)

    2012-03-28

    200,000 people were infected with an antibiotic -resistant strain of Salmonella caused by an inadvertent contamination at a single northern Illinois dairy...Fenecia et al., 1999). The disease has also been correlated with alteration of protective endogenous microflora by broad-spectrum antibiotics after... equine antitoxin, and discharged after 9 days. While not a naturally occurring form of the disease, inhalational botulism has implications as a

  20. Defining the Attributes of a CBRN Human Response Model: Findings and Conclusions

    Science.gov (United States)

    2009-08-01

    countermeasures, such as vaccination or antibiotic prophylaxis; the spread of disease from the release of an agent that is human-to-human contagious; or...pre- or post-exposure use of antibiotics , antivirals, immunoglobulins/antitoxins, and active immunoprophylaxis by immunization. In the context of... equine encephalitis, pertussis, measles, hepatitis, VHF, rabies, plague, burkholderia, and SEB. Two noted that biological agents might be less of a

  1. Human monoclonal antibodies directed against toxins A and B prevent Clostridium difficile-induced mortality in hamsters.

    Science.gov (United States)

    Babcock, Gregory J; Broering, Teresa J; Hernandez, Hector J; Mandell, Robert B; Donahue, Katherine; Boatright, Naomi; Stack, Anne M; Lowy, Israel; Graziano, Robert; Molrine, Deborah; Ambrosino, Donna M; Thomas, William D

    2006-11-01

    Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea, and recent outbreaks of strains with increased virulence underscore the importance of identifying novel approaches to treat and prevent relapse of Clostridium difficile-associated diarrhea (CDAD). CDAD pathology is induced by two exotoxins, toxin A and toxin B, which have been shown to be cytotoxic and, in the case of toxin A, enterotoxic. In this report we describe fully human monoclonal antibodies (HuMAbs) that neutralize these toxins and prevent disease in hamsters. Transgenic mice carrying human immunoglobulin genes were used to isolate HuMAbs that neutralize the cytotoxic effects of either toxin A or toxin B in cell-based in vitro neutralization assays. Three anti-toxin A HuMAbs (3H2, CDA1, and 1B11) could all inhibit the enterotoxicity of toxin A in mouse intestinal loops and the in vivo toxicity in a systemic mouse model. Four anti-toxin B HuMAbs (MDX-1388, 103-174, 1G10, and 2A11) could neutralize cytotoxicity in vitro, although systemic toxicity in the mouse could not be neutralized. Anti-toxin A HuMAb CDA1 and anti-toxin B HuMAb MDX-1388 were tested in the well-established hamster model of C. difficile disease. CDA1 alone resulted in a statistically significant reduction of mortality in hamsters; however, the combination treatment offered enhanced protection. Compared to controls, combination therapy reduced mortality from 100% to 45% (P<0.0001) in the primary disease hamster model and from 78% to 32% (P<0.0001) in the less stringent relapse model.

  2. Bacterial Programmed Cell Death as a Population Phenomenon

    Science.gov (United States)

    2013-06-11

    response to take place. The SOS response is permitted in E. coli strains carrying prophage lambda. One of the few genes expressed by phage λ in its... antibiotics due to the expected diversity of individual microbiota. References 1)Aizenman E, Engelberg-Kulka H, Glaser G (1996) An Escherichia coli... phage λ repressors. Proc Natl. Acad. Sci. USA 81, 1375-1379. 19)Mittenhuber, G. (1999). Occurrence of mazEF-like antitoxin/toxin systems in bacteria. J

  3. Dynamic Metabolic Adjustments and Genome Plasticity Are Implicated in the Heat Shock Response of the Extremely Thermoacidophilic Archaeon Sulfolobus solfataricus†

    Science.gov (United States)

    Tachdjian, Sabrina; Kelly, Robert M.

    2006-01-01

    Approximately one-third of the open reading frames encoded in the Sulfolobus solfataricus genome were differentially expressed within 5 min following an 80 to 90°C temperature shift at pH 4.0. This included many toxin-antitoxin loci and insertion elements, implicating a connection between genome plasticity and metabolic regulation in the early stages of stress response. PMID:16740961

  4. Allergic, Immunological and Infectious Disease Problems in Aerospace Medicine Held in Rome, Italy on 21 - 25 October 1991 (Les Problemes Causes par les Maladies Allergiques, Immunologiques et Contagieuses en Mdecine Aerospatiale).

    Science.gov (United States)

    1992-04-01

    R., Muylle, L., 1992. "HAV Cholera - - - infections in Belgium". in: Meningoc. meningitis in epidemics.trekkers, Sahel "Proceedings, Second Conference...toxoid and can be saline solution and a similar mixture with 0.1 ml of routinely used as a booster in adults, standard Tetanus Antitoxin containing 0.1...and supernatants diluted 1 : 10 with phosphate None of the subjects treated with live attenuated oral vaccine buffer saline (PBS). reported noteworthy

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

    OpenAIRE

    Zasada, Aleksandra A.; 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...

  6. Analysis of non-typeable Haemophilous influenzae VapC1 mutations reveals structural features required for toxicity and flexibility in the active site.

    Directory of Open Access Journals (Sweden)

    Brooke Hamilton

    Full Text Available Bacteria have evolved mechanisms that allow them to survive in the face of a variety of stresses including nutrient deprivation, antibiotic challenge and engulfment by predator cells. A switch to dormancy represents one strategy that reduces energy utilization and can render cells resistant to compounds that kill growing bacteria. These persister cells pose a problem during treatment of infections with antibiotics, and dormancy mechanisms may contribute to latent infections. Many bacteria encode toxin-antitoxin (TA gene pairs that play an important role in dormancy and the formation of persisters. VapBC gene pairs comprise the largest of the Type II TA systems in bacteria and they produce a VapC ribonuclease toxin whose activity is inhibited by the VapB antitoxin. Despite the importance of VapBC TA pairs in dormancy and persister formation, little information exists on the structural features of VapC proteins required for their toxic function in vivo. Studies reported here identified 17 single mutations that disrupt the function of VapC1 from non-typeable H. influenzae in vivo. 3-D modeling suggests that side chains affected by many of these mutations sit near the active site of the toxin protein. Phylogenetic comparisons and secondary mutagenesis indicate that VapC1 toxicity requires an alternative active site motif found in many proteobacteria. Expression of the antitoxin VapB1 counteracts the activity of VapC1 mutants partially defective for toxicity, indicating that the antitoxin binds these mutant proteins in vivo. These findings identify critical chemical features required for the biological function of VapC toxins and PIN-domain proteins.

  7. 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.

  8. Clostridium tetani growth and toxin production in the intestines of germfree rats.

    OpenAIRE

    Wells, C L; Balish, E

    1983-01-01

    Germfree rats were challenged orally and intrarectally with spores of Clostridium tetani. Although C. tetani spores remained viable in the intestinal tract, they were unable to germinate. Germfree rats were then challenged orally with vegetative cells of C. tetani. Vegetative cells were able to colonize the intestinal tract, replicate, and produce toxin. Tetanus antitoxin, but no tetanus toxin, was detected in the sera of monoassociated rats.

  9. Primer aislamiento de Clostridium tetani a partir de suelos de la Meseta Central de Costa Rica

    OpenAIRE

    Rodríguez, Evelyn; Gamboa, María del Mar; Fernández, Bernal

    2016-01-01

    Clínical evidence has long pointed to the existence of Clostridium tetani in Costa Rica. Thirty soil samples were studied for clostridia, and two yielded six strains of C. tetani, four of which proved to be toxigenic when mice were inoculated intraperitoneally with the culture supemates, These four isolates could be neutralized when their toxic supemates were admixed with tetanus antitoxin. Clínical evidence has long pointed to the existence of Clostridium tetani in Costa Rica. Thirty soil...

  10. [Intoxication of botulinum toxin].

    Science.gov (United States)

    Chudzicka, Aleksandra

    2015-09-01

    Botulinum toxin is an egzotoxin produced by Gram positive bacteria Clostridium botulinum. It is among the most potent toxins known. The 3 main clinical presentations of botulism are as follows: foodborne botulism, infant botulism and wound botulism. The main symptom of intoxication is flat muscles paralysis. The treatment is supportive care and administration of antitoxin. In prevention the correct preparing of canned food is most important. Botulinum toxin is accepted as a biological weapon.

  11. Two programmed cell death systems in Escherichia coli: an apoptotic-like death is inhibited by the mazEF-mediated death pathway.

    Directory of Open Access Journals (Sweden)

    Ariel Erental

    Full Text Available In eukaryotes, the classical form of programmed cell death (PCD is apoptosis, which has as its specific characteristics DNA fragmentation and membrane depolarization. In Escherichia coli a different PCD system has been reported. It is mediated by the toxin-antitoxin system module mazEF. The E. coli mazEF module is one of the most thoroughly studied toxin-antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. mazEF-mediated cell death is a population phenomenon requiring the quorum-sensing pentapeptide NNWNN designated Extracellular Death Factor (EDF. mazEF is triggered by several stressful conditions, including severe damage to the DNA. Here, using confocal microscopy and FACS analysis, we show that under conditions of severe DNA damage, the triggered mazEF-mediated cell death pathway leads to the inhibition of a second cell death pathway. The latter is an apoptotic-like death (ALD; ALD is mediated by recA and lexA. The mazEF-mediated pathway reduces recA mRNA levels. Based on these results, we offer a molecular model for the maintenance of an altruistic characteristic in cell populations. In our model, the ALD pathway is inhibited by the altruistic EDF-mazEF-mediated death pathway.

  12. mazEF-mediated programmed cell death in bacteria: "what is this?".

    Science.gov (United States)

    Ramisetty, Bhaskar Chandra Mohan; Natarajan, Bhargavi; Santhosh, Ramachandran Sarojini

    2015-02-01

    Toxin-antitoxin (TA) systems consist of a bicistronic operon, encoding a toxin and an antitoxin. They are widely distributed in the prokaryotic kingdom, often in multiple numbers. TAs are implicated in contradicting phenomena of persistence and programmed cell death (PCD) in bacteria. mazEF TA system, one of the widely distributed type II toxin-antitoxin systems, is particularly implicated in PCD of Escherichia coli. Nutrient starvation, antibiotic stress, heat shock, DNA damage and other kinds of stresses are shown to elicit mazEF-mediated-PCD. ppGpp and extracellular death factor play a central role in regulating mazEF-mediated PCD. The activation of mazEF system is achieved through inhibition of transcription or translation of mazEF loci. Upon activation, MazF cleaves RNA in a ribosome-independent fashion and subsequent processes result in cell death. It is hypothesized that PCD aids in perseverance of the population during stress; the surviving minority of the cells can scavenge the nutrients released by the dead cells, a kind of "nutritional-altruism." Issues regarding the strains, reproducibility of experimental results and ecological plausibility necessitate speculation. We review the molecular mechanisms of the activation of mazEF TA system, the consequences leading to cell death and the pros and cons of the altruism hypothesis from an ecological perspective.

  13. Viral evasion of a bacterial suicide system by RNA-based molecular mimicry enables infectious altruism.

    Science.gov (United States)

    Blower, Tim R; Evans, Terry J; Przybilski, Rita; Fineran, Peter C; Salmond, George P C

    2012-01-01

    Abortive infection, during which an infected bacterial cell commits altruistic suicide to destroy the replicating bacteriophage and protect the clonal population, can be mediated by toxin-antitoxin systems such as the Type III protein-RNA toxin-antitoxin system, ToxIN. A flagellum-dependent bacteriophage of the Myoviridae, ΦTE, evolved rare mutants that "escaped" ToxIN-mediated abortive infection within Pectobacterium atrosepticum. Wild-type ΦTE encoded a short sequence similar to the repetitive nucleotide sequence of the RNA antitoxin, ToxI, from ToxIN. The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination. Expression of the pseudo-ToxI repeats during ΦTE infection allowed the phage to replicate, unaffected by ToxIN, through RNA-based molecular mimicry. This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system. Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

  14. Viral evasion of a bacterial suicide system by RNA-based molecular mimicry enables infectious altruism.

    Directory of Open Access Journals (Sweden)

    Tim R Blower

    Full Text Available Abortive infection, during which an infected bacterial cell commits altruistic suicide to destroy the replicating bacteriophage and protect the clonal population, can be mediated by toxin-antitoxin systems such as the Type III protein-RNA toxin-antitoxin system, ToxIN. A flagellum-dependent bacteriophage of the Myoviridae, ΦTE, evolved rare mutants that "escaped" ToxIN-mediated abortive infection within Pectobacterium atrosepticum. Wild-type ΦTE encoded a short sequence similar to the repetitive nucleotide sequence of the RNA antitoxin, ToxI, from ToxIN. The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination. Expression of the pseudo-ToxI repeats during ΦTE infection allowed the phage to replicate, unaffected by ToxIN, through RNA-based molecular mimicry. This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system. Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

  15. The ClpXP protease is responsible for the degradation of the Epsilon antidote to the Zeta toxin of the streptococcal pSM19035 plasmid.

    Science.gov (United States)

    Brzozowska, Iwona; Zielenkiewicz, Urszula

    2014-03-14

    Most bacterial genomes contain different types of toxin-antitoxin (TA) systems. The ω-ε-ζ proteinaceous type II TA cassette from the streptococcal pSM19035 plasmid is a member of the ε/ζ family, which is commonly found in multiresistance plasmids and chromosomes of various human pathogens. Regulation of type II TA systems relies on the proteolysis of antitoxin proteins. Under normal conditions, the Epsilon antidote neutralizes the Zeta toxin through the formation of a tight complex. In this study, we show, using both in vivo and in vitro analyses, that the ClpXP protease is responsible for Epsilon antitoxin degradation. Using in vivo studies, we examined the stability of the plasmids with active or inactive ω-ε-ζ TA cassettes in B. subtilis mutants that were defective for different proteases. Using in vitro assays, the degradation of purified His6-Epsilon by the His6-LonBs, ClpPBs, and ClpXBs proteases from B. subtilis was analyzed. Additionally, we showed that purified Zeta toxin protects the Epsilon protein from rapid ClpXP-catalyzed degradation.

  16. A novel membrane-bound toxin for cell division, CptA (YgfX), inhibits polymerization of cytoskeleton proteins, FtsZ and MreB, in Escherichia coli.

    Science.gov (United States)

    Masuda, Hisako; Tan, Qian; Awano, Naoki; Yamaguchi, Yoshihiro; Inouye, Masayori

    2012-03-01

    Nearly all free-living bacteria carry toxin-antitoxin (TA) systems on their genomes, through which cell growth and death are regulated. Toxins target a variety of essential cellular functions, including DNA replication, translation, and cell division. Here, we identified a novel toxin, YgfX, on the Escherichia coli genome. The toxin, consisting of 135 residues, is composed of the N-terminal membrane domain, which encompasses two transmembrane segments, and the C-terminal cytoplasmic domain. Upon YgfX expression, the cells were initially elongated and then the middle portion of the cells became inflated to form a lemon shape. YgfX was found to interact with MreB and FtsZ, two essential cytoskeletal proteins in E. coli. The cytoplasmic domain [YgfX(C)] was found to be responsible for the YgfX toxicity, as purified YgfX(C) was found to block the polymerization of FtsZ and MreB in vitro. YgfY, located immediately upstream of YgfX, was shown to be the cognate antitoxin; notably, YgfX is the first membrane-associating toxin in bacterial TA systems. We propose to rename the toxin and the antitoxin as CptA and CptB (for Cytoskeleton Polymerization inhibiting Toxin), respectively.

  17. Toxicity and immunogenicity of Enterotoxigenic Escherichia coli heat-labile and heat-stable toxoid fusion 3xSTa(A14Q)-LT(S63K/R192G/L211A) in a murine model.

    Science.gov (United States)

    Zhang, Chengxian; Knudsen, David E; Liu, Mei; Robertson, Donald C; Zhang, Weiping

    2013-01-01

    Diarrhea is the second leading cause of death to young children. Enterotoxigenic Escherichia coli (ETEC) are the most common bacteria causing diarrhea. Adhesins and enterotoxins are the virulence determinants in ETEC diarrhea. Adhesins mediate bacterial attachment and colonization, and enterotoxins including heat-labile (LT) and heat-stable type Ib toxin (STa) disrupt fluid homeostasis in host cells that leads to fluid hyper-secretion and diarrhea. Thus, adhesins and enterotoxins have been primarily targeted in ETEC vaccine development. A recent study reported toxoid fusions with STa toxoid (STa(P13F)) fused at the N- or C-terminus, or inside the A subunit of LT(R192G) elicited neutralizing antitoxin antibodies, and suggested application of toxoid fusions in ETEC vaccine development (Liu et al., Infect. Immun. 79:4002-4009, 2011). In this study, we generated a different STa toxoid (STa(A14Q)) and a triple-mutant LT toxoid (LT(S63K/R192G/L211A), tmLT), constructed a toxoid fusion (3xSTa(A14Q)-tmLT) that carried 3 copies of STa(A14Q) for further facilitation of anti-STa immunogenicity, and assessed antigen safety and immunogenicity in a murine model to explore its potential for ETEC vaccine development. Mice immunized with this fusion antigen showed no adverse effects, and developed antitoxin antibodies particularly through the IP route. Anti-LT antibodies were detected and were shown neutralizing against CT in vitro. Anti-STa antibodies were also detected in the immunized mice, and serum from the IP immunized mice neutralized STa toxin in vitro. Data from this study indicated that toxoid fusion 3xSTa(A14Q)-tmLT is safe and can induce neutralizing antitoxin antibodies, and provided helpful information for vaccine development against ETEC diarrhea.

  18. Toxicity and immunogenicity of Enterotoxigenic Escherichia coli heat-labile and heat-stable toxoid fusion 3xSTa(A14Q-LT(S63K/R192G/L211A in a murine model.

    Directory of Open Access Journals (Sweden)

    Chengxian Zhang

    Full Text Available Diarrhea is the second leading cause of death to young children. Enterotoxigenic Escherichia coli (ETEC are the most common bacteria causing diarrhea. Adhesins and enterotoxins are the virulence determinants in ETEC diarrhea. Adhesins mediate bacterial attachment and colonization, and enterotoxins including heat-labile (LT and heat-stable type Ib toxin (STa disrupt fluid homeostasis in host cells that leads to fluid hyper-secretion and diarrhea. Thus, adhesins and enterotoxins have been primarily targeted in ETEC vaccine development. A recent study reported toxoid fusions with STa toxoid (STa(P13F fused at the N- or C-terminus, or inside the A subunit of LT(R192G elicited neutralizing antitoxin antibodies, and suggested application of toxoid fusions in ETEC vaccine development (Liu et al., Infect. Immun. 79:4002-4009, 2011. In this study, we generated a different STa toxoid (STa(A14Q and a triple-mutant LT toxoid (LT(S63K/R192G/L211A, tmLT, constructed a toxoid fusion (3xSTa(A14Q-tmLT that carried 3 copies of STa(A14Q for further facilitation of anti-STa immunogenicity, and assessed antigen safety and immunogenicity in a murine model to explore its potential for ETEC vaccine development. Mice immunized with this fusion antigen showed no adverse effects, and developed antitoxin antibodies particularly through the IP route. Anti-LT antibodies were detected and were shown neutralizing against CT in vitro. Anti-STa antibodies were also detected in the immunized mice, and serum from the IP immunized mice neutralized STa toxin in vitro. Data from this study indicated that toxoid fusion 3xSTa(A14Q-tmLT is safe and can induce neutralizing antitoxin antibodies, and provided helpful information for vaccine development against ETEC diarrhea.

  19. Evidence of increased carriage of Corynebacterium spp. in healthy individuals with low antibody titres against diphtheria toxoid.

    Science.gov (United States)

    Bergamini, M; Fabrizi, P; Pagani, S; Grilli, A; Severini, R; Contini, C

    2000-08-01

    This study evaluated whether a correlation exists between carriage of corynebacteria and the lack of immunity to diphtheria toxoid. Samples of both nasal and pharyngeal secretions were taken from 500 apparently healthy subjects of both sexes and of all ages and inoculated onto Tinsdale's medium. A serum sample was also taken for ELISA test to determine the titre of diphtheria toxin antibodies. None of the subjects carried Corynebacterium diphtheriae. Ninety-three strains of Corynebacterium spp. were isolated from 93 subjects and 86 of these were classified to species or group level by biochemical tests. C. xerosis was the most common (25.8%) followed by C. pseudodiphthericum (16.1%), C. jeikeium and C. striatum (both 10.8%), and C. urealyticum (9.7%). Three other species accounted for approximately 20% of strains and seven were unclassified as biochemically atypical corynebacteria. Non-protective antibodies to diphtheria toxin were found in 80 of the 93 subjects and a strong statistical association was demonstrated between carriage of corynebacteria and non-protective levels of anti-toxin antibodies. The remaining 13 subjects had protective levels of antitoxin antibodies. In contrast, only 45 of the 407 non-colonized subjects had non-protective antitoxin titres. The prevalence of carriage increased with age among males as did the percentage of non-protected subjects. The prevalence of female carriers of corynebacteria was significantly lower. Serum samples from 12 subjects with different antibody titres to diphtheria toxoid reacted to varying degrees with whole-cell lysates of a number of species of corynebacteria. The results suggest that a causal relationship may exist between nasopharyngeal carriage of corynebacteria and a low anti-diphtheria toxin immune response.

  20. 新佐剂--Montanide ISA 50 V2在马免疫中的应用研究%Application of a new adjuvant-Montanide ISA 50 V2 in horse immunization program

    Institute of Scientific and Technical Information of China (English)

    刘卫涛; 柳洲; 李育合; 谢小荣; 孟祥玉; 张廷芬; 高建军

    2013-01-01

    Objective To improve the titers of antitoxin serum prepared in horses immunization program .Methods Mon-tanide ISA 50V2 was used as a new adjuvant in immunization program for rabbits and horses .Montanide SA 50V2 and Clostridium botulinum type A and F toxoids antigens were emulsified to O /W emulsion, and then using this emulsion to im-munize the horses , and compared with the conventional adjuvant ( FIA) with the same antigen of clostridium botulinum type A and F toxoids .Results The immune effect was the best when two emulsions were alternately used ,the titer of antitoxin serum was the highest .Conclusion Montanide ISA 50V2 could be used as a adjuvant in horses immunization program for production of higher titers of antitoxin serum .%目的研究新佐剂-Montanide ISA 50V2在马免疫中的应用,制定合理的免疫程序,提高马免疫血清效价。方法用Montanide ISA 50V2乳化A型和F型肉毒类毒素制备成油乳抗原,免疫家兔和马,并与福氏不完全佐剂进行对比。结果在马免疫程序中,Montanide ISA 50V2与福氏不完全佐剂交替使用效果更好,免疫效价较高。结论在进一步试验确证后,将Montanide ISA 50V2作为佐剂应用于马免疫中,以获得更高的免疫血清的效价。

  1. 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

    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...... 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...

  2. Cutaneous Corynebacterium Diphtheriae: A Traveller’s Disease?

    Directory of Open Access Journals (Sweden)

    A Berih

    1995-01-01

    Full Text Available A Canadian soldier incurred a nonhealing traumatic skin ulcer while on duty in Somalia. The diagnosis of localized cutaneous diphtheria was confirmed by isolation of a toxigenic strain of Corynebacterium diphtheriae from the ulcer. The patient was placed in isolation and treated with erythromycin and penicillin for 10 days without antitoxin. He was released when two consecutive daily cultures were negative. Public health officials evaluated his wife, two children and close contacts for carriage, but no carriers or secondary cases were identified. Cutaneous diphtheria as a diagnostic and management patient problem and potential public health problem are discussed.

  3. Biotechnological Trends in Spider and Scorpion Antivenom Development

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard; Solà, Mireia; Jappe, Emma Christine;

    2016-01-01

    Spiders and scorpions are notorious for their fearful dispositions and their ability to inject venom into prey and predators, causing symptoms such as necrosis, paralysis, and excruciating pain. Information on venom composition and the toxins present in these species is growing due to an interest...... at an increasingly faster pace. In this review, the current knowledge of spider and scorpion venoms is presented, followed by a discussion of all published biotechnological efforts within development of spider and scorpion antitoxins based on small molecules, antibodies and fragments thereof, and next generation...

  4. [Streptococcal toxic shock syndrome or Kawasaki disease? Two case studies of children with group A streptococcal pneumonia empyema].

    Science.gov (United States)

    Bosland, A; Arlaud, K; Rousset-Rouvière, C; Fouilloux, V; Paut, O; Dubus, J-C; Bosdure, E

    2011-12-01

    We report 2 cases of children with group A streptococcus pyogenes pleuropneumonia, in one child associated with Kawasaki disease and in the other with streptococcal toxic shock syndrome. These 2 features, with theoretically well-defined clinical and biological criteria, are difficult to differentiate in clinical practice, however, likely due to their pathophysiological links. In case of clinical doubt, an echocardiography needs to be performed to search for coronary involvement and treatment including intravenous immunoglobulins, and an antibiotic with an anti-toxin effect such as clindamycin has to be started early.

  5. Inhibiting bacterial toxins by channel blockage.

    Science.gov (United States)

    Bezrukov, Sergey M; Nestorovich, Ekaterina M

    2016-03-01

    Emergent rational drug design techniques explore individual properties of target biomolecules, small and macromolecule drug candidates, and the physical forces governing their interactions. In this minireview, we focus on the single-molecule biophysical studies of channel-forming bacterial toxins that suggest new approaches for their inhibition. We discuss several examples of blockage of bacterial pore-forming and AB-type toxins by the tailor-made compounds. In the concluding remarks, the most effective rationally designed pore-blocking antitoxins are compared with the small-molecule inhibitors of ion-selective channels of neurophysiology.

  6. Utilización del ácido caprílico en la obtención de inmunoglobulina G de caballo antitoxina tetánica

    OpenAIRE

    2014-01-01

    Las inmunoglobulinas de caballo antitoxina tetánica purificadas, con altas concentración de anticuerpos y actividad biológica, se utilizan como sueros heterólogosy sueros estándar para la producción de la vacuna correspondiente. No obstante, la selección de agentes precipitantes para la purificación de las mismas constituye un problema en términos de pureza, rendimiento y de economía, aún por resolver. En este estudio se describieron las condiciones para el fraccionamiento de plasma antitoxin...

  7. 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.

  8. Toxin MqsR Cleaves Single-Stranded mRNA with Various 5 Ends

    Science.gov (United States)

    2016-08-24

    Escherichia coli RNase E catalytic domain and implications for RNA turnover. Nature 437:1187–1191. Cheng, H.-Y., V. W. C. Soo, S. Islam , M. J. McAnulty, M. J...The phage abortive infection system, ToxIN, functions as a protein– RNA toxin–antitoxin pair. Proc. Natl. Acad. Sci. USA 106:894–899. Gerdes, K., F...multimeric RNase E and RNase G by 5′- monophosphorylated RNA. Proc. Natl. Acad. Sci. USA 101:9211–9216. Keren, I., D. Shah, A. Spoering, N. Kaldalu

  9. Development of a Vaccine Against Experimental Cholera and Escherichia coli Diarrheal Disease

    OpenAIRE

    Rappaport, R. S.; Bonde, G.

    1981-01-01

    The results of the present investigation indicate a simple approach to the development of a single-vaccine formula which may ultimately be used to confer protection against both cholera and certain types of Escherichia coli diarrheal disease in humans and domestic animals. The design of the vaccine is based on the well-documented ability of cholera antitoxin to neutralize both cholera and heat-labile E. coli enterotoxins (CT and LT, respectively) and on the ability of killed E. coli to enhanc...

  10. 创伤性休克患者破伤风抗毒素皮内试验的观察

    Institute of Scientific and Technical Information of China (English)

    徐桂琴; 何忠杰; 程尉新

    2004-01-01

    作者在急诊科为创伤患者进行破伤风抗毒素试验(tetanus antitoxin test,TAT)时发现,处于休克期的患者局部皮丘无任何改变,更无一例出现全身症状。因此,我们于1998年2月至2000年2月对160例创伤性休克病人进行了TAT皮内试验的观察和对比分析,现将结果报告如下。

  11. THE EFFECT OF ANESTHESIA AND OF SEDATIVES ON THE SERUM THERAPY OF EXPERIMENTAL BOTULISM.

    Science.gov (United States)

    Bronfenbrenner, J J; Weiss, H

    1924-03-31

    When guinea pigs are fed large amounts of botulinus toxin, they develop symptoms of intoxication within 6 hours and die usually within 12 hours after the feeding of toxin. If very large amounts of toxin are introduced intraperitoneally, the animals may show symptoms of intoxication at the end of the 1st hour and die usually within 2 hours following the administration of toxin. If these animals are placed under anesthesia following the administration of toxin, the intoxication proceeds much more slowly. Anesthesia, by ether, is effective in this manner whether the toxin is given per os or intraperitoneally. The life of guinea pigs kept under ether after the administration of lethal amounts of toxin is prolonged by a period approximately equal to that during which the administration of the anesthetic is continued. When anesthesia is discontinued, the intoxication proceeds at its usual rate. It appears, therefore, that administration of ether delays the rate of intoxication but does not alter the toxin or the nature of the mechanism of intoxication. Anesthesia thus delays the progress of intoxication not only when administered immediately after the intake of toxin, but when administered much later after the intoxication has already progressed far enough to cause definite objective symptoms of poisoning. At this late stage of intoxication, the unsupported serum therapy of botulism in guinea pigs usually remains without effect. If, however, the animals are anesthetized at this time and kept under the influence of ether for some time, antitoxin therapy becomes effective. Indeed the antitoxin treatment can be delayed further for several hours, provided the animal is kept under anesthesia during the interval. Results of a similar nature were obtained with luminal sodium, nitrous oxide-oxygen mixture, and morphine used in place of ether. In view of the fact that the published data indicate that botulinus antitoxin has thus far failed to give beneficial results in the

  12. The SOS response is permitted in Escherichia coli strains deficient in the expression of the mazEF pathway.

    Science.gov (United States)

    Kalderon, Ziva; Kumar, Sathish; Engelberg-Kulka, Hanna

    2014-01-01

    The Escherichia coli (E. coli) SOS response is the largest, most complex, and best characterized bacterial network induced by DNA damage. It is controlled by a complex network involving the RecA and LexA proteins. We have previously shown that the SOS response to DNA damage is inhibited by various elements involved in the expression of the E. coli toxin-antitoxin mazEF pathway. Since the mazEF module is present on the chromosomes of most E. coli strains, here we asked: Why is the SOS response found in so many E. coli strains? Is the mazEF module present but inactive in those strains? We examined three E. coli strains used for studies of the SOS response, strains AB1932, BW25113, and MG1655. We found that each of these strains is either missing or inhibiting one of several elements involved in the expression of the mazEF-mediated death pathway. Thus, the SOS response only takes place in E. coli cells in which one or more elements of the E. coli toxin-antitoxin module mazEF or its downstream pathway is not functioning.

  13. Difteria: situação imunitária de uma população infantil urbana de São Paulo, SP, Brasil Diphtheria: immunity in an infant population in the city of S. Paulo, SP, Brazil

    Directory of Open Access Journals (Sweden)

    Hideyo Iizuka

    1980-12-01

    Full Text Available A verificação do teor de anticorpos antidiftéricos provenientes de 130 crianças de 7 a 10 anos de idade, do município de São Paulo, Brasil, revelou 31, 14 e 5% de indivíduos susceptíveis nas idades de 7, 8 e 9 anos, respectivamente. Todas as crianças de 10 anos de idade apresentaram proteção contra a difteria, revelando teor de antitoxina circulante em níveis superiores a 0,01 UI/ml. O teor médio de antitoxina diftérica encontrada variou de 0,0385 a 0,1315 UI/ml de soro, na população examinada.An ascertainment of the level diphtheria antibodies in 130 children, 7 to 10 years old, in the city of S. Paulo (Brazil, revealed susceptibility in 31% of the 7-year-olds, 14% in the eight-year-olds, and 5% in the nine-year-olds. All ten-year-olds had protective circulating antitoxin at levels superior to 0.01 IU/ml. Analysis of the results thus showed that susceptibility varies inversely to age. In the population examined, the mean diphtheric antitoxin content oscillated between 0.0385 and 0.1315 IU/ml of serum.

  14. Screening for Escherichia coli K-12 genes conferring glyoxal resistance or sensitivity by transposon insertions.

    Science.gov (United States)

    Lee, Changhan; Kim, Jihong; Kwon, Minsuk; Lee, Kihyun; Min, Haeyoung; Kim, Seong Hun; Kim, Dongkyu; Lee, Nayoung; Kim, Jiyeun; Kim, Doyun; Ko, Changmin; Park, Chankyu

    2016-09-01

    Glyoxal (GO) belongs to the reactive electrophilic species generated in vivo in all organisms. In order to identify targets of GO and their response mechanisms, we attempted to screen for GO-sensitive mutants by random insertions of TnphoA-132. The genes responsible for GO susceptibility were functionally classified as the following: (i) tRNA modification; trmE, gidA and truA, (ii) DNA repair; recA and recC, (iii) toxin-antitoxin; mqsA and (iv) redox metabolism; yqhD and caiC In addition, an insertion in the crp gene, encoding the cAMP responsive transcription factor, exhibits a GO-resistant phenotype, which is consistent with the phenotype of adenylate cyclase (cya) mutant showing GO resistance. This suggests that global regulation involving cAMP is operated in a stress response to GO. To further characterize the CRP-regulated genes directly associated with GO resistance, we created double mutants deficient in both crp and one of the candidate genes including yqhD, gloA and sodB The results indicate that these genes are negatively regulated by CRP as confirmed by real-time RT-PCR. We propose that tRNA as well as DNA are the targets of GO and that toxin/antitoxin, antioxidant and cAMP are involved in cellular response to GO.

  15. Nanoparticle-detained toxins for safe and effective vaccination

    Science.gov (United States)

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

    2013-12-01

    Toxoid vaccines--vaccines based on inactivated bacterial toxins--are routinely used to promote antitoxin immunity for the treatment and prevention of bacterial infections. Following chemical or heat denaturation, inactivated toxins can be administered to mount toxin-specific immune responses. However, retaining faithful antigenic presentation while removing toxin virulence remains a major challenge and presents a trade-off between efficacy and safety in toxoid development. Here, we show a nanoparticle-based toxin-detainment strategy that safely delivers non-disrupted pore-forming toxins for immune processing. Using erythrocyte membrane-coated nanoparticles and staphylococcal α-haemolysin, we demonstrate effective virulence neutralization via spontaneous particle entrapment. Compared with vaccination with heat-denatured toxin, mice vaccinated with the nanoparticle-detained toxin showed superior protective immunity against toxin-mediated adverse effects. We find that the non-disruptive detoxification approach benefited the immunogenicity and efficacy of toxoid vaccines. We anticipate that this study will open new possibilities in the preparation of antitoxin vaccines against the many virulence factors that threaten public health.

  16. Study on the use of an enzyme-linked immunosorbent assay in determining human antibodies to diphtheria toxin as compared with a reference toxin neutralization assay.

    Science.gov (United States)

    Skoura, L; Efstratiou, A; Tsakris, A; Pournaras, S; George, R C; Douboyas, J

    1999-07-01

    Serum samples from 156 Greek persons were assessed by an IgG-specific enzyme-linked immunosorbent assay (ELISA) and a reference tissue culture toxin-neutralization (TN) assay for the quantitation of diphtheria toxin antibodies. By the reference method, 7.7% of the persons were susceptible to diphtheria (antitoxin or = 0.1 IU/ ml), while the corresponding figures were 17.9, 36.5 and 45.5% when they were tested by the immunoassay. None of the samples been susceptible by the TN assay were found to have some protection when tested by ELISA. However, three (6.7%) of the 45 samples showing a basic protection with TN, were fully protective when titrated by the immunoassay. In addition, 31 (31.3%) of the 99 samples been fully protective by the bioassay, were found to be either basically protective or susceptible by means of the ELISA. Overall, validity features of the immunoassay were: sensitivity 68.7%, specificity 94.7%, positive predictive value 95.8% and negative predictive value 63.5%. The ELISA tested in our study could be used to determine diphtheria antitoxin in individuals needed a booster immunization (susceptible or basic protective samples), although it might falsely include in the above categories samples that are within the fully protective levels of antibodies.

  17. Characterization of botulinum neurotoxin type A neutralizing monoclonal antibodies and influence of their half-lives on therapeutic activity.

    Directory of Open Access Journals (Sweden)

    Christelle Mazuet

    Full Text Available Botulinum toxins, i.e. BoNT/A to/G, include the most toxic substances known. Since botulism is a potentially fatal neuroparalytic disease with possible use as a biowarfare weapon (Centers for Disease Control and Prevention category A bioterrorism agent, intensive efforts are being made to develop vaccines or neutralizing antibodies. The use of active fragments from non-human immunoglobulins (F(ab'(2, Fab', scFv, chemically modified or not, may avoid side effects, but also largely modify the in vivo half-life and effectiveness of these reagents. We evaluated the neutralizing activity of several monoclonal anti-BoNT/A antibodies (mAbs. F(ab'(2 fragments, native or treated with polyethyleneglycol (PEG, were prepared from selected mAbs to determine their half-life and neutralizing activity as compared with the initial mAbs. We compared the protective efficiency of the different biochemical forms of anti-toxin mAbs providing the same neutralizing activity. Among fourteen tested mAbs, twelve exhibited neutralizing activity. Fragments from two of the best mAbs (TA12 and TA17, recognizing different epitopes, were produced. These two mAbs neutralized the A1 subtype of the toxin more efficiently than the A2 or A3 subtypes. Since mAb TA12 and its fragments both exhibited the greatest neutralizing activity, they were further evaluated in the therapeutic experiments. These showed that, in a mouse model, a 2- to 4-h interval between toxin and antitoxin injection allows the treatment to remain effective, but also suggested an absence of correlation between the half-life of the antitoxins and the length of time before treatment after botulinum toxin A contamination. These experiments demonstrate that PEG treatment has a strong impact on the half-life of the fragments, without affecting the effectiveness of neutralization, which was maintained after preparation of the fragments. These reagents may be useful for rapid treatment after botulinum toxin A

  18. 毒素蛋白基因mazF在基因修饰系统中的应用进展%Application Progress ofmazF Gene in Genetic Modification System

    Institute of Scientific and Technical Information of China (English)

    石杨; 董会娜; 张大伟

    2014-01-01

    毒素-抗毒素系统(Toxin-antitoxin system,TA系统)存在于大部分细菌中。mazEF是大肠杆菌中一种毒素-抗毒素系统。毒素基因mazF编码的MazF毒素蛋白可以特异性地剪切自由mRNA的ACA序列,从而抑制蛋白合成、引起细胞生长停滞;近些年,许多学者利用mazF基因作为反向筛选标记对不同种微生物建立了无标记或无痕的基因修饰系统,并实现了不同菌株的基因组修饰。主要综述了大肠杆菌mazF基因作为反向筛选基因的应用原理及其在不同种类微生物的基因修饰系统中的应用进展,然后对mazF基因及其他毒素基因在基因修饰系统中的应用进行了展望。%Toxin-antitoxin system(TA)exists in most of the genetic material of bacteria.mazEF is a kind of toxin-antitoxin system in E. coli. MazF encoded bymazF is an mRNA interferase that specifically cleaves free mRNAs at ACA sequences, resulting in inhibited protein synthesis and cell growth arrest. Recently, some scholars have usedmazF as a counter-selection marker in genetic modification systems, and achieved modifications of the genome in various strains. In this article, the research advances of E. coli mazF used as counter-selection marker in bacterial genetic modification were reviewed, followed by application of other toxin gene. Finally, perspective of the possible new pathways for developing new genetic modification methods were addressed.

  19. CAUSE OF IMMEDIATE DEATH BY LARGE DOSES OF BOTULINUS TOXIN.

    Science.gov (United States)

    Bronfenbrenner, J J; Schlesinger, M J; Orr, P F

    1924-06-30

    Parenteral introduction of amounts of the culture filtrate of Bacillus botulinus greatly in excess of the minimum lethal dose has been observed to cause the practically immediate death of mice. This result is due to the presence in the filtrates of a chemical poison possessing properties distinct from those of the contained botulinus toxin which itself acts only after a well defined period of incubation. This chemical poison is not neutralized by botulinus antitoxin; it is effective only when large amounts of the culture filtrate are given; it is thermostable, not being destroyed when heated in the autoclave in a sealed tube, though when it is heated in an open container its toxicity diminishes with a coincidental volatilization of basic material. The volatile substance can be identified as ammonia. Death resulting from the injection of comparatively large amounts of ammonium salts (0.1 gm.) is easily distinguished from that due to botulism, both through the character of the symptoms and the absence of an incubation period. However, when the amount of toxic salts injected is smaller (0.01 gm.), the symptoms of poisoning are not so characteristic and death may be delayed long enough to suggest a period of incubation similar to that observed in botulism (Table IV). This circumstance is of importance in connection with the examination of partly decomposed food products in which the presence of botulinus toxin is suspected. As a rule such suspected material is injected in massive doses (0.5 to 1 cc.) in mice. It is conceivable that such spoiled foods may be contaminated with common putrefactive bacteria yielding ammonia during their growth and thus may cause death of the test animals. If in such tests mice passively protected by the preliminary injection of an excess of antitoxin be used in addition to normal animals, the chances of an error in the interpretation of the results will be materially reduced, though not ruled out. Unfortunately for such a procedure

  20. Outbreak of Botulism After Consumption of Illicit Prison-Brewed Alcohol in a Maximum Security Prison--Arizona, 2012.

    Science.gov (United States)

    Yasmin, Seema; Adams, Laura; Briggs, Graham; Weiss, Joli; Bisgard, Kris; Anderson, Shoana; Tsang, Clarisse; Henke, Evan; Vasiq, Muhammad; Komatsu, Ken

    2015-10-01

    The authors investigated the second botulism outbreak to occur in a maximum security prison in Arizona within a 4-month period. Botulism was confirmed in eight men aged 20 to 35 years who reported sharing a single batch of pruno made with potatoes. Initial symptoms included blurred vision, slurred speech, muscle weakness, ptosis, and dysphagia. All patients received heptavalent botulinum antitoxin, seven required mechanical ventilation, and all survived. The median incubation period was 29 hours. Sera from all patients and leftover pruno tested positive for botulinum toxin type A. Botulism should be considered among prisoners with cranial nerve palsies and descending, symmetric flaccid paralysis. Prison-brewed alcohol, particularly when made with potatoes, can be a vehicle for botulism and is associated with outbreaks of botulism in prisons.

  1. 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...

  2. 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...

  3. Corynebacterium diphtheriae infections currently and in the past.

    Science.gov (United States)

    Zasada, Aleksandra Anna

    2015-01-01

    Along with the introduction of common obligatory vaccinations against diphtheria, the disease has been limited in developed countries. However, diphtheria is still endemic in developing countries. Due to a growing popularity of visiting these countries, there is a risk of importation of the disease to Europe. Studies revealed that over 60% of persons aged >40 years in the Polish population do not have a protective level of antibodies against diphtheria. Furthermore, an access to diphtheria antitoxin, which is essential in diphtheria treatment, is now hardly accessible in Europe. On the other hand, in many countries, including Poland, new infections caused by non-toxigenic Corynebacterium diphtheriae have been emerged. Such infections are frequently manifested by bacteraemia and endocarditis with a high fatality rate, amounting even to 41%.

  4. The art of blocking ADP-ribosyltransferases (ARTs): nanobodies as experimental and therapeutic tools to block mammalian and toxin ARTs.

    Science.gov (United States)

    Menzel, Stephan; Rissiek, Björn; Haag, Friedrich; Goldbaum, Fernando A; Koch-Nolte, Friedrich

    2013-08-01

    In 1901, the first Nobel Prize in Physiology or Medicine was awarded to Emil von Behring for his ground-breaking discovery of serum therapy: serum from horses vaccinated with toxin-containing culture medium of Corynebacterium diphtheriae contained life-saving 'antitoxins'. The molecular nature of the ADP-ribosylating toxin and the neutralizing antibodies were unraveled only 50 years later. Today, von Behring's antibody therapy is being refined with a new generation of recombinant antibodies and antibody fragments. Nanobodies, which are single-domain antibodies derived from the peculiar heavy-chain antibodies of llamas and other camelids, are emerging as a promising new class of highly specific enzyme inhibitors. In this review, we illustrate the potential of nanobodies as tools to block extracellular and intracellular ADP-ribosyltransferases (ARTs), using the toxin-related membrane-bound mammalian ecto-enzyme ARTC2 and the actin-ADP-ribosylating Salmonella virulence plasmid factor B toxin of Salmonella enterica as examples.

  5. ccdAB system and the encoding toxin protein-CcdB%ccdAB系统及其编码的毒素蛋白CcdB

    Institute of Scientific and Technical Information of China (English)

    贾卉; 井申荣

    2009-01-01

    ccdAB系统(control of cell division or death system)是目前已知的一种毒素-抗毒素系统(toxin-antitoxin system,TA系统),存在于致病性大肠杆菌F质粒及染色体骨架上,由ccdA和ccdB两个基因组成.质粒上的ccdAB系统编码一种毒素蛋白CcdB,在缺乏抗毒素的情况下,CcdB使细胞内促旋酶中毒,从而干扰DNA的合成,杀伤宿主细胞.本文对ccdAB系统的结构和功能,以及所编码CcdB的作用机制进行了综述.

  6. Breast milk reduces the risk of illness in children of mothers with cholera

    DEFF Research Database (Denmark)

    Qureshi, Katja; Mølbak, Kåre; Sandström, Anita

    2006-01-01

    BACKGROUND: A protective effect of breastfeeding against cholera has been demonstrated in areas endemic of cholera. To assess the protection offered by breast milk from mothers living in an area that had been free from cholera for 7 years, we investigated mothers with cholera and their children...... during an epidemic with Vibrio cholerae El Tor in the capital of Guinea-Bissau. METHODS: Eighty mothers with clinical cholera and their children were identified, and interviewed. Blood samples for vibriocidal and antitoxin antibodies were collected from mother-and-child pairs. Breast milk samples were...... collected from lactating mothers.Cholera was defined as acute watery diarrhea during the epidemic and a vibriocidal reciprocal titer of 20 or above. RESULTS: Three (7%) of 42 breastfed children had cholera as defined above compared with 9 (24%) of 38 nonbreastfed children (RR for breastfed children, 0...

  7. The Workshop on Animal Botulism in Europe

    DEFF Research Database (Denmark)

    Skarin, Hanna; Tevell Åberg, Annica; Woudstra, Cédric

    2013-01-01

    A workshop on animal botulism was held in Uppsala, Sweden, in June 2012. Its purpose was to explore the current status of the disease in Europe by gathering the European experts in animal botulism and to raise awareness of the disease among veterinarians and others involved in biopreparedness....... Animal botulism is underreported and underdiagnosed, but an increasing number of reports, as well as the information gathered from this workshop, show that it is an emerging problem in Europe. The workshop was divided into 4 sessions: animal botulism in Europe, the bacteria behind the disease, detection...... conclusions drawn from these discussions were that there is an urgent need to replace the mouse bioassay for botulinum toxin detection with an in vitro test and that there is a need for a European network to function as a reference laboratory, which could also organize a European supply of botulinum antitoxin...

  8. [The molecular physiological and genetic mechanisms underlying the superb efficacy of quinolones].

    Science.gov (United States)

    Long, Quan-xin; He, Ying; Xie, Jian-ping

    2012-08-01

    The fluoroquinolones are the most widely used broad-spectrum antibiotics, accounting for 18% of global antibacterial market share. They can kill bacteria rapidly with variety of derivatives available. Different quinolones vary significantly in rate and spectrum of killing, oxygen requirement for metabolism and reliance upon protein synthesis. Further understanding the sophisticated mechanisms of action of this important antibiotic family based on the molecular genetic response of bacteria can facilitate the discovery of better quinolone derivatives. Factors such as SOS response, bacterial toxin-antitoxin system, programmed death, chromosome fragmentation and reactive oxygen have been implicated in the action to some extent. "Two steps characteristic" of quinolones killing is also emphasized, which might inspire future better quinolones modification.

  9. Antibacterial action of quinolones: from target to network.

    Science.gov (United States)

    Cheng, Guyue; Hao, Haihong; Dai, Menghong; Liu, Zhenli; Yuan, Zonghui

    2013-08-01

    Quinolones are widely used broad-spectrum antibacterials with incomplete elucidated mechanism of action. Here, molecular basis for the antibacterial action of quinolones, from target to network, is fully discussed and updated. Quinolones trap DNA gyrase or topoisomerase IV to form reversible drug-enzyme-DNA cleavage complexes, resulting in bacteriostasis. Cell death arises from chromosome fragmentation in protein synthesis-dependent or -independent pathways according to distinguished quinolone structures. In the former pathway, irreversible oxidative DNA damage caused by reactive oxygen species kills bacteria eventually. Toxin-antitoxin mazEF is triggered as an additional lethal action. Bacteria survive and develop resistance by SOS and other stress responses. Enlarged knowledges of quinolone actions and bacterial response will provide new targets for drug design and approaches to prevent bacterial resistance.

  10. How World War 1 changed global attitudes to war and infectious diseases.

    Science.gov (United States)

    Shanks, G Dennis

    2014-11-08

    World War 1 was a key transition point towards scientific medicine. Medical officers incorporated Louis Pasteur's discoveries into their understanding of microorganisms as the cause of infectious diseases, which were therefore susceptible to rational control and treatment measures even in the pre-antibiotic era. Typhoid vaccination led to the successful evasion of the disastrous epidemics of previous wars. The incidence of tetanus was probably decreased by giving millions of doses of horse antitoxin to wounded soldiers. Quinine treated but could not control malaria; its use required mass compulsion. Tuberculosis was not a great military problem during World War 1, although mortality in civilian populations increased substantially. Treatment of sexually transmitted infections remained a matter of aversive conditioning, with invasive antiseptics used in the absence of antibiotics. Pandemic influenza in 1918-19 killed more people than died during the entire war, showing how much remained beyond the capability of the scientists and doctors who fought infectious diseases during World War 1.

  11. Special considerations for prophylaxis for and treatment of anthrax in pregnant and postpartum women.

    Science.gov (United States)

    Meaney-Delman, Dana; Zotti, Marianne E; Creanga, Andreea A; Misegades, Lara K; Wako, Etobssie; Treadwell, Tracee A; Messonnier, Nancy E; Jamieson, Denise J

    2014-02-01

    In August 2012, the Centers for Disease Control and Prevention, in partnership with the Association of Maternal and Child Health Programs, convened a meeting of national subject matter experts to review key clinical elements of anthrax prevention and treatment for pregnant, postpartum, and lactating (P/PP/L) women. National experts in infectious disease, obstetrics, maternal fetal medicine, neonatology, pediatrics, and pharmacy attended the meeting, as did representatives from professional organizations and national, federal, state, and local agencies. The meeting addressed general principles of prevention and treatment for P/PP/L women, vaccines, antimicrobial prophylaxis and treatment, clinical considerations and critical care issues, antitoxin, delivery concerns, infection control measures, and communication. The purpose of this meeting summary is to provide updated clinical information to health care providers and public health professionals caring for P/PP/L women in the setting of a bioterrorist event involving anthrax.

  12. Two cases of type A infant botulism in Grenoble, France: no honey for infants.

    Science.gov (United States)

    Hoarau, Gautier; Pelloux, Isabelle; Gayot, Armelle; Wroblewski, Isabelle; Popoff, Michel-Robert; Mazuet, Christelle; Maurin, Max; Croizé, Jacques

    2012-03-01

    We report two severe cases of infant botulism diagnosed at Grenoble University Hospital, France, respectively in 2006 and 2009. Both cases were characterized by a delay in diagnosis, severe neurological manifestations and extended period of hospitalization in intensive care unit, but a complete recovery. Infant botulism is a rare but life-threatening disease. It primarily affects infants, and the main risk factor is honey ingestion. Diagnosis should be systematically evoked by pediatricians in infants suffering from constipation, fatigue, muscle weakness, difficult feeding and altered cry, but before the onset of generalized flaccid paralysis, so as to administer specific treatment (BabyBIG®, a human derived botulinum antitoxin) at an early stage of the disease when it is most effective. In conclusion, parents should be aware of the role of honey as a source of spores of Clostridium botulinum and therefore infant botulism in the first year of life.

  13. [Scarlet fever epidemic during year 1929 in Chile].

    Science.gov (United States)

    Laval R, Enrique

    2009-04-01

    Scarlet fever is endemic in Chile, with relatively low morbidity and periodic exacerbations every 4 or 5 years, which can become epidemics. From 1921 to 1927, the number of patients hospitalized in the country fluctuated from 15 to 65 per year, until an epidemic involving nearly 3.000 patients started at the end of 1928 and continued during all 1929. 978 patients died, 537 (52.5%) were from Santiago. Public Health authorities confronted this emergency with prevention, prophylaxis, isolation and treatment measures and 558 beds were disposed for patient hospitalization. Vaccination trials were undertaken and specific treatment with antitoxins was used in patients with toxic clinical cases, having satisfactory results. Convalescent patients were controlled in order to stop the spread of the infection. After approximately 3 years, this disease returned to its regular endemicity.

  14. 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

    have developed a genetic system for S. islandicus LAL14/1 and created ¿pyrEF and ¿CRISPR_1 mutants using double cross-over and pop-in/pop-out approaches, respectively. Thus, LAL14/1 is a promising model to study virus-host interactions and the CRISPR/Cas defence mechanism in Archaea....... 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...

  15. The Role of Reactive Oxygen Species in Antibiotic-Mediated Killing of Bacteria.

    Science.gov (United States)

    Van Acker, Heleen; Coenye, Tom

    2017-01-12

    Recently, it was proposed that there is a common mechanism behind the activity of bactericidal antibiotics, involving the production of reactive oxygen species (ROS). However, the involvement of ROS in antibiotic-mediated killing has become the subject of much debate. In the present review, we provide an overview of the data supporting the ROS hypothesis; we also present data that explain the contradictory results often obtained when studying antibiotic-induced ROS production. For this latter aspect we will focus on the importance of taking the experimental setup into consideration and on the importance of some technical aspects of the assays typically used. Finally, we discuss the link between ROS production and toxin-antitoxin modules, and present an overview of implications for treatment.

  16. Biotechnological Trends in Spider and Scorpion Antivenom Development

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard; Solà, Mireia; Jappe, Emma Christine

    2016-01-01

    Spiders and scorpions are notorious for their fearful dispositions and their ability to inject venom into prey and predators, causing symptoms such as necrosis, paralysis, and excruciating pain. Information on venom composition and the toxins present in these species is growing due to an interest...... in using bioactive toxins from spiders and scorpions for drug discovery purposes and for solving crystal structures of membrane-embedded receptors. Additionally, the identification and isolation of a myriad of spider and scorpion toxins has allowed research within next generation antivenoms to progress...... at an increasingly faster pace. In this review, the current knowledge of spider and scorpion venoms is presented, followed by a discussion of all published biotechnological efforts within development of spider and scorpion antitoxins based on small molecules, antibodies and fragments thereof, and next generation...

  17. Single-mode tapered optical fiber loop immunosensor II: assay of anti-cholera toxin immunoglobulins

    Science.gov (United States)

    Marks, Robert S.; Hale, Zoe M.; Levine, Myron M.; Lowe, C. R.; Payne, Frank P.

    1994-07-01

    An evanescent wave immunoassay for cholera antitoxin immunoglobulins was performed using a single mode tapered optical fiber loop sensor. The transducer was silanized with 3- glycidoxypropyltrimethoxysilane and chemically modified to link covalently either cholera toxin B subunit or a synthetic peptide derived from it, CTP3. The sensor was exposed to seral fluids, obtained from human volunteers having been exposed to live virulent Vibrio cholerae 01 and shown to produce rice-water stools. Other toxins of interest, such as Clostridium botulinum toxin A, have been tested on similar systems. The bound unlabelled immunoglobulins were then exposed to a mixture of FITC-anti-IgG and TRITC-anti-IgA, without requirement for a separation step. The emanating fluorescent emissions of fluorescein and rhodamine, excited by the input laser light, were coupled back into the guided mode of the tapered fiber, and used to determine the concentrations of the complementary antigens.

  18. Genotyping, evolution and epidemiological findings of Rickettsia species.

    Science.gov (United States)

    Merhej, Vicky; Angelakis, Emmanouil; Socolovschi, Cristina; Raoult, Didier

    2014-07-01

    Rickettsiae are obligate intracellular bacteria that can cause mild to life-threatening diseases, including epidemic typhus, one of the oldest pernicious diseases of mankind. Clinical awareness of rickettsial diseases and molecular diagnosis have shown that rickettsioses should be viewed as new emerging and reemerging diseases. Rickettsia has been shown to be a large genus with a worldwide distribution, a very diverse host range, including hosts that have no relationship with vertebrate. Genomic studies have demonstrated genome reduction due to gene loss associated with increased pathogenicity and horizontal DNA acquisition according to a sympatric mode of evolution in hosts that contain several organisms. This article presents a review of genotyping techniques and examines the principle of genotype determination in terms of taxonomic strategies and detection methods. This article summarizes the epidemiological and pathological features of Rickettsia and discusses the genomic findings that help the understanding of the evolution of pathogenicity including the deleterious mutations of repair systems and the toxin-antitoxin systems.

  19. Short-term booster effect of diphtheria toxoid in initially long-term protected individuals.

    Science.gov (United States)

    Danilova, Elena; Shiryayev, Alexey; Skogen, Vegard; Kristoffersen, Einar Klaeboe; Sjursen, Haakon

    2005-02-10

    The main objective of this study was to investigate the booster antibody response in individuals with initially high levels of diphtheria antitoxin. Sixty individuals eligible for the routine booster by the age of 18 years each received a single dose of 5 Lf of diphtheria toxoid in diphtheria-tetanus vaccine. A double antigen ELISA was used for the assessment of the antibody levels. Chaotropic disruption in paired ELISA was used to test antibody avidity. The ratio between initial and maximum antibody concentrations after 1 month was >10 times higher and after 6 months still four times higher in those with initial antibody levels antibody levels >/=1 IU/ml a two-fold decrease was observed after 6 months compared to the initial levels. Thus, vaccination of individuals with initial long-term protection against diphtheria (antibody levels >/=1 IU/ml) is unnecessary and should be avoided.

  20. Scorpion antivenom effect of micropropagated Aristolochia elegans.

    Science.gov (United States)

    Izquierdo, Alejandro Mora; Zapata, Elsa Ventura; Jiménez-Ferrer, J Enrique; Muñoz, Crescencio Bazaldúa; Aparicio, Antonio Jiménez; Torres, Kalina Bermúdez; Torres, Lidia Osuna

    2010-08-01

    Aristolochia elegans Mast. (Aristolochiaceae) has been used to treat scorpion envenoming in Mexican traditional medicine. In vitro studies of the pharmacological activity of raw extracts from A. elegans roots have shown activity against scorpion bite. The aim of the present study was to determine for the first time the antagonistic effect of hexane and methanol extracts of the aerial parts and roots from micropropagated A. elegans plants in a model of isolated guinea-pig ileum contracted by scorpion bite. Results showed that the methanol extracts of aerial organs (74%) and roots (65%) of micropropagated plants have a similar antitoxin activity against scorpion poisoning to hexane extracts of wild plants (65%). These results suggest that using methanol extracts from the micropropagated plant material instead of wild plant root extracts from A. elegans is an alternative for treatment against scorpion bite symptoms, and will contribute to the conservation of this medicinal species.

  1. 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

    2016-01-01

    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......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...

  2. [Literature memories about diphtheria: Mark Twain, W.G. Sebald and Stendhal syndrome].

    Science.gov (United States)

    Ledermann, Walter

    2013-02-01

    Memories of W.G. Sebald from the diphtheria he suffered as a child, gave rise to a discussion about the origin of classic clinical descriptions and the traps memory tends. Good examples of the latter are some experiences of Stendhal, who must also be distrusted given his hypersensitivity, which gave name to a psychosomatic syndrome. Mark Twain, a more practical man, brings us back to reality with a funny story about the terror the disease caused in the late nineteenth century. This leads us to remember isolation measures and topical treatments from the period immediately preceding the antitoxin. They included manual removal of the pseudo membranes, maneuver that led Marañón to misinterpret a painting by Goya on a scene of "Lazarillo de Tormes".

  3. 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.

  4. Regeneration and gene transformation systems of Robinia pseudoacacia 'Idaho' mediated by Agrobacterium tumefaciens

    Institute of Scientific and Technical Information of China (English)

    Li Min; Cai Zao; Sun De-you; Yin Wei-lun; Chen Shou-yi; Wang Hua-fang

    2006-01-01

    Robinia pseudoacacia 'Idaho' is one of several multi-purpose trees used in ornamental, soil and water conservation, fodder and nectar sources. Plant abiotic stress tolerance transformed by genes could meet the requirements for reclamation of arid or alkalid lands and vegetation restoration. For this paper, we studied the effects of auxin and cytokine on Idaho locust in vitro regeneration and the establishment of gene transformation systems for plants mediated by Agrobacterium tumefaciens. Results showed that the ratios of cytokinin and auxin were the major factors affecting adventitious bud differentiation on a MS medium; the concentration of 0.5inhibit rooting. The most effective antitoxin for screening transgenic Idaho locust shoots was G418 and the most sensitive concentration of it was 8 mg·L-1.

  5. TAT皮试液配置方法及皮试液浓度分析

    Institute of Scientific and Technical Information of China (English)

    杨芳

    2012-01-01

    目的 探讨破伤风抗毒素(Tetanus Antitoxin)皮试液配置方法及皮试液浓度,减轻患者痛苦,提高护理质量.方法 对保定市急救中心260例需注射破伤风抗毒素患者进行常规及实验组皮试随机观察.结果 常规组阳性率83.08%,实验组阳性率22.31%.结论 选择正确稀释液,降低皮试液浓度,可有效降低TAT皮试阳性率.

  6. 西藏地区急性肉毒杆菌中毒抢救成功一例

    Institute of Scientific and Technical Information of China (English)

    惠升; 刘智广; 普布次仁

    2016-01-01

    肉毒杆菌(Clostridium botulinum),亦称肉毒梭菌,革兰阳性、粗短杆状、严格厌氧,可产生一种剧毒的肉毒杆菌毒素(Botulinum toxin)。该毒素可抑制神经介质-乙酰胆碱的释放,严重影响人神经电生理功能,中毒者常因呼吸衰竭而死亡[1]。急性肉毒杆菌中毒病例在西藏地区鲜有报道,其病情复杂、恶化快,早期明确诊断,尽早应用肉毒抗毒素(Botulinum antitoxins)和应用机械通气治疗是抢救成功的关键。

  7. 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.

  8. Improved vacuum sealing drainage in the treatment of gas gangrene: a case report.

    Science.gov (United States)

    Liu, Zhaofa; Zhao, Dewei; Wang, Benjie

    2015-01-01

    In this case, improved vacuum sealing drainage was used for gas gangrene treatment, which is different from traditional therapies of gas gangrene and this is the first report of using improved vacuum sealing drainage to treat gas gangrene. The patient was a 12-year-old Asian Male who was presented to the Emergency Department with a one-day history of left femoral progressing swelling, paining and fevering. Four days ago, rusty iron bars were plugged into the muscle of the left femoral when he played. Then he was taken to the local clinic and injected with tetanus antitoxin. A diagnosis of gas gangrene was made and modified vacuum sealing drainage device was used after thorough debridement. After two weeks' treatment, left femoral was kept and gas gangrene was cured successfully.

  9. Pitfalls to avoid when using phage display for snake toxins.

    Science.gov (United States)

    Laustsen, Andreas Hougaard; Lauridsen, Line Præst; Lomonte, Bruno; Andersen, Mikael Rørdam; Lohse, Brian

    2017-02-01

    Antivenoms against bites and stings from snakes, spiders, and scorpions are associated with immunological side effects and high cost of production, since these therapies are still derived from the serum of hyper-immunized production animals. Biotechnological innovations within envenoming therapies are thus warranted, and phage display technology may be a promising avenue for bringing antivenoms into the modern era of biologics. Although phage display technology represents a robust and high-throughput approach for the discovery of antibody-based antitoxins, several pitfalls may present themselves when animal toxins are used as targets for phage display selection. Here, we report selected critical challenges from our own phage display experiments associated with biotinylation of antigens, clone picking, and the presence of amber codons within antibody fragment structures in some phage display libraries. These challenges may be detrimental to the outcome of phage display experiments, and we aim to help other researchers avoiding these pitfalls by presenting their solutions.

  10. Zebrafish (Danio rerio) bioassay for visceral toxicosis of catfish and botulinum neurotoxin serotype E.

    Science.gov (United States)

    Chatla, Kamalakar; Gaunt, Patricia; Petrie-Hanson, Lora; Hohn, Claudia; Ford, Lorelei; Hanson, Larry

    2014-03-01

    Visceral toxicosis of catfish (VTC), a sporadic disease of cultured channel catfish (Ictalurus punctatus) often with high mortality, is caused by botulinum neurotoxin serotype E (BoNT/E). Presumptive diagnosis of VTC is based on characteristic clinical signs and lesions, and the production of these signs and mortality after sera from affected fish is administered to sentinel catfish. The diagnosis is confirmed if the toxicity is neutralized with BoNT/E antitoxin. Because small catfish are often unavailable, the utility of adult zebrafish (Danio rerio) was evaluated in BoNT/E and VTC bioassays. Channel catfish and zebrafish susceptibilities were compared using trypsin-activated BoNT/E in a 96-hr trial by intracoelomically administering 0, 1.87, 3.7, 7.5, 15, or 30 pg of toxin per gram of body weight (g-bw) of fish. All of the zebrafish died at the 7.5 pg/g-bw and higher, while the catfish died at the 15 pg/g-bw dose and higher. To test the bioassay, sera from VTC-affected fish or control sera were intracoelomically injected at a dose of 10 µl per zebrafish and 20 µl/g-bw for channel catfish. At 96 hr post-injection, 78% of the zebrafish and 50% of the catfish receiving VTC sera died, while no control fish died. When the VTC sera were preincubated with BoNT/E antitoxin, they became nontoxic to zebrafish. Histology of zebrafish injected with either VTC serum or BoNT/E demonstrated renal necrosis. Normal catfish serum was toxic to larval zebrafish in immersion exposures, abrogating their utility in VTC bioassays. The results demonstrate bioassays using adult zebrafish for detecting BoNT/E and VTC are sensitive and practical.

  11. Efficacy demonstration of tetanus vaccines by double antigen ELISA.

    Science.gov (United States)

    Rosskopf, U; Noeske, K; Werner, E

    2005-09-01

    This paper describes a double antigen ELISA (DAE) for rapid, specific and reliable assessment of the antitetanus immune status of horses and sheep. Compared with the indirect ELISA, the double antigen ELISA has the advantage of species-independent testing of sera. Thanks to its test design, it is more specific since the detected antibodies are forced to bind tetanus toxoid twice. In addition, it is very sensitive to tetanus antibodies, enabling the detection of low antibody titres, in range which is relevant for the assessment of the protective status (tetanus toxin neutralising antibodies). The detection limit of the DAE for tetanus antibodies is in the order of 10(-4) EU/ml. A comparison of in vitro results of individual sera with in vivo titres showed that horse sera with titres of 0.04 and 0.05 EU/ml in the DAE showed titres of > 0.05 IU and 0.034 IU/ml respectively during in vivo testing thus indicating good agreement. For tested sheep sera which were rated > 0.05 IU/ml in vivo, the corresponding titre in the DAE was 0.24 EU/ml. Clear tetanus antitoxin establishment of protective ELISA limits requires further comparative examination of sera with low titres (marketing authorisation procedures of tetanus vaccines ad us. vet. As a consequence, the toxin neutralisation test (still being the standard method of choice for quantifying tetanus toxin neutralising antitoxin titres) could be replaced, since it requires too great a number of animals per test and involves considerable suffering for the animals. The test described here reduces the use of mice and guinea pigs within vaccine efficacy testing. In addition, it involves less exposure of the laboratory personnel to toxin.

  12. VapC6, a ribonucleolytic toxin regulates thermophilicity in the crenarchaeote Sulfolobus solfataricus.

    Science.gov (United States)

    Maezato, Yukari; Daugherty, Amanda; Dana, Karl; Soo, Edith; Cooper, Charlotte; Tachdjian, Sabrina; Kelly, Robert M; Blum, Paul

    2011-07-01

    The phylum Crenarchaeota includes hyperthermophilic micro-organisms subjected to dynamic thermal conditions. Previous transcriptomic studies of Sulfolobus solfataricus identified vapBC6 as a heat-shock (HS)-inducible member of the Vap toxin-antitoxin gene family. In this study, the inactivation of the vapBC6 operon by targeted gene disruption produced two recessive phenotypes related to fitness, HS sensitivity and a heat-dependent reduction in the rate of growth. In-frame vapBC6 deletion mutants were analyzed to examine the respective roles of each protein. Since vapB6 transcript abundance was elevated in the vapC6 deletion, the VapC6 toxin appears to regulate abundance of its cognate antitoxin. In contrast, vapC6 transcript abundance was reduced in the vapB6 deletion. A putative intergenic terminator may underlie these observations by coordinating vapBC6 expression. As predicted by structural modeling, recombinant VapC6 produced using chaperone cosynthesis exhibited heat-dependent ribonucleolytic activity toward S. solfataricus total RNA. This activity could be blocked by addition of preheated recombinant VapB6. In vivo transcript targets were identified by assessing the relative expression of genes that naturally respond to thermal stress in VapBC6-deficient cells. Preferential increases were observed for dppB-1 and tetR, and preferential decreases were observed for rpoD and eIF2 gamma. Specific VapC6 ribonucleolytic action could also be demonstrated in vitro toward RNAs whose expression increased in the VapBC6-deficient strain during heat shock. These findings provide a biochemical mechanism and identify cellular targets underlying VapBC6-mediated control over microbial growth and survival at temperature extremes.

  13. Immunogenicity of a low-dose diphtheria, tetanus and acellular pertussis combination vaccine with either inactivated or oral polio vaccine compared to standard-dose diphtheria, tetanus, acellular pertussis when used as a pre-school booster in UK children: A 5-year follow-up of a randomised controlled study.

    Science.gov (United States)

    John, T; Voysey, M; Yu, L M; McCarthy, N; Baudin, M; Richard, P; Fiquet, A; Kitchin, N; Pollard, A J

    2015-08-26

    This serological follow up study assessed the kinetics of antibody response in children who previously participated in a single centre, open-label, randomised controlled trial of low-dose compared to standard-dose diphtheria booster preschool vaccinations in the United Kingdom (UK). Children had previously been randomised to receive one of three combination vaccines: either a combined adsorbed tetanus, low-dose diphtheria, 5-component acellular pertussis and inactivated polio vaccine (IPV) (Tdap-IPV, Repevax(®); Sanofi Pasteur MSD); a combined adsorbed tetanus, low-dose diphtheria and 5-component acellular pertussis vaccine (Tdap, Covaxis(®); Sanofi Pasteur MSD) given concomitantly with oral polio vaccine (OPV); or a combined adsorbed standard-dose diphtheria, tetanus, 2-component acellular pertussis and IPV (DTap-IPV, Tetravac(®); Sanofi Pasteur MSD). Blood samples for the follow-up study were taken at 1, 3 and 5 years after participation in the original trial (median, 5.07 years of age at year 1), and antibody persistence to each vaccine antigen measured against defined serological thresholds of protection. All participants had evidence of immunity to diphtheria with antitoxin concentrations greater than 0.01IU/mL five years after booster vaccination and 75%, 67% and 79% of children who received Tdap-IPV, Tdap+OPV and DTap-IPV, respectively, had protective antitoxin levels greater than 0.1IU/mL. Long lasting protective immune responses to tetanus and polio antigens were also observed in all groups, though polio responses were lower in the sera of those who received OPV. Low-dose diphtheria vaccines provided comparable protection to the standard-dose vaccine and are suitable for use for pre-school booster vaccination.

  14. An autonomous in vivo dual selection protocol for boolean genetic circuits.

    Science.gov (United States)

    Beneš, David; Sosík, Petr; Rodríguez-Patón, Alfonso

    2015-01-01

    Success in synthetic biology depends on the efficient construction of robust genetic circuitry. However, even the direct engineering of the simplest genetic elements (switches, logic gates) is a challenge and involves intense lab work. As the complexity of biological circuits grows, it becomes more complicated and less fruitful to rely on the rational design paradigm, because it demands many time-consuming trial-and-error cycles. One of the reasons is the context-dependent behavior of small assembly parts (like BioBricks), which in a complex environment often interact in an unpredictable way. Therefore, the idea of evolutionary engineering (artificial directed in vivo evolution) based on screening and selection of randomized combinatorial genetic circuit libraries became popular. In this article we build on the so-called dual selection technique. We propose a plasmid-based framework using toxin-antitoxin pairs together with the relaxase conjugative protein, enabling an efficient autonomous in vivo evolutionary selection of simple Boolean circuits in bacteria (E. coli was chosen for demonstration). Unlike previously reported protocols, both on and off selection steps can run simultaneously in various cells in the same environment without human intervention; and good circuits not only survive the selection process but are also horizontally transferred by conjugation to the neighbor cells to accelerate the convergence rate of the selection process. Our directed evolution strategy combines a new dual selection method with fluorescence-based screening to increase the robustness of the technique against mutations. As there are more orthogonal toxin-antitoxin pairs in E. coli, the approach is likely to be scalable to more complex functions. In silico experiments based on empirical data confirm the high search and selection capability of the protocol.

  15. Expression of toxin co-regulated pilus subunit A (TCPA) of Vibrio cholerae and its immunogenic epitopes fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

    Science.gov (United States)

    Sharma, Manoj Kumar; Singh, Nirmal Kumar; Jani, Dewal; Sisodia, Rama; Thungapathra, M; Gautam, J K; Meena, L S; Singh, Yogendra; Ghosh, Amit; Tyagi, Akhilesh Kumar; Sharma, Arun Kumar

    2008-02-01

    For protection against cholera, it is important to develop efficient vaccine capable of inducing anti-toxin as well as anti-colonizing immunity against Vibrio cholerae infections. Earlier, expression of cholera toxin B subunit (CTB) in tomato was reported by us. In the present investigation, toxin co-regulated pilus subunit A (TCPA), earlier reported to be an antigen capable of providing anti-colonization immunity, has been expressed in tomato. Further, to generate more potent combinatorial antigens, nucleotides encoding P4 or P6 epitope of TCPA were fused to cholera toxin B subunit gene (ctxB) and expressed in tomato. Presence of transgenes in the tomato genome was confirmed by PCR and expression of genes was confirmed at transcript and protein level. TCPA, chimeric CTB-P4 and CTB-P6 proteins were also expressed in E. coli. TCPA protein expressed in E. coli was purified to generate anti-TCPA antibodies in rabbit. Immunoblot and G(M1)-ELISA verified the synthesis and assembly of pentameric chimeric proteins in fruit tissue of transgenic tomato plants. The chimeric protein CTB-P4 and CTB-P6 accumulated up to 0.17 and 0.096% of total soluble protein (TSP), respectively, in tomato fruits. Whereas expression of TCPA, CTB-P4 and CTB-P6 in E. coli can be utilized for development of conventional vaccine, expression of these antigens which can provide both anti-toxin as well as anti-colonization immunity, has been demonstrated in plants, in a form which is potentially capable of inducing immune response against cholera infection.

  16. Clinical applications of the equine antitetanus immunoglobulin F(ab′)2%马破伤风免疫球蛋白F(ab′)2及其临床应用前景

    Institute of Scientific and Technical Information of China (English)

    丁天然; 张永信

    2012-01-01

      Despite the high-quality therapy and nursing care nowadays, mortality caused by tetanus still reaches 10 percent. Tetanus antitoxin has been used in the clinic for many years, the pharmaceutical preparation of which contains equine antitetanus immunoglobulin F(ab′)2 and immunoglobulin G. With the improvement of the preparation techonology, a new preparation“equine antitetanus immunoglobulin F(ab′)2”comes to the stage. Equine antitetanus immunoglobulin F(ab′)2 has a higher content of F(ab′)2 and a lower content of immunoglobulin G which causes allergy. With its distinct qualities and promising clinical prospect, equine antitetanus immunoglobulin F(ab′)2 can replace tetanus antitoxin in clinical applications.%  破伤风在现今医疗条件下的死亡率仍高达10%。长期以来,破伤风防治主要依赖破伤风抗毒素(TAT),后者主要含有马破伤风免疫球蛋白F(ab′)2和特异性抗体免疫球蛋白G(IgG)。经过多年的工艺改进,新制品马破伤风免疫球蛋白F(ab′)2因提高了可中和破伤风毒素的有效成分F(ab′)2的含量,同时降低了会引起过敏反应的主要成分IgG的含量、使不良反应发生率降低,具有良好的临床应用前景,是TAT的升级换代制品。

  17. 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.

  18. Sequencing and phylogenetic analysis of neurotoxin gene from an environmental isolate of Clostridium sp.: comparison with other clostridial neurotoxins.

    Science.gov (United States)

    Dixit, Aparna; Alam, Syed Imteyaz; Singh, Lokendra

    2006-07-01

    A Clostridium sp. isolated from intestine of decaying fish exhibited 99% sequence identity with C. tetani at 16S rRNA level. It produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E) as well as tetanus antitoxin. The gene fragments for light chain, C-terminal and N-terminal regions of the heavy chain of the toxin were amplified using three reported primer sets for tetanus neurotoxin (TeNT). The neurotoxin gene fragments were cloned in Escherichia coli and sequenced. The sequences obtained exhibited approximately 98, 99 and 98% sequence identity with reported gene sequences of TeNT/LC, TeNT/HC and TeNT/HN, respectively. The phylogenetic interrelationship between the neurotoxin gene of Clostridium sp. with previously reported gene sequences of Clostridium botulinum A to G and C. tetani was examined by analysis of differences in the nucleotide sequences. Six amino acids were substituted at four different positions in the light chain of neurotoxin from the isolate when compared with the reported closest sequence of TeNT. Of these, four were located in the beta15 motif at a solvent inaccessible, buried region of the protein molecule. One of these substitutions were on the solvent accessible surface residue of alpha1 motif, previously shown to have strong sequence conservation. A substitution of two amino acids observed in N-terminal region of heavy chain were buried residues, located in the beta21 and beta37 motifs showing variability in other related sequences. The C-terminal region responsible for binding to receptor was conserved, showing no changes in the amino acid sequence.

  19. Stochastic induction of persister cells by HipA through (p)ppGpp-mediated activation of mRNA endonucleases.

    Science.gov (United States)

    Germain, Elsa; Roghanian, Mohammad; Gerdes, Kenn; Maisonneuve, Etienne

    2015-04-21

    The model organism Escherichia coli codes for at least 11 type II toxin-antitoxin (TA) modules, all implicated in bacterial persistence (multidrug tolerance). Ten of these encode messenger RNA endonucleases (mRNases) inhibiting translation by catalytic degradation of mRNA, and the 11th module, hipBA, encodes HipA (high persister protein A) kinase, which inhibits glutamyl tRNA synthetase (GltX). In turn, inhibition of GltX inhibits translation and induces the stringent response and persistence. Previously, we presented strong support for a model proposing (p)ppGpp (guanosine tetra and penta-phosphate) as the master regulator of persistence. Stochastic variation of [(p)ppGpp] in single cells induced TA-encoded mRNases via a pathway involving polyphosphate and Lon protease. Polyphosphate activated Lon to degrade all known type II antitoxins of E. coli. In turn, the activated mRNases induced persistence and multidrug tolerance. However, even though it was known that activation of HipA stimulated (p)ppGpp synthesis, our model did not explain how hipBA induced persistence. Here we show that, in support of and consistent with our initial model, HipA-induced persistence depends not only on (p)ppGpp but also on the 10 mRNase-encoding TA modules, Lon protease, and polyphosphate. Importantly, observations with single cells convincingly show that the high level of (p)ppGpp caused by activation of HipA does not induce persistence in the absence of TA-encoded mRNases. Thus, slow growth per se does not induce persistence in the absence of TA-encoded toxins, placing these genes as central effectors of bacterial persistence.

  20. Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines-part 1.

    Science.gov (United States)

    Winsnes, R; Sesardic, D; Daas, A; Behr-Gross, M-E

    2004-01-01

    A collaborative study on the evaluation of an alternative functional assay, the Vero cell method, to the Ph. Eur. in vivo challenge procedures for potency determination of diphtheria toxoid in 6 different combined vaccines was initiated in January 2001. The study was an extension of a previous study for the validation of serological methods for potency testing of tetanus toxoid vaccines for human use. To allow interim evaluation of test results and to monitor study progress, the project was divided into three consecutive phases. The results of Phase I and II studies are presented in this report. Pre-validation (Phase I) study, performed in two laboratories, indicated that comparable diphtheria potency estimates were obtained in the Ph. Eur. direct intradermal challenge assay in guinea pigs, in Vero cell assay and in indirect ELISA for five vaccines of different potencies (range of estimates: ca. 20-200 IU/ml). The correlation coefficients between the challenge assay and the Vero cell assay corresponded to those between the challenge assay and ELISA, confirming that the antibodies play an important role in protection and that predominantly protective/neutralising antibodies are present in guinea pigs, at the time point investigated. It was observed, for Vero cell assays, that about 16-35 (9-28 in Phase II study) fold lower titre of individual serum samples were obtained when using equine, rather than guinea pig reference serum. The study also provided preliminary information that sera from the same guinea pigs may be used for potency determination of both diphtheria and tetanus toxoid components of vaccines. In Phase II, another five laboratories analysed a subset of the vaccines included in Phase I study plus an additional vaccine. Four laboratories performed the lethal challenge assay and one laboratory carried out the intradermal challenge assay. All laboratories also performed the Vero cell assay and both ELISA for diphtheria antitoxin and ELISA for tetanus

  1. Molecular mechanisms of bacterial persistence and phenotypic antibiotic resistance%细菌持留与抗生素表型耐药机制

    Institute of Scientific and Technical Information of China (English)

    崔鹏; 许涛; 张文宏; 张颖

    2016-01-01

    Bacterial persistence refers to a state of reduced metabolic activity that endows a subpopulation of isogenic bacteria with multidrug tolerance. Persisters are phenotypic variants but not mutants. Since its discovery in 1944, bacterial persistence has not received enough attention until recently when its implications in persistent infections and biofilm infections become apparent. Much research has been done in recent years to investigate the mechanisms underlying bacterial persistence and phenotypic antibiotic resistance. The mechanisms of bacterial persistence are complex and the following pathways are involved in persister formation: toxin-antitoxin systems, reduced metabolism, energy production, protein and nucleic acid synthesis, DNA repair and protection, protein degradation, transporters/efflux systems, and transcriptional regulators etc. Although persistence mechanisms are conserved in terms of the gene function and pathways involved among different bacterial species, they may vary in gene homology and relative importance of a given pathway. For example, Escherichia coli toxin-antitoxin systems play an important role in persister formation, while Staphylococcus aureus persister formation does not appear to use toxin-antitoxin systems. Here we provide an update on recent progress in persistence mechanisms using E. coli and S. aureus as models, as well as discuss approaches in the treatment of persistent bacterial infections.%持留菌是细菌群体中一小部分具有表型耐药的细菌。自1944年被发现后,近几十年来因其在慢性持续性感染和生物膜感染中的重要作用而得到越来越多的重视。已有的研究结果表明,细菌持留的机理复杂,涉及的相关信号通路有毒素-抗毒素系统、细胞能量代谢及蛋白核酸合成等生理状态的降低、DNA保护修复系统、蛋白酶系统、反式翻译、外排泵系统等。虽然不同细菌的持留机理有一定的相似性和保守性,但

  2. Scientific treatment and prevention of tetanus%破伤风的科学防治

    Institute of Scientific and Technical Information of China (English)

    沈银忠; 张永信

    2012-01-01

      尽管破伤风已不再是一种多发病,但因破伤风杆菌广泛存在于环境中且引起其感染的外伤性因素依然很多、病死率又高,故破伤风仍是威胁人类健康的主要疾病之一。破伤风的诊断主要依赖外伤史和临床表现,早期识别并给予科学的治疗是改善预后的关键,而关键的治疗措施包括对伤口的彻底处理以及控制肌肉痉挛和肺部感染等。破伤风抗毒素是防治破伤风的重要药物,但其明显的不良反应限制了临床应用。新型抗毒素免疫制剂如人破伤风免疫球蛋白和马破伤风免疫球蛋白F(ab′)2将在今后破伤风的防治中发挥重要作用。预防接种是预防破伤风的主要措施。推广新法接生、外伤后正确处理伤口等也有助于预防破伤风。外伤后应首先清洁伤口,而后根据伤口情况及患者受伤前的免疫接种史来决定如何进行抗毒素预防接种。%  Tetanus is no longer a common disease nowadays, however, it is still a serious disease threatening the human health and has a high mortality because Clostridium tetani is distributed widely in the environment, and there exist many traumatic factors which can cause tetanus. The diagnosis of tetanus is entirely dependent on the history of trauma and clinical manifestations. Early identification and scientific treatment is the key to improve the prognosis. The key measure of treatment includes the wound thoroughly cleaned and controls of muscle cramps and pulmonary infection. Tetanus antitoxin is an important drug to prevent and treat tetanus but its side effect restricts the clinical usage. A new type antitoxin immune agent such as tetanus immunoglobulin will play an important role in prevention and treatment of tetanus in the future. Immunization is a main measure to prevent tetanus. Popularizing hospital delivery and correct treating wounds post-traumatic are also benefit for preventing tetanus. After trauma

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

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    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

  4. Long-Term Protection against Diphtheria in the Netherlands after 50 Years of Vaccination: Results from a Seroepidemiological Study.

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    E M Swart

    Full Text Available To evaluate the National Immunisation Programme (NIP a population-based cross-sectional seroepidemiological study was performed in the Netherlands. We assessed diphtheria antitoxin levels in the general Dutch population and in low vaccination coverage (LVC areas where a relatively high proportion of orthodox Protestants live who decline vaccination based on religious grounds. Results were compared with a nationwide seroepidemiological study performed 11 years earlier.In 2006/2007 a national serum bank was established. Blood samples were tested for diphtheria antitoxin IgG concentrations using a multiplex immunoassay for 6383 participants from the national sample (NS and 1518 participants from LVC municipalities. A cut-off above 0.01 international units per ml (IU/ml was used as minimum protective level.In the NS 91% of the population had antibody levels above 0.01 IU/ml compared to 88% in the 1995/1996 serosurvey (p<0.05. On average, 82% (vs. 78% in the 1995/1996 serosurvey, p<0.05 of individuals from the NS born before introduction of diphtheria vaccination in the NIP and 46% (vs. 37% in the 1995/1996 serosurvey, p = 0.11 of orthodox Protestants living in LVC areas had antibody levels above 0.01 IU/ml. Linear regression analysis among fully immunized individuals (six vaccinations without evidence of revaccination indicated a continuous decline in antibodies in both serosurveys, but geometric mean antibodies remained well above 0.01 IU/ml in all age groups.The NIP provides long-term protection against diphtheria, although antibody levels decline after vaccination. As a result of natural waning immunity, a substantial proportion of individuals born before introduction of diphtheria vaccination in the NIP lack adequate levels of diphtheria antibodies. Susceptibility due to lack of vaccination is highest among strictly orthodox Protestants. The potential risk of spread of diphtheria within the geographically clustered orthodox Protestant community

  5. [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

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

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    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

  7. Replacement of the in vivo neutralisation test for efficacy demonstration of tetanus vaccines ad us. vet.

    Science.gov (United States)

    Rosskopf, Ute; Noeske, Kerstin; Werner, Esther

    2005-01-01

    The bacterium Clostridium (C.) tetani is an ubiquitous pathogen. This anaerobic, gram-positive bacterium can form spores and can be found in the whole environment. It enters the body via injuries of the skin and wounds where it releases the neurotoxin "tetanospasmin" (= tetanus toxin). The animals most susceptible to tetanus infection are horses and sheep. Only active immunisation by tetanus vaccine provides effective protection against tetanus intoxication. The marketing authorisation requirements stipulate that efficacy of tetanus vaccines ad us. vet. must be demonstrated in all target animal species via determination of neutralising tetanus serum antitoxin concentrations. The standard method used for this purpose is still the toxin neutralisation test (TNT), as it quantifies the tetanus toxin-neutralising effect of tetanus serum antibodies in vivo. In this test, tetanus toxin is added to dilutions of serum from vaccinated horse and sheep. The serum dilutions are then administered to mice or guinea pigs, which are observed for toxic symptoms. Against the background of animal protection, the goal of one project of the Paul-Ehrlich-Institut (Bundesministerium fuer Bildung und Forschung (Federal Ministry for Education and Research), 0312636) was to establish an alternative to the toxin neutralisation test, enabling the testing of efficacy of tetanus vaccines with serological in vitro methods. For this purpose, a so-called double antigen ELISA (DAE) was established which enables the testing of sera of different species in one assay. In addition, the sera were tested in an indirect ELISA for horses and sheep separately. Altogether, ten groups of horses and eight groups of sheep were immunised with ten animals per group each. The tetanus vaccines comprised almost all products authorised for the German market at the start of the project. 564 horse sera and 257 sheep sera were tested using the two ELISA methods. Some sera were also tested in vivo. The kinetics of

  8. Effect of schedule on reactogenicity and antibody persistence of acellular and whole-cell pertussis vaccines: value of laboratory tests as predictors of clinical performance.

    Science.gov (United States)

    Miller, E; Ashworth, L A; Redhead, K; Thornton, C; Waight, P A; Coleman, T

    1997-01-01

    The performance of four acellular pertussis vaccines containing between two and five pertussis antigens combined with diphtheria and tetanus toxoids was compared with that of British whole-cell diphtheria/tetanus/pertussis (DTP) vaccine both in laboratory assays for potency, toxicity and immunogenicity, and for reactogenicity and immunogenicity in infants. Clinical responses were evaluated in double blind randomized Phase II trials using 3/5/9 month and 2/3/4 month schedules. The acellular DTPs had much lower toxicity than whole-cell DTP in laboratory tests and were significantly less pyrogenic than whole-cell DTP under both schedules. Local reactions were not consistently lower in acellular than whole-cell vaccinees and varied with the source of the diphtheria and tetanus antigens used. Differences in endotoxin level and content of active pertussis toxin (PT) between acellular DTP vaccines were not clinically significant. The reactogenicity advantage of the acellular vaccines was substantially reduced under the 2/3/4 month schedule due to the reduced reactogenicity of the whole-cell DTP vaccine when given at a younger age. There was no relationship between antigen content measured in micrograms per dose and ELISA antibody responses to filamentous haemagglutinin (FHA) and PT in infants, nor was murine immunogenicity predictive of immunogenicity in humans. Antibody response to PT was attenuated in the whole-cell group under the 2/3/4 month schedule but was unaffected in the group receiving acellular vaccines with individually purified components; antibody response to pertactin (69 kDa antigen) was similar in recipients of the whole-cell and component acellular vaccines under the 2/3/4 month schedule. PT antibody persistence until 4-5 years of age was significantly better in recipients of the component acellular than either the whole-cell vaccine or the co-purified acellular vaccine under the 3/5/9 month schedule. However, diphtheria antitoxin levels were reduced in

  9. 处置突发公共卫生事件的部队官兵预防破伤风发病研究:软组织损伤1782例分析%Study of tetanus prevention of troops in public health event-1 782 patients with soft tissue injury

    Institute of Scientific and Technical Information of China (English)

    侯振才; 刘晓冰; 刘禄明; 李雪莲; 王丕明

    2011-01-01

    目的 突发公共卫生事件中破伤风发病研究及相关风险因素的分析,探讨应急突发公共卫生事件中软组织损伤破伤风的防控.方法 对l 782例软组织损伤者.采用碘伏、生理盐水、双氧水擦拭冲洗伤口.并行特殊缝合术及包扎;破伤风抗毒素皮试阳性者脱敏注射或注射人破伤风免疫球蛋白.结果 术后观察7天,随诊60天,无一例破伤风发病;破伤风抗毒素皮试附性率占46.2%(823/1782).结论 突发公共卫生事件中,特别是洪涝灾害,破伤风杆菌污染最多,对极易感染软组织损伤的人群,多措并举进行伤口处置是可靠有效的防控措施,推荐在突发公共卫生事件中应用破伤风免疫球蛋白.%Objective To investigate the prevalence and risk factors of tetanus in public health event and explore the approach to prevent and control the disease. Methods 1,782 patients experiencing soft tissue injury were treated with iodophored NS, hydrogen peroxide, specific suture, and tetanus anti-toxin or human tetanus immunoglobulin when there was a positive skin test.Results There is not even a single case of infection on a seven-day observation followed by a 60-day post visit. The positive skin test demonstrated as 46.2% (823/1782). Conclusion Most likely tetanus spread in flood. Debridement combined with tetanus anti-toxin is a reliable measure to prevent tetanus infection, recommending of tetanus immune globulin application in massed public health event.

  10. 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.

  11. Escherichia coli Quorum-Sensing EDF, A Peptide Generated by Novel Multiple Distinct Mechanisms and Regulated by trans-Translation

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    Sathish Kumar

    2016-01-01

    Full Text Available Eshcerichia coli mazEF is a stress-induced toxin-antitoxin module mediating cell death and requiring a quorum-sensing (QS extracellular death factor (EDF, the pentapeptide NNWNN. Here we uncovered several distinct molecular mechanisms involved in its generation from the zwf mRNA encoding glucose-6-phosphate dehydrogenase. In particular, we show that, under stress conditions, the endoribonuclease MazF cleaves specific ACA sites, thereby generating a leaderless zwf mRNA which is truncated 30 codons after the EDF-encoding region. Since the nascent ribosome peptide exit tunnel can accommodate up to 40 amino acids, this arrangement allows the localization of the EDF residues inside the tunnel when the ribosome is stalled at the truncation site. Moreover, ribosome stalling activates the trans-translation system, which provides a means for the involvement of ClpPX in EDF generation. Furthermore, the trans-translation is described as a regulatory system that attenuated the generation of EDF, leading to low levels of EDF in the single cell. Therefore, the threshold EDF molecule concentration required is achieved only by the whole population, as expected for QS.

  12. 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.

  13. Dual Toxic-Peptide-Coding Staphylococcus aureus RNA under Antisense Regulation Targets Host Cells and Bacterial Rivals Unequally

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    Marie-Laure Pinel-Marie

    2014-04-01

    Full Text Available Produced from the pathogenicity islands of Staphylococcus aureus clinical isolates, stable SprG1 RNA encodes two peptides from a single internal reading frame. These two peptides accumulate at the membrane, and inducing their expression triggers S. aureus death. Replacement of the two initiation codons by termination signals reverses this toxicity. During growth, cis-antisense RNA SprF1 is expressed, preventing mortality by reducing SprG1 RNA and peptide levels. The peptides are secreted extracellularly, where they lyse human host erythrocytes, a process performed more efficiently by the longer peptide. The two peptides also inactivate Gram-negative and -positive bacteria, with the shorter peptide more effective against S. aureus rivals. Two peptides are secreted from an individual RNA containing two functional initiation codons. Thus, we present an unconventional type I toxin-antitoxin system expressed from a human pathogen producing two hemolytic and antibacterial peptides from a dual-coding RNA, negatively regulated by a dual-acting antisense RNA.

  14. Mechanisms of post-transcriptional gene regulation in bacterial biofilms

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    Viveka eVadyvaloo

    2014-03-01

    Full Text Available Abstract Biofilms are characterized by a dense multicellular community of microorganisms that can be formed by the attachment of bacteria to an inert surface and to each other. The development of biofilm involves the initial attachment of planktonic bacteria to a surface, followed by replication, cell-to-cell adhesion to form microcolonies, maturation and detachment. Mature biofilms are embedded in a self-produced extracellular polymeric matrix composed primarily of bacterial-derived exopolysaccharides, specialized proteins, adhesins and occasionally DNA. Because the synthesis and assembly of biofilm matrix components is an exceptionally complex process, the transition between its different phases requires the coordinate expression and simultaneous regulation of many genes by complex genetic networks involving all levels of gene regulation. The finely controlled intracellular level of the chemical second messenger molecule, cyclic-di-GMP is central to the post-transcriptional mechanisms governing the switch between the motile planktonic lifestyle and the sessile biofilm forming state in many bacteria. Several other post-transcriptional regulatory mechanisms are known to dictate biofilm development and assembly and these include RNA-binding proteins, small non-coding RNAs, toxin-antitoxin systems, riboswitches and RNases. Post-transcriptional regulation is therefore a powerful molecular mechanism employed by bacteria to rapidly adjust to the changing environment and to fine tune gene expression to the developmental needs of the cell. In this review, we discuss post-transcriptional mechanisms that influence the biofilm developmental cycle in a variety of pathogenic bacteria.

  15. Veterinary immunology as colonial science: method and quantification in the investigation of horsesickness in South Africa, C. 1905-1945.

    Science.gov (United States)

    Gilfoyle, Daniel

    2006-01-01

    This article examines the practice of veterinary immunology in South Africa during the first half of the twentieth century through an analysis of research into a horsesickness vaccine at the Onderstepoort Veterinary Institute. From the early 1900s, Arnold Theiler prioritized research into horsesickness, by then defined as an insect-borne disease caused by an ultravisible virus. He succeeded in devising a means of prophylaxis using a simultaneous injection of infective blood and immune serum, but he discovered antigenically different strains of the virus, which could overcome the immunity produced by his treatment. The practical value of Theiler's methods was further limited by difficulties in standardizing the biological material used in immunization, the results of which remained too erratic for application on a large scale. No further advances were made until the 1930s, by which time Onderstepoort had been drawn more closely into international scientific networks. Using techniques derived from research into yellow fever in America and canine distemper in Britain, the Onderstepoort scientist Raymond Alexander invented a method of immunization that utilized the propagation of the horsesickness virus in the brains of mice. Alexander's methods, which were characterized by successful technical adaptation and innovation, depended upon methods of quantification first developed by Paul Ehrlich to standardize diphtheria antitoxin during the 1890s. During the 1940s, vaccination expanded rapidly in South Africa, and Onderstepoort later exported the vaccine and associated technology to other countries affected by horsesickness.

  16. Persister formation in Staphylococcus aureus is associated with ATP depletion

    Energy Technology Data Exchange (ETDEWEB)

    Conlon, Brian P.; Rowe, Sarah E.; Gandt, Autumn Brown; Nuxoll, Austin S.; Donegan, Niles P.; Zalis, Eliza A.; Clair, Geremy; Adkins, Joshua N.; Cheung, Ambrose L.; Lewis, Kim

    2016-04-18

    Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics1. Persisters are associated with chronic bacterial infection and antibiotic treatment failure. In Escherichia coli, toxin/antitoxin (TA) modules are responsible for persister formation. The mechanism of persister formation in Gram positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting TA modules in S. aureus did not affect the level of persisters. Here we show that S. aureus persisters are produced due to a stochastic entrance to stationary phase accompanied by a drop in intracellular ATP. Cells expressing stationary state markers are present throughout the growth phase, increasing in frequency with cell density. Cell sorting revealed that expression of stationary markers was associated with a 100-1000 fold increased likelihood of survival to antibiotic challenge. We find that the antibiotic tolerance of these cells is due to a drop in intracellular ATP. The ATP level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotic treatment.

  17. Inhibition of Shiga toxin 2 (Stx2) in apple juices and its resistance to pasteurization.

    Science.gov (United States)

    Rasooly, Reuven; Do, Paula M; Levin, Carol E; Friedman, Mendel

    2010-06-01

    In the present study, we evaluated Shiga toxin (Stx2) activity in apple juices by measuring a decrease in dehydrogenase activity of Vero cells with the microculture tetrazolium (MTT) assay. Freshly prepared juice from Red Delicious apples and Golden Delicious apples inhibited the biological activity of the bacterial toxin Stx2 produced by E. coli O157:H7 strains. Studies with immunomagnetic beads bearing specific antibodies against the toxin revealed that Stx2 activity was restored when removed from the apple juice. SDS gel electrophoresis revealed no difference (P juices. These results suggest that Stx2 may be reversibly bound to small molecular weight constituents in the juice. The Stx2 toxin was not inactivated on exposure to heat programs (63 degrees C for 30 min, 72 degrees C for 15 s, 89 degrees C for 1 s) commonly used to pasteurize apple juice, but lost all activity when exposed to 100 degrees C for 5 min. The results suggest that pasteurization of apple juice used to inactivate E. coli O157:H7 has no effect on Stx2, and that food-compatible and safe antitoxin compounds can be used to inhibit the biological activity of the Shiga toxin.

  18. Genome Analyses of Icelandic Strains of Sulfolobus islandicus, Model Organisms for Genetic and Virus-Host Interaction Studies

    DEFF Research Database (Denmark)

    Guo, Li; Brügger, Kim; Liu, Chao;

    2011-01-01

    The genomes of two Sulfolobus islandicus strains obtained from Icelandic solfataras were sequenced and analyzed. Strain REY15A is a host for a versatile genetic toolbox. It exhibits a genome of minimal size, is stable genetically, and is easy to grow and manipulate. Strain HVE10/4 shows a broad h...... conjugative plasmids, which have integrated at a few tRNA genes lacking introns. This provides a possible rationale for the presence of the introns.......The genomes of two Sulfolobus islandicus strains obtained from Icelandic solfataras were sequenced and analyzed. Strain REY15A is a host for a versatile genetic toolbox. It exhibits a genome of minimal size, is stable genetically, and is easy to grow and manipulate. Strain HVE10/4 shows a broad...... in gene content and gene order occur. These include gene clusters involved in specific metabolic pathways, multiple copies of VapBC antitoxin-toxin gene pairs, and in strain HVE10/4, a 50-kb region rich in glycosyl transferase genes. The variable region also contains most of the insertion sequence (IS...

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Bioterrorism in Canada: An Economic Assessment of Prevention and Postattack Response

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    Ronald St John

    2001-01-01

    Full Text Available The present paper calculates the human and economic consequences of a bioterrorist attack on Canadian soil using aerosolized Bacillus anthracis and Clostridium botulinum. The study assumed that 100,000 people in a Canadian suburban neighbourhood were exposed over a 2 h period to an infectious dose of one of the agents. Using an epidemic curve based on the epidemiology and management of anthrax and botulinum poisoning, the costs of intervention and treatment after an attack were compared with the costs of preparedness before a bioterrorist attack. The results show that an investment in planning and preparedness to manage the consequences of an attack can reduce morbidity, mortality and economic costs. The sooner that an intervention program is instituted, the more significant are the health and economic benefits. The greatest benefits were realized when postattack intervention was initiated before day 3 after the event. The economic impact of a bioterrorist attack in Canada could range from $6.4 billion/100,000 exposed to B anthracis to $8.6 billion/100,000 exposed in an attack using C botulinum. Without the benefit of an effective consequence management program, predicted deaths totalled 32,875 from anthrax and 30,000 from botulinum toxin. Rapid implementation of a postattack prophylaxis program that includes the stockpiling of antibiotics, vaccines and antitoxins; training of first responders in the diagnosis, handling and treatment of pathogens; and the general enhancement of Canada's response capability would reduce both human and economic losses.

  1. Effect of clpP and clpC deletion on persister cell number in Staphylococcus aureus.

    Science.gov (United States)

    Springer, Matthew T; Singh, Vineet K; Cheung, Ambrose L; Donegan, Niles P; Chamberlain, Neal R

    2016-08-01

    Staphylococcus aureus is responsible for a wide variety of infections that include superficial skin and soft tissue infections, septicaemia, central nervous system infections, endocarditis, osteomyelitis and pneumonia. Others have demonstrated the importance of toxin-antitoxin (TA) modules in the formation of persisters and the role of the Clp proteolytic system in the regulation of these TA modules. This study was conducted to determine the effect of clpP and clpC deletion on S. aureus persister cell numbers following antibiotic treatment. Deletion of clpP resulted in a significant decrease in persister cells following treatment with oxacillin and erythromycin but not with levofloxacin and daptomycin. Deletion of clpC resulted in a decrease in persister cells following treatment with oxacillin. These differences were dependent on the antibiotic class and the CFU ml-1 in which the cells were treated. Persister revival assays for all the bacterial strains in these studies demonstrated a significant delay in resumption of growth characteristic of persister cells, indicating that the surviving organisms in this study were not likely due to spontaneous antibiotic resistance. Based on our results, ClpP and possibly ClpC play a role in persister cell formation or maintenance, and this effect is dependent on antibiotic class and the CFU ml-1 or the growth phase of the cells.

  2. 饮酒时间对破伤风抗毒素皮试影响的临床观察

    Institute of Scientific and Technical Information of China (English)

    冯莉

    2007-01-01

    破伤风是常和创伤相关联的一种特异性感染,是一种严重的疾病,但是可以预防的。破伤风抗毒素(Tetanus AntitoxinTAT)是破伤风类毒素免疫马的血浆经胃酶消化后用盐析法制得的液体或冻干抗毒素球蛋白制剂,用于治疗及预防破伤风。然而对人体而言,TAT是一种异体蛋白质,具有抗原性,人体注射后很容易发生过敏反应,轻者皮疹,重者休克。因此在临床使用前应做TAT皮试,准确判断TAY皮试结果至关重要。若皮试阳性需采用脱敏注射,注射后引起的过敏反应给护理人员及病人造成极大的威胁与烦恼,严重时导致病人死亡。因此,对324例外伤伴饮酒的病人做TAT皮试临床观察及分析。现报告如下。

  3. 白藜芦醇在创伤及脓毒症中的应用研究进展%Applied research progress of resveratrol on trauma and sepsis

    Institute of Scientific and Technical Information of China (English)

    李香琴; 范霞; 梁华平

    2016-01-01

    白藜芦醇是一种天然抗毒素,广泛存在于葡萄、花生、坚果等食物中,具有抗炎、抗氧化、抗衰老、抗肿瘤、免疫调节等多种生物活性.近年来大量研究结果显示白藜芦醇对创伤及脓毒症诱发的损伤展现出良好的疗效.本文主要针对白藜芦醇在创伤-失血性休克、创伤性脑损伤、脊髓损伤、缺血再灌注损伤及脓毒症损伤中的保护作用及其作用机制进行概述.%Resveratrol is a natural antitoxin which widely present in grapes,peanuts,nuts and other foods with anti-inflammatory,anti-oxidation,anti-aging,anti-tumor,immunomodulatory and other biological activities.In recent years,a large number of studies have showed that resveratrol have great effect on trauma and sepsis-induced damage.This paper mainly focuses on the protective effects of resveratrol and its potential mechanisms on trauma-hemorrhagic shock,traumatic brain injury,spinal cord injury,ischemiec reperfusion injury and sepsis injury.

  4. In vitro Determination of Extracellular Proteins from Xylella fastidiosa

    Science.gov (United States)

    Mendes, Juliano S.; Santiago, André S.; Toledo, Marcelo A. S.; Horta, Maria A. C.; de Souza, Alessandra A.; Tasic, Ljubica; de Souza, Anete P.

    2016-01-01

    The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa. Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa. Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (3–30 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components. PMID:28082960

  5. Extraction of BoNT/A, /B, /E, and /F with a single, high affinity monoclonal antibody for detection of botulinum neurotoxin by Endopep-MS.

    Directory of Open Access Journals (Sweden)

    Suzanne R Kalb

    Full Text Available Botulinum neurotoxins (BoNTs are extremely potent toxins that are capable of causing respiratory failure leading to long-term intensive care or death. The best treatment for botulism includes serotype-specific antitoxins, which are most effective when administered early in the course of the intoxication. Early confirmation of human exposure to any serotype of BoNT is an important public health goal. In previous work, we focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating the seven serotypes (BoNT/A-G in buffer and BoNT/A, /B, /E, and /F (the four serotypes that commonly affect humans in clinical samples. We have previously reported the success of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. However, to check for any one of the four serotypes of BoNT/A, /B, /E, or /F, each sample is split into 4 aliquots, and tested for the specific serotypes separately. The discovery of a unique monoclonal antibody that recognizes all four serotypes of BoNT/A, /B, /E and /F allows us to perform simultaneous detection of all of them. When applied in conjunction with the Endopep-MS assay, the detection limit for each serotype of BoNT with this multi-specific monoclonal antibody is similar to that obtained when using other serotype-specific antibodies.

  6. Clostridium difficile-induced colitis in mice is independent of leukotrienes.

    Science.gov (United States)

    Trindade, Bruno C; Theriot, Casey M; Leslie, Jhansi L; Carlson, Paul E; Bergin, Ingrid L; Peters-Golden, Marc; Young, Vincent B; Aronoff, David M

    2014-12-01

    Clostridium difficile is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis in healthcare settings. However, the host factors involved in the intestinal inflammatory response and pathogenesis of C. difficile infection (CDI) are largely unknown. Here we investigated the role of leukotrienes (LTs), a group of pro-inflammatory lipid mediators, in CDI. Notably, the neutrophil chemoattractant LTB4, but not cysteinyl (cys) LTs, was induced in the intestine of C57BL/6 mice infected with either C. difficile strain VPI 10463 or strain 630. Genetic or pharmacological ablation of LT production did not ameliorate C. difficile colitis or clinical signs of disease in infected mice. Histological analysis demonstrated that intestinal neutrophilic inflammation, edema and tissue damage in mice during acute and severe CDI were not modulated in the absence of LTs. In addition, CDI induced a burst of cytokines in the intestine of infected mice in a LT-independent manner. Serum levels of anti-toxin A immunoglobulin (Ig) G levels were also not modulated by endogenous LTs. Collectively, our results do not support a role for LTs in modulating host susceptibility to CDI in mice.

  7. The natural history of ADP-ribosyltransferases and the ADP-ribosylation system.

    Science.gov (United States)

    Aravind, L; Zhang, Dapeng; de Souza, Robson F; Anand, Swadha; Iyer, Lakshminarayan M

    2015-01-01

    Catalysis of NAD(+)-dependent ADP-ribosylation of proteins, nucleic acids, or small molecules has evolved in at least three structurally unrelated superfamilies of enzymes, namely ADP-ribosyltransferase (ART), the Sirtuins, and probably TM1506. Of these, the ART superfamily is the most diverse in terms of structure, active site residues, and targets that they modify. The primary diversification of the ART superfamily occurred in the context of diverse bacterial conflict systems, wherein ARTs play both offensive and defensive roles. These include toxin-antitoxin systems, virus-host interactions, intraspecific antagonism (polymorphic toxins), symbiont/parasite effectors/toxins, resistance to antibiotics, and repair of RNAs cleaved in conflicts. ARTs evolving in these systems have been repeatedly acquired by lateral transfer throughout eukaryotic evolution, starting from the PARP family, which was acquired prior to the last eukaryotic common ancestor. They were incorporated into eukaryotic regulatory/epigenetic control systems (e.g., PARP family and NEURL4), and also used as defensive (e.g., pierisin and CARP-1 families) or immunity-related proteins (e.g., Gig2-like ARTs). The ADP-ribosylation system also includes other domains, such as the Macro, ADP-ribosyl glycohydrolase, NADAR, and ADP-ribosyl cyclase, which appear to have initially diversified in bacterial conflict-related systems. Unlike ARTs, sirtuins appear to have a much smaller presence in conflict-related systems.

  8. Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Tobias Dörr

    2010-02-01

    Full Text Available Bacteria induce stress responses that protect the cell from lethal factors such as DNA-damaging agents. Bacterial populations also form persisters, dormant cells that are highly tolerant to antibiotics and play an important role in recalcitrance of biofilm infections. Stress response and dormancy appear to represent alternative strategies of cell survival. The mechanism of persister formation is unknown, but isolated persisters show increased levels of toxin/antitoxin (TA transcripts. We have found previously that one or more components of the SOS response induce persister formation after exposure to a DNA-damaging antibiotic. The SOS response induces several TA genes in Escherichia coli. Here, we show that a knockout of a particular SOS-TA locus, tisAB/istR, had a sharply decreased level of persisters tolerant to ciprofloxacin, an antibiotic that causes DNA damage. Step-wise administration of ciprofloxacin induced persister formation in a tisAB-dependent manner, and cells producing TisB toxin were tolerant to multiple antibiotics. TisB is a membrane peptide that was shown to decrease proton motive force and ATP levels, consistent with its role in forming dormant cells. These results suggest that a DNA damage-induced toxin controls production of multidrug tolerant cells and thus provide a model of persister formation.

  9. Toxin YafQ increases persister cell formation by reducing indole signalling.

    Science.gov (United States)

    Hu, Ying; Kwan, Brian W; Osbourne, Devon O; Benedik, Michael J; Wood, Thomas K

    2015-04-01

    Persister cells survive antibiotic and other environmental stresses by slowing metabolism. Since toxins of toxin/antitoxin (TA) systems have been postulated to be responsible for persister cell formation, we investigated the influence of toxin YafQ of the YafQ/DinJ Escherichia coli TA system on persister cell formation. Under stress, YafQ alters metabolism by cleaving transcripts with in-frame 5'-AAA-G/A-3' sites. Production of YafQ increased persister cell formation with multiple antibiotics, and by investigating changes in protein expression, we found that YafQ reduced tryptophanase levels (TnaA mRNA has 16 putative YafQ cleavage sites). Consistently, TnaA mRNA levels were also reduced by YafQ. Tryptophanase is activated in the stationary phase by the stationary-phase sigma factor RpoS, which was also reduced dramatically upon production of YafQ. Tryptophanase converts tryptophan into indole, and as expected, indole levels were reduced by the production of YafQ. Corroborating the effect of YafQ on persistence, addition of indole reduced persistence. Furthermore, persistence increased upon deleting tnaA, and persistence decreased upon adding tryptophan to the medium to increase indole levels. Also, YafQ production had a much smaller effect on persistence in a strain unable to produce indole. Therefore, YafQ increases persistence by reducing indole, and TA systems are related to cell signalling.

  10. 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.

    Directory of Open Access Journals (Sweden)

    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.

  11. A processed noncoding RNA regulates an altruistic bacterial antiviral system.

    Science.gov (United States)

    Blower, Tim R; Pei, Xue Y; Short, Francesca L; Fineran, Peter C; Humphreys, David P; Luisi, Ben F; Salmond, George P C

    2011-02-01

    The ≥ 10³⁰ bacteriophages on Earth relentlessly drive adaptive coevolution, forcing the generation of protective mechanisms in their bacterial hosts. One such bacterial phage-resistance system, ToxIN, consists of a protein toxin (ToxN) that is inhibited in vivo by a specific RNA antitoxin (ToxI); however, the mechanisms for this toxicity and inhibition have not been defined. Here we present the crystal structure of the ToxN-ToxI complex from Pectobacterium atrosepticum, determined to 2.75-Å resolution. ToxI is a 36-nucleotide noncoding RNA pseudoknot, and three ToxI monomers bind to three ToxN monomers to generate a trimeric ToxN-ToxI complex. Assembly of this complex is mediated entirely through extensive RNA-protein interactions. Furthermore, a 2'-3' cyclic phosphate at the 3' end of ToxI, and catalytic residues, identify ToxN as an endoRNase that processes ToxI from a repetitive precursor but is regulated by its own catalytic product.

  12. Multitasking SecB chaperones in bacteria

    Directory of Open Access Journals (Sweden)

    Ambre eSala

    2014-12-01

    Full Text Available Protein export in bacteria is facilitated by the canonical SecB chaperone, which binds to unfolded precursor proteins, maintains them in a translocation competent state and specifically cooperates with the translocase motor SecA to ensure their proper targeting to the Sec translocon at the cytoplasmic membrane. Besides its key contribution to the Sec pathway, SecB chaperone tasking is critical for the secretion of the Sec-independent heme-binding protein HasA and actively contributes to the cellular network of chaperones that control general proteostasis in Escherichia coli, as judged by the significant interplay found between SecB and the Trigger Factor, DnaK and GroEL chaperones. Although SecB is mainly a proteobacterial chaperone associated with the presence of an outer membrane and outer membrane proteins, secB-like genes are also found in Gram-positive bacteria as well as in certain phages and plasmids, thus suggesting alternative functions. In addition, a SecB-like protein is also present in the major human pathogen M. tuberculosis where it specifically controls a stress-responsive toxin-antitoxin (TA system. This review focuses on such very diverse chaperone functions of SecB, both in E. coli and in other unrelated bacteria.

  13. 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.

  14. Clinical evaluation strategies for a live attenuated tetravalent dengue vaccine.

    Science.gov (United States)

    Precioso, Alexander Roberto; Palacios, Ricardo; Thomé, Beatriz; Mondini, Gabriella; Braga, Patrícia; Kalil, Jorge

    2015-12-10

    Butantan Institute is a public Brazilian biomedical research-manufacturer center affiliated to the São Paulo State Secretary of Health. Currently, Butantan is one of the main public producers of vaccines, antivenoms, and antitoxins in Latin America. The partnership between Butantan and the National Institutes of Health (NIH) of the United Sates has been one of the longest and most successful partnerships in the development and manufacturing of new vaccines. Recently, Butantan Institute has developed and manufactured a lyophilized tetravalent live attenuated dengue vaccine with the four dengue viruses attenuated and licensed from the Laboratory of Infectious Diseases at The National Institutes of Allergy and Infectious Diseases (LID/NIAID/NIH). The objective of this paper is to describe the clinical evaluation strategies of a live attenuated tetravalent dengue vaccine (Butantan-DV) developed and manufactured by Butantan Institute. These clinical strategies will be used to evaluate the Butantan-DV Phase III trial to support the Butantan-DV licensure for protection against any symptomatic dengue caused by any serotype in people aged 2 to 59 years.

  15. Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection.

    Science.gov (United States)

    Pardo-López, Liliana; Soberón, Mario; Bravo, Alejandra

    2013-01-01

    Bacillus thuringiensis bacteria are insect pathogens that produce different Cry and Cyt toxins to kill their hosts. Here we review the group of three-domain Cry (3d-Cry) toxins. Expression of these 3d-Cry toxins in transgenic crops has contributed to efficient control of insect pests and a reduction in the use of chemical insecticides. The mode of action of 3d-Cry toxins involves sequential interactions with several insect midgut proteins that facilitate the formation of an oligomeric structure and induce its insertion into the membrane, forming a pore that kills midgut cells. We review recent progress in our understanding of the mechanism of action of these Cry toxins and focus our attention on the different mechanisms of resistance that insects have evolved to counter their action, such as mutations in cadherin, APN and ABC transporter genes. Activity of Cry1AMod toxins, which are able to form toxin oligomers in the absence of receptors, against different resistant populations, including those affected in the ABC transporter and the role of dominant negative mutants as antitoxins, supports the hypothesis that toxin oligomerization is a limiting step in the Cry insecticidal activity. Knowledge of the action of 3d-Cry toxin and the resistance mechanisms to these toxins will set the basis for a rational design of novel toxins to overcome insect resistance, extending the useful lifespan of Cry toxins in insect control programs.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. Natural Inhibitors of Snake Venom Metalloendopeptidases: History and Current Challenges

    Science.gov (United States)

    Bastos, Viviane A.; Gomes-Neto, Francisco; Perales, Jonas; Neves-Ferreira, Ana Gisele C.; Valente, Richard H.

    2016-01-01

    The research on natural snake venom metalloendopeptidase inhibitors (SVMPIs) began in the 18th century with the pioneering work of Fontana on the resistance that vipers exhibited to their own venom. During the past 40 years, SVMPIs have been isolated mainly from the sera of resistant animals, and characterized to different extents. They are acidic oligomeric glycoproteins that remain biologically active over a wide range of pH and temperature values. Based on primary structure determination, mammalian plasmatic SVMPIs are classified as members of the immunoglobulin (Ig) supergene protein family, while the one isolated from muscle belongs to the ficolin/opsonin P35 family. On the other hand, SVMPIs from snake plasma have been placed in the cystatin superfamily. These natural antitoxins constitute the first line of defense against snake venoms, inhibiting the catalytic activities of snake venom metalloendopeptidases through the establishment of high-affinity, non-covalent interactions. This review presents a historical account of the field of natural resistance, summarizing its main discoveries and current challenges, which are mostly related to the limitations that preclude three-dimensional structural determinations of these inhibitors using “gold-standard” methods; perspectives on how to circumvent such limitations are presented. Potential applications of these SVMPIs in medicine are also highlighted. PMID:27571103

  18. Efficacy of ETI-204 monoclonal antibody as an adjunct therapy in a New Zealand white rabbit partial survival model for inhalational anthrax.

    Science.gov (United States)

    Biron, Bethany; Beck, Katie; Dyer, David; Mattix, Marc; Twenhafel, Nancy; Nalca, Aysegul

    2015-04-01

    Inhalational anthrax is characterized by extensive bacteremia and toxemia as well as nonspecific to mild flu-like symptoms, until the onset of hypotension, shock, and mortality. Without treatment, the mortality rate approaches 100%. Antibiotic treatment is not always effective, and alternative treatments are needed, such as monotherapy for antibiotic-resistant inhalational anthrax or as an adjunct therapy in combination with antibiotics. The Bacillus anthracis antitoxin monoclonal antibody (MAb) ETI-204 is a high-affinity chimeric deimmunized antibody which targets the anthrax toxin protective antigen (PA). In this study, a partial protection New Zealand White (NZW) rabbit model was used to evaluate the protective efficacy of the adjunct therapy with the MAb. Following detection of PA in the blood, NZW rabbits were administered either an antibiotic (doxycycline) alone or the antibiotic in conjunction with ETI-204. Survival was evaluated to compare the efficacy of the combination adjunct therapy with that of an antibiotic alone in treating inhalational anthrax. Overall, the results from this study indicate that a subtherapeutic regimen consisting of an antibiotic in combination with an anti-PA MAb results in increased survival compared to the antibiotic alone and would provide an effective therapeutic strategy against symptomatic anthrax in nonvaccinated individuals.

  19. Safety and immunogenicity of escalating dosages of a single oral administration of peru-15 pCTB, a candidate live, attenuated vaccine against enterotoxigenic Escherichia coli and Vibrio cholerae.

    Science.gov (United States)

    Chen, Wilbur H; Garza, Jose; Choquette, Monique; Hawkins, Jennifer; Hoeper, Amy; Bernstein, David I; Cohen, Mitchell B

    2015-01-01

    Enterotoxigenic Escherichia coli (ETEC) organisms are a leading cause of infectious diarrhea in developing countries. A live, attenuated cholera strain that expresses high levels of the nontoxic B subunit of cholera toxin, which might also serve as an ETEC protective antigen, was evaluated for safety, excretion, and immunogenicity in healthy volunteers. We enrolled four inpatient dose-escalation cohorts of 15 to 16 eligible subjects to randomly (3:1) receive a single oral dose of vaccine or placebo (buffer alone), evaluating 1 ×10(7), 1 ×10(8), 1 ×10(9), and 1 ×10(10) CFU of the vaccine. The vaccine was well tolerated, although some subjects experienced moderate diarrhea. The serum Inaba vibriocidal antibody response appeared to display a dose-response relationship with increasing dosages of vaccine, plateauing at the 10(9)-CFU dosage. The serum antitoxin (cholera toxin and heat-labile enterotoxin) antibody seroconversion rate (4-fold increase over baseline) also appeared to display a dose-response relationship. The vaccine strain was excreted in stool cultures, displaying a dose-response relationship. A single oral dose of Peru-15 pCTB at dosages up to 1 ×10(10) CFU was safe and immunogenic in this first-in-human trial. These encouraging data support the ongoing clinical development of this candidate combined cholera and ETEC vaccine. (This study has been registered at ClinicalTrials.gov under registration no. NCT00654108.).

  20. The pharmaceuticalisation of security: Molecular biomedicine, antiviral stockpiles, and global health security.

    Science.gov (United States)

    Elbe, Stefan

    2014-12-01

    Pharmaceuticals are now critical to the security of populations. Antivirals, antibiotics, next-generation vaccines, and antitoxins are just some of the new 'medical countermeasures' that governments are stockpiling in order to defend their populations against the threat of pandemics and bioterrorism. How has security policy come to be so deeply imbricated with pharmaceutical logics and solutions? This article captures, maps, and analyses the 'pharmaceuticalisation' of security. Through an in-depth analysis of the prominent antiviral medication Tamiflu, it shows that this pharmaceutical turn in security policy is intimately bound up with the rise of a molecular vision of life promulgated by the biomedical sciences. Caught in the crosshairs of powerful commercial, political, and regulatory pressures, governments are embracing a molecular biomedicine promising to secure populations pharmaceutically in the twenty-first century. If that is true, then the established disciplinary view of health as a predominantly secondary matter of 'low' international politics is mistaken. On the contrary, the social forces of health and biomedicine are powerful enough to influence the core practices of international politics - even those of security. For a discipline long accustomed to studying macrolevel processes and systemic structures, it is in the end also our knowledge of the minute morass of molecules that shapes international relations.

  1. Comparative Genomics of Acetobacterpasteurianus Ab3, an Acetic Acid Producing Strain Isolated from Chinese Traditional Rice Vinegar Meiguichu.

    Science.gov (United States)

    Xia, Kai; Li, Yudong; Sun, Jing; Liang, Xinle

    2016-01-01

    Acetobacter pasteurianus, an acetic acid resistant bacterium belonging to alpha-proteobacteria, has been widely used to produce vinegar in the food industry. To understand the mechanism of its high tolerance to acetic acid and robust ability of oxidizing ethanol to acetic acid (> 12%, w/v), we described the 3.1 Mb complete genome sequence (including 0.28 M plasmid sequence) with a G+C content of 52.4% of A. pasteurianus Ab3, which was isolated from the traditional Chinese rice vinegar (Meiguichu) fermentation process. Automatic annotation of the complete genome revealed 2,786 protein-coding genes and 73 RNA genes. The comparative genome analysis among A. pasteurianus strains revealed that A. pasteurianus Ab3 possesses many unique genes potentially involved in acetic acid resistance mechanisms. In particular, two-component systems or toxin-antitoxin systems may be the signal pathway and modulatory network in A. pasteurianus to cope with acid stress. In addition, the large numbers of unique transport systems may also be related to its acid resistance capacity and cell fitness. Our results provide new clues to understanding the underlying mechanisms of acetic acid resistance in Acetobacter species and guiding industrial strain breeding for vinegar fermentation processes.

  2. Molecular characterization of a 21.4 kilobase antibiotic resistance plasmid from an α-hemolytic Escherichia coli O108:H- human clinical isolate.

    Directory of Open Access Journals (Sweden)

    Fay E Dawes

    Full Text Available This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-. DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes.

  3. 一株C型肉毒梭菌的分离和鉴别%The isolation and identification of a type C strain of clostridium botulinum

    Institute of Scientific and Technical Information of China (English)

    冉枭; 钟权涛; 傅思武

    2013-01-01

    OBJECTIVE The study is examined for distribution of clostridium botulinum in Northwest,China.METHODS All soils from Gansu Province were washed by 0.2% gelatin phosphate buffer,and then carried out with the methods of bacteria-proliferating,isolating culture repeatedly.The isolated strain was identified by toxin determination and biochemical characteristics.RESULTS 2 of the 175 soil specimens appeared typical symptoms of botulism,and only 1 could be completely neutralized by type C botulinum antitoxin.CONCLUSION The strain is identified as type C clostridium botulinum.%目的 为了解肉毒梭菌在我国西北地区的分布情况.方法 对从甘肃省采集的所有土壤样品用0.2%的明胶磷酸盐缓冲液冲洗后,反复增菌、分离培养,并对分离菌株进行毒素测定和生化特性分析.结果 在采集的175份样品中有2份样品出现典型的肉毒中毒症状,1株与C型肉毒抗毒素完全中和.结论 分离得到的菌株经鉴定为C型肉毒梭菌.

  4. The SecB-like chaperone Rv1957 from Mycobacterium tuberculosis: crystallization and X-ray crystallographic analysis.

    Science.gov (United States)

    Lu, Zuokun; Wang, Han; Yu, TingTing

    2016-06-01

    Protein export is important in all bacteria, and bacteria have evolved specialized export machineries to fulfil this task. In Mycobacterium tuberculosis, the causative agent of tuberculosis, the general secretion pathway (Sec pathway) is conserved and is essential in performing the export of proteins. The bacterial Sec pathway post-translationally exports unfolded proteins out of the cytoplasm, and the core of the Sec pathway is composed of a heterotrimeric membrane-embedded channel, SecYEG, and two cytosolic components, SecA and SecB. SecB functions by stabilizing unfolded proteins, maintaining them in an export-competent state. Although SecB is mainly found in Proteobacteria, a SecB-like protein, Rv1957, that controls a stress-response toxin-antitoxin system, is found in M. tuberculosis. Rv1957 can also functionally replace the Escherichia coli SecB chaperone both in vivo and in vitro. In this work, the production, crystallization and X-ray crystallographic analysis of Rv1957 are reported. Notably, diffraction-quality crystals were obtained only at high concentrations of dimethyl sulfoxide, i.e. about 12%(v/v). The crystals of Rv1957 belonged to space group P212121, with unit-cell parameters a = 64.5, b = 92.0, c = 115.4 Å.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Čelešnik, Helena; Tanšek, Anja; Tahirović, Aneja; Vižintin, Angelika; Mustar, Jernej; Vidmar, Vita; Dolinar, Marko

    2016-01-01

    In recent years, photosynthetic autotrophic cyanobacteria have attracted interest for biotechnological applications for sustainable production of valuable metabolites. Although biosafety issues can have a great impact on public acceptance of cyanobacterial biotechnology, biosafety of genetically modified cyanobacteria has remained largely unexplored. We set out to incorporate biocontainment systems in the model cyanobacteriumSynechocystissp. PCC 6803. Plasmid-encoded safeguards were constructed using the nonspecific nuclease NucA fromAnabaenacombined with different metal-ion inducible promoters. In this manner, conditional lethality was dependent on intracellular DNA degradation for regulated autokilling as well as preclusion of horizontal gene transfer. In cells carrying the suicide switch comprising thenucAgene fused to a variant of thecopMpromoter, efficient inducible autokilling was elicited. Parallel to nuclease-based safeguards, cyanobacterial toxin/antitoxin (TA) modules were examined in biosafety switches. Rewiring ofSynechocystisTA pairsssr1114/slr0664andslr6101/slr6100for conditional lethality using metal-ion responsive promoters resulted in reduced growth, rather than cell killing, suggesting cells could cope with elevated toxin levels. Overall, promoter properties and translation efficiency influenced the efficacy of biocontainment systems. Several metal-ion promoters were tested in the context of safeguards, and selected promoters, including anrsBvariant, were characterized by beta-galactosidase reporter assay.

  7. A NEW APPROACH TO BACTERIAL VACCINES.

    Science.gov (United States)

    GREENBERG, L

    1963-08-31

    Immunizing antigens against only 10 bacterial diseases-cholera, diphtheria, paratyphoid, pertussis, plague, scarlet fever, staphylococcal disease, tetanus, tuberculosis and typhoid-have been licensed for sale in Canada and the United States. Convincing evidence of efficacy is available for only four of these-diphtheria and tetanus toxoids, and pertussis and typhoid vaccines.The principles which determine the efficacy of different immunizing antigens are not always the same. Toxoids, for example, stimulate the formation of antitoxin-producing mechanisms which can neutralize toxins produced by invading organisms, thereby rendering them harmless. Conversely, vaccines stimulate the formation of antibacterial mechanisms which stop the growth of organisms before they can produce disease.Use of enzyme-lysed vaccines for prevention of staphylococcal disease represents a new approach in vaccine research. Animal tests have shown lysed vaccines to be 10 to 100 times less toxic, and about eight times more effective, than whole bacterial vaccines. Studies with lysed vaccines for other diseases are now in progress.

  8. Single-Cell Analysis of the Plasmablast Response to Vibrio cholerae Demonstrates Expansion of Cross-Reactive Memory B Cells

    Science.gov (United States)

    Kauffman, Robert C.; Bhuiyan, Taufiqur R.; Nakajima, Rie; Mayo-Smith, Leslie M.; Rashu, Rasheduzzaman; Hoq, Mohammad Rubel; Chowdhury, Fahima; Khan, Ashraful Islam; Rahman, Atiqur; Bhaumik, Siddhartha K.; Harris, Levelle; O'Neal, Justin T.; Trost, Jessica F.; Alam, Nur Haq; Jasinskas, Algis; Dotsey, Emmanuel; Kelly, Meagan; Charles, Richelle C.; Xu, Peng; Kováč, Pavol; Calderwood, Stephen B.; Ryan, Edward T.; Felgner, Phillip L.; Qadri, Firdausi

    2016-01-01

    ABSTRACT We characterized the acute B cell response in adults with cholera by analyzing the repertoire, specificity, and functional characteristics of 138 monoclonal antibodies (MAbs) generated from single-cell-sorted plasmablasts. We found that the cholera-induced responses were characterized by high levels of somatic hypermutation and large clonal expansions. A majority of the expansions targeted cholera toxin (CT) or lipopolysaccharide (LPS). Using a novel proteomics approach, we were able to identify sialidase as another major antigen targeted by the antibody response to Vibrio cholerae infection. Antitoxin MAbs targeted both the A and B subunits, and most were also potent neutralizers of enterotoxigenic Escherichia coli heat-labile toxin. LPS-specific MAbs uniformly targeted the O-specific polysaccharide, with no detectable responses to either the core or the lipid moiety of LPS. Interestingly, the LPS-specific antibodies varied widely in serotype specificity and functional characteristics. One participant infected with the Ogawa serotype produced highly mutated LPS-specific antibodies that preferentially bound the previously circulating Inaba serotype. This demonstrates durable memory against a polysaccharide antigen presented at the mucosal surface and provides a mechanism for the long-term, partial heterotypic immunity seen following cholera. PMID:27999163

  9. 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.

  10. Insights into dynamics of mobile genetic elements in hyperthermophilic environments from five new Thermococcus plasmids.

    Science.gov (United States)

    Krupovic, Mart; Gonnet, Mathieu; Hania, Wajdi Ben; Forterre, Patrick; Erauso, Gaël

    2013-01-01

    Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1), with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA) systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.

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

    Directory of Open Access Journals (Sweden)

    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. Differential Rickettsial Transcription in Bloodfeeding and Non-Bloodfeeding Arthropod Hosts

    Science.gov (United States)

    Verhoeve, Victoria I.; Jirakanwisal, Krit; Utsuki, Tadanobu; Macaluso, Kevin R.

    2016-01-01

    Crucial factors influencing the epidemiology of Rickettsia felis rickettsiosis include pathogenesis and transmission. Detection of R. felis DNA in a number of arthropod species has been reported, with characterized isolates, R. felis strain LSU and strain LSU-Lb, generated from the cat flea, Ctenocephalides felis, and the non-hematophagous booklouse, Liposcelis bostrychophila, respectively. While it is realized that strain influence on host biology varies, the rickettsial response to these distinct host environments remained undefined. To identify a panel of potential rickettsial transmission determinants in the cat flea, the transcriptional profile for these two strains of R. felis were compared in their arthropod hosts using RNAseq. Rickettsial genes with increased transcription in the flea as compared to the booklouse were identified. Genes previously associated with bacterial virulence including LPS biosynthesis, Type IV secretion system, ABC transporters, and a toxin-antitoxin system were selected for further study. Transcription of putative virulence-associated genes was determined in a flea infection bioassay for both strains of R. felis. A host-dependent transcriptional profile during bloodfeeding, specifically, an increased expression of selected transcripts in newly infected cat fleas and flea feces was detected when compared to arthropod cell culture and incubation in vertebrate blood. Together, these studies have identified novel, host-dependent rickettsial factors that likely contribute to successful horizontal transmission by bloodfeeding arthropods. PMID:27662479

  13. Report: seroprevalence of corynebacterium diphtheriae among vaccinated population of Rawalpindi/Islamabad, Pakistan.

    Science.gov (United States)

    Faryal, Rani; Noreen, Zobia; Tahir, Faheem; Rehman, Zahidur

    2013-05-01

    Diphtheria is a communicable disease of global significance, and its outbreaks have to be reported to the world community under the International Health Regulations (IHR). A pilot seroepidemiological survey was conducted to assess immunity status of diphtheria among healthy individuals of Rawalpindi/Islamabad (Pakistan), who had been administered at least one dose of the vaccine against the disease, as part of childhood vaccination. The study group comprised of 128 healthy subjects, grouped according to the decade representing their age. Antidiphtheria IgG levels were measured by Enzyme Linked Immunosorbent Assay (ELISA) method. The studied sample showed 100% prevalence of diphtheria antitoxin, confirming prior vaccination; however 49.2% exhibited only minimal protection against diphtheria. Full protection was observed in a significantly higher (p=0.013) percentage of males (54.45%) as compared to female subjects (33.33%). Maximum level of serum antibodies were seen in 1-10 year age group (0.195+0.031 IU/mL), which was significantly higher than that recorded in the age group of 11-20 (p=0.024) and above 30 years (p=0.0064). The present results emphasize the need for periodical booster immunization in adolescents and adults, after primary childhood immunization.

  14. Lack of significant differences in immunity against diphtheria between populations of Eastern and Western regions of Poland.

    Science.gov (United States)

    Chudnicka, Alina; Walory, Jarosław

    2003-01-01

    Incidents of diphtheria in countries which were formerly part of the Soviet Union (Ukraine, Russia and Belorus) resulted in the need to evaluate thoroughly the effectiveness of preventive vaccination in Poland, especially in the border regions of the country where the biggest migration of population can be observed. The aim of this work was a comparison of the immunity to diphtheria in two geographically different regions of Poland--eastern (Lublin) and western (Zielona Gora) ones. It showed immunoprophylaxis to diphtheria that was implemented on these areas. Diphtheria antitoxin level (IgG) was determined with application of the ELISA method in 1236 (529/707) people. No significant differences were found in the level of antibodies in the groups 19 years of age in people below the protective titre (0.1 IU/ml). The difference occurring in the interval between 2nd and 18th year of life (in western Poland 7.6% and in eastern Poland 16%) may result from different implementation of the vaccination program in these regions (booster doses). Recommendations for vaccination to diphtheria in people over 25 years of age should be implemented especially in the frontier regions of Poland adjoining countries threatened with diphtheria occurrence.

  15. An antibody induction method in mice for the potency assays of diphtheria and tetanus components in combined vaccines.

    Science.gov (United States)

    Maheshwari, S C; Sharma, S B; Kumar, A; Sokhey, J

    1998-09-01

    Eleven batches of Adsorbed Diphtheria-Tetanus (DT) vaccines and thirteen batches of Adsorbed Diphtheria-Pertussis-Tetanus (DTP) vaccines were tested for the potency of diphtheria and tetanus components by an Antibody Induction Method (AIM) developed in mice. The potency results obtained were found comparable and did not show any statistically significant difference with those obtained by WHO recommended lethal challenge tests for diphtheria in guinea pigs and for tetanus in mice. AIM in mice is more economical as both diphtheria and tetanus components of combined vaccine can be tested in the same experiment and the procedure also eliminates the use of guinea pigs required in the lethal challenge/conventional tests. The data obtained while testing tetanus component by the conventional antibody induction (IP) method in guinea pigs suggests that minimum requirements laid down in i.p. is too low which may be fixed as at least 3 out of 9 guinea pig sera and should contain > or = 4 units of tetanus antitoxin per ml.

  16. The role of bacteriocins as selfish genetic elements.

    Science.gov (United States)

    Inglis, R Fredrik; Bayramoglu, Bihter; Gillor, Osnat; Ackermann, Martin

    2013-06-23

    Bacteria produce a wide arsenal of toxic compounds in order to kill competing species. Bacteriocins, protein-based toxins produced by nearly all bacteria, have generally been considered a ubiquitous anti-competitor strategy, used to kill competing bacterial strains. Some of these bacteriocins are encoded on plasmids, which also code for closely linked immunity compounds (thereby rendering toxin producing cells immune to their own toxin). However, the production of bacteriocins can also be interpreted as a means to promote plasmid stability by preferentially selecting for cells carrying the plasmid. If, for example, a cell were to lose the plasmid, it would no longer produce the immunity compound and would be killed by its bacteriocin-producing clone mates. In this respect, bacteriocins can be regarded as similar to previously described toxin-antitoxin systems that are able promote the stable transmission of plasmids to daughter cells. In order to test this prediction, we carried out an experimental evolution study using the bacterium Escherichia coli, finding that bacteriocins can indeed select for the stable maintenance of plasmids. This suggests that bacteriocins can act primarily as selfish genetic elements promoting their own transmission in the population, which may help explain their unique ecology and evolution.

  17. The highly conserved MraZ protein is a transcriptional regulator in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Eraso, Jesus M.; Markillie, Lye Meng; Mitchell, Hugh D.; Taylor, Ronald C.; Orr, Galya; Margolin, William

    2014-05-05

    The mraZ and mraW genes are highly conserved in bacteria, both in sequence and location at the head of the division and cell wall (dcw) gene cluster. Although MraZ has structural similarity to the AbrB transition state regulator and the MazE antitoxin, and MraW is known to methylate ribosomal RNA, mraZ and mraW null mutants have no detectable growth phenotype in any species tested to date, hampering progress in understanding their physiological role. Here we show that overproduction of Escherichia coli MraZ perturbs cell division and the cell envelope, is more lethal at high levels or in minimal growth medium, and that MraW antagonizes these effects. MraZGFP localizes to the nucleoid, suggesting that it binds DNA. Indeed, purified MraZ directly binds a region upstream from its own promoter containing three direct repeats to regulate its own expression and that of downstream cell division and cell wall genes. MraZ-LacZ fusions are repressed by excess MraZ but not when DNA binding by MraZ is inhibited. RNAseq analysis indicates that MraZ is a global transcriptional regulator with numerous targets in addition to dcw genes. One of these targets, mioC, is directly bound by MraZ in a region with three direct repeats.

  18. [Management of severe invasive group A streptococcal infections].

    Science.gov (United States)

    Faye, A; Lorrot, M; Bidet, Ph; Bonacorsi, S; Cohen, R

    2014-11-01

    The group A streptococcus (GAS) is the 5(th) responsible pathogen of invasive infections in children in France. These particularly severe diseases are dominated in children by soft tissue infection, isolated bacteremia but also osteoarthritis. Other complications are rare in France such as lung infections, necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). More unusual localizations such as meningitis, neonatal infections, severe ear and throat and gastrointestinal infections and vascular disorders are also described. Based on published series, mortality ranging from 0-8 % of cases, is high but still lower than that observed in adults. Probabilistic antibiotherapy includes a β-lactam with anti-SGA but also anti-staphylococcal (predominantly methi-S in France) activity such as clavulanic acid- amoxicillin followed by amoxicillin as soon as identification of SGA is performed. The addition of an anti-toxin antibiotic such as clindamycin is recommended particularly in NF or STSS or clinical signs suggestive of toxin production by the SGA (rash, gastrointestinal signs, hemodynamic disorders). The use of intravenous polyvalent immunoglobulins must also be discussed in NF and STSS. In all cases surgery should be discussed. The prognosis of these potentially very severe infections is related to their early diagnosis and treatment. A better understanding of the pathophysiology of these infections may optimize their management but also their prevention.

  19. Biotechnological Trends in Spider and Scorpion Antivenom Development

    Science.gov (United States)

    Laustsen, Andreas Hougaard; Solà, Mireia; Jappe, Emma Christine; Oscoz, Saioa; Lauridsen, Line Præst; Engmark, Mikael

    2016-01-01

    Spiders and scorpions are notorious for their fearful dispositions and their ability to inject venom into prey and predators, causing symptoms such as necrosis, paralysis, and excruciating pain. Information on venom composition and the toxins present in these species is growing due to an interest in using bioactive toxins from spiders and scorpions for drug discovery purposes and for solving crystal structures of membrane-embedded receptors. Additionally, the identification and isolation of a myriad of spider and scorpion toxins has allowed research within next generation antivenoms to progress at an increasingly faster pace. In this review, the current knowledge of spider and scorpion venoms is presented, followed by a discussion of all published biotechnological efforts within development of spider and scorpion antitoxins based on small molecules, antibodies and fragments thereof, and next generation immunization strategies. The increasing number of discovery and development efforts within this field may point towards an upcoming transition from serum-based antivenoms towards therapeutic solutions based on modern biotechnology. PMID:27455327

  20. Biotechnological Trends in Spider and Scorpion Antivenom Development

    Directory of Open Access Journals (Sweden)

    Andreas Hougaard Laustsen

    2016-07-01

    Full Text Available Spiders and scorpions are notorious for their fearful dispositions and their ability to inject venom into prey and predators, causing symptoms such as necrosis, paralysis, and excruciating pain. Information on venom composition and the toxins present in these species is growing due to an interest in using bioactive toxins from spiders and scorpions for drug discovery purposes and for solving crystal structures of membrane-embedded receptors. Additionally, the identification and isolation of a myriad of spider and scorpion toxins has allowed research within next generation antivenoms to progress at an increasingly faster pace. In this review, the current knowledge of spider and scorpion venoms is presented, followed by a discussion of all published biotechnological efforts within development of spider and scorpion antitoxins based on small molecules, antibodies and fragments thereof, and next generation immunization strategies. The increasing number of discovery and development efforts within this field may point towards an upcoming transition from serum-based antivenoms towards therapeutic solutions based on modern biotechnology.

  1. The role of DNA restriction-modification systems in the biology of Bacillus anthracis

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    Ramakrishnan eSitaraman

    2016-01-01

    Full Text Available Restriction-modification (R-M systems are widespread among prokaryotes and, depending on their type, may be viewed as selfish genetic elements that persist as toxin-antitoxin modules or as cellular defense systems against phage infection. Studies in the last decade have made it amply clear that these two options do not exhaust the list of possible biological roles for R-M systems. Their presence in a cell may also have a bearing on other processes such as horizontal gene transfer and gene regulation. From genome sequencing and experimental data, we know that Bacillus anthracis encodes at least three methylation-dependent (typeIV restriction endonucleases, and an orphan DNA methyltransferase. In this article, we first present an outline of our current knowledge of R-M systems in Bacillus anthracis. Based on available DNA sequence data, and on our current understanding of the functions of similar genes in other systems, we conclude with hypotheses on the possible roles of the three restriction endonucleases and the orphan DNA methyltransferase.

  2. Bacterial Programmed Cell Death Mediated by mazEF System Under Stressful Conditions%mazEF系统介导胁迫诱导细菌细胞程序性死亡

    Institute of Scientific and Technical Information of China (English)

    庄强; 宁德刚

    2009-01-01

    mazEF是细菌染色体上的"毒素-抗毒素系统"基因(Toxin-antitoxin system,TA系统),可介导胁迫诱导细菌细胞程序性死亡.本文介绍了mazEF系统的遗传结构特征、生理生化功能、环境胁迫激活mazEF系统介导的细菌细胞程序性死亡的机制,参与细胞死亡过程中的细胞信号和细胞因子的调控,以及关于mazEF系统介导的细菌细胞程序性死亡理论的争论,提出了进一步丰富和完善细菌细胞程序性死亡理论亟待解决的问题.

  3. The Mechanisms of Virulence Regulation by Small Noncoding RNAs in Low GC Gram-Positive Pathogens

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    Stephanie Pitman

    2015-12-01

    Full Text Available The discovery of small noncoding regulatory RNAs (sRNAs in bacteria has grown tremendously recently, giving new insights into gene regulation. The implementation of computational analysis and RNA sequencing has provided new tools to discover and analyze potential sRNAs. Small regulatory RNAs that act by base-pairing to target mRNAs have been found to be ubiquitous and are the most abundant class of post-transcriptional regulators in bacteria. The majority of sRNA studies has been limited to E. coli and other gram-negative bacteria. However, examples of sRNAs in gram-positive bacteria are still plentiful although the detailed gene regulation mechanisms behind them are not as well understood. Strict virulence control is critical for a pathogen’s survival and many sRNAs have been found to be involved in that process. This review outlines the targets and currently known mechanisms of trans-acting sRNAs involved in virulence regulation in various gram-positive pathogens. In addition, their shared characteristics such as CU interaction motifs, the role of Hfq, and involvement in two-component regulators, riboswitches, quorum sensing, or toxin/antitoxin systems are described.

  4. Starvation, together with the SOS response, mediates high biofilm-specific tolerance to the fluoroquinolone ofloxacin.

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    Steve P Bernier

    Full Text Available High levels of antibiotic tolerance are a hallmark of bacterial biofilms. In contrast to well-characterized inherited antibiotic resistance, molecular mechanisms leading to reversible and transient antibiotic tolerance displayed by biofilm bacteria are still poorly understood. The physiological heterogeneity of biofilms influences the formation of transient specialized subpopulations that may be more tolerant to antibiotics. In this study, we used random transposon mutagenesis to identify biofilm-specific tolerant mutants normally exhibited by subpopulations located in specialized niches of heterogeneous biofilms. Using Escherichia coli as a model organism, we demonstrated, through identification of amino acid auxotroph mutants, that starved biofilms exhibited significantly greater tolerance towards fluoroquinolone ofloxacin than their planktonic counterparts. We demonstrated that the biofilm-associated tolerance to ofloxacin was fully dependent on a functional SOS response upon starvation to both amino acids and carbon source and partially dependent on the stringent response upon leucine starvation. However, the biofilm-specific ofloxacin increased tolerance did not involve any of the SOS-induced toxin-antitoxin systems previously associated with formation of highly tolerant persisters. We further demonstrated that ofloxacin tolerance was induced as a function of biofilm age, which was dependent on the SOS response. Our results therefore show that the SOS stress response induced in heterogeneous and nutrient-deprived biofilm microenvironments is a molecular mechanism leading to biofilm-specific high tolerance to the fluoroquinolone ofloxacin.

  5. Comparative Genomics of Carriage and Disease Isolates of Streptococcus pneumoniae Serotype 22F Reveals Lineage-Specific Divergence and Niche Adaptation.

    Science.gov (United States)

    Cleary, David W; Devine, Vanessa T; Jefferies, Johanna M C; Webb, Jeremy S; Bentley, Stephen D; Gladstone, Rebecca A; Faust, Saul N; Clarke, Stuart C

    2016-04-21

    Streptococcus pneumoniae is a major cause of meningitis, sepsis, and pneumonia worldwide. Pneumococcal conjugate vaccines have been part of the United Kingdom's childhood immunization program since 2006 and have significantly reduced the incidence of disease due to vaccine efficacy in reducing carriage in the population. Here we isolated two clones of 22F (an emerging serotype of clinical concern, multilocus sequence types 433 and 698) and conducted comparative genomic analysis on four isolates, paired by Sequence Type (ST) with one of each pair being derived from carriage and the other disease (sepsis). The most compelling observation was of nonsynonymous mutations in pgdA, encoding peptidoglycan N-acetylglucosamine deacetylase A, which was found in the carriage isolates of both ST433 and 698. Deacetylation of pneumococcal peptidoglycan is known to enable resistance to lysozyme upon invasion. Althought no other clear genotypic signatures related to disease or carriage could be determined, additional intriguing comparisons between the two STs were possible. These include the presence of an intact prophage, in addition to numerous additional phage insertions, within the carriage isolate of ST433. Contrasting gene repertoires related to virulence and colonization, including bacteriocins, lantibiotics, and toxin--antitoxin systems, were also observed.

  6. Induction of neutralizing antibodies in mice immunized with scorpion toxins detoxified by liposomal entrapment

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    S.G. Fonseca

    1997-07-01

    Full Text Available The possibility of producing neutralizing antibodies against the lethal effects of scorpion toxins was evaluated in the mouse model by immunization with an immunogen devoid of toxicity. A toxic fraction (5 mg from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes. The liposomes were treated for 1 h at 37oC with a 1% (w/w trypsin solution in 0.2 M sodium carbonate buffer, pH 8.3. This treatment led to a strong reduction in venom toxicity. Immunization was performed as follows: mice were injected sc with 20 µg of the liposome-entrapped toxic fraction on days 1 and 21 and a final injection (20 µg was administered ip on day 36. After injection of the immunogen, all mice developed an IgG response which was shown to be specific for the toxic antigen. The antibodies were measured 10 days after the end of the immunization protocol. In an in vitro neutralization assay we observed that pre-incubation of a lethal dose of the toxic fraction with immune serum strongly reduced its toxicity. In vivo protection assays showed that mice with anti-toxin antibodies could resist the challenge with the toxic fraction, which killed, 30 min after injection, all non-immune control mice

  7. 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.

  8. A case of treating tetanus by Professor FENG Wu-jin%冯五金教授治破伤风验案一则

    Institute of Scientific and Technical Information of China (English)

    王艳蓉

    2014-01-01

    Tetanus is an acute specific infectious diseases generated by clostridium tetani invading the cuts of our bodies. Antibacterial, tetanus antitoxin(TAT) and immune globulin are usually used for tetanus. TCM has its own procedures and therapies for tetanus. The thesis will share famous doctor’s experience in a case of treating tetanus with Wuhu Zhuifeng San.%破伤风是一种急性特异性感染性疾病,是由破伤风杆菌经创口入侵人体后引起的。临床常用抗菌药物、破伤风抗毒素与免疫球蛋白预防和治疗。祖国医学对其有一套有别于其他证候的诊疗程序和理论。本文借导师冯五金教授运用五虎追风散治疗破伤风的一则验案,与大家分享名医经验。

  9. 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.

  10. Investigations into an Outbreak of Botulism Caused by Clostridium botulinum Type C/D in Laying Hens.

    Science.gov (United States)

    Skarin, Hanna; Lindgren, Ylva; Jansson, Désirée S

    2015-06-01

    This case report describes a recent botulism outbreak in commercial laying hens with a history of increased mortality and flaccid paralysis. Routine diagnostic gross examination and microscopy from seven hens were inconclusive, but botulinum neurotoxin (BoNT) in peripheral blood was neutralized with both type C and type D antitoxins in the mouse bioassay. During a farm visit, 10 additional hens from a 34-wk-old flock on the farm were selected for clinical examination and further sampling. Nine hens were observed in sternal recumbency, with flaccid paralysis of the neck, drooping wings and tail, inability to escape, and bilateral ptosis, and one hen showed nonspecific clinical signs. Samples from cecum and liver were collected, and the gene coding for BoNT was detected by PCR in all 10 cecal samples and in four of the liver samples. Clostridium botulinum mosaic type C/D was isolated from 5 out of 10 hens from either cecum or liver, and the isolates were subjected to pulsed-field gel electrophoresis subtyping. All five isolates produced the same banding pattern, which was identical or showed >90% similarity to isolates from three different outbreaks on broiler farms in Sweden and Denmark during the 2007-10 period. However, they were clearly distinguishable from the predominantly reported pulsotype associated with avian botulism outbreaks in Europe. The authors are unaware of any previous report of C. botulinum mosaic type C/D isolates from laying hens.

  11. Hepatitis and Jaundice Associated with Immunization against Certain Virus Diseases: (Section of Comparative Medicine).

    Science.gov (United States)

    Findlay, G M; Maccallum, F O

    1938-05-01

    (1) Among 3,100 persons immunized against yellow fever with virus and immune serum over a period of five years, 89 cases of jaundice have been traced.(2) The symptoms are those of a hepatitis and closely resemble those produced by common infective hepatic jaundice, cases of which have frequently been noted as occurring in the same areas.(3) The average period between the time of inoculation and the development of hepatitis is between two and three months.(4) Attention is directed to the occurrence of hepatitis in horses, usually two to three months after immunization against the viruses of horse sickness and equine encephalomyelitis, and also after the injection of horse serum containing antitoxins against Cl. welchii toxins. Similar symptoms were observed, though to a lesser extent, in normal horses.(5) The only factor common to the inoculated horses and men was the injection of homologous proteins, either in sera or in tissue extracts.(6) The only theories which at present explain the observed facts are that either (1) a hepatotoxic virus is introduced with the virus inoculum or that (2) two factors combine to induce the hepatitis (a) a hepatotoxic substance present in the homologous sera or tissue extract injected and (b) an infective agent which, in the case at least of human beings, is probably the causal agent of common infective hepatic jaundice.

  12. 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.

  13. Insights into dynamics of mobile genetic elements in hyperthermophilic environments from five new Thermococcus plasmids.

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    Mart Krupovic

    Full Text Available Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1, with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.

  14. 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

  15. Single-Cell Analysis of the Plasmablast Response to Vibrio cholerae Demonstrates Expansion of Cross-Reactive Memory B Cells

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    Robert C. Kauffman

    2016-12-01

    Full Text Available We characterized the acute B cell response in adults with cholera by analyzing the repertoire, specificity, and functional characteristics of 138 monoclonal antibodies (MAbs generated from single-cell-sorted plasmablasts. We found that the cholera-induced responses were characterized by high levels of somatic hypermutation and large clonal expansions. A majority of the expansions targeted cholera toxin (CT or lipopolysaccharide (LPS. Using a novel proteomics approach, we were able to identify sialidase as another major antigen targeted by the antibody response to Vibrio cholerae infection. Antitoxin MAbs targeted both the A and B subunits, and most were also potent neutralizers of enterotoxigenic Escherichia coli heat-labile toxin. LPS-specific MAbs uniformly targeted the O-specific polysaccharide, with no detectable responses to either the core or the lipid moiety of LPS. Interestingly, the LPS-specific antibodies varied widely in serotype specificity and functional characteristics. One participant infected with the Ogawa serotype produced highly mutated LPS-specific antibodies that preferentially bound the previously circulating Inaba serotype. This demonstrates durable memory against a polysaccharide antigen presented at the mucosal surface and provides a mechanism for the long-term, partial heterotypic immunity seen following cholera.

  16. Polyphosphate, cyclic AMP, guanosine tetraphosphate, and c-di-GMP reduce in vitro Lon activity

    Science.gov (United States)

    Osbourne, Devon O; Soo, Valerie WC; Konieczny, Igor; Wood, Thomas K

    2014-01-01

    Lon protease is conserved from bacteria to humans and regulates cellular processes by degrading different classes of proteins including antitoxins, transcriptional activators, unfolded proteins, and free ribosomal proteins. Since we found that Lon has several putative cyclic diguanylate (c-di-GMP) binding sites and since Lon binds polyphosphate (polyP) and lipid polysaccharide, we hypothesized that Lon has an affinity for phosphate-based molecules that might regulate its activity. Hence we tested the effect of polyP, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), guanosine tetraphosphate (ppGpp), c-di-GMP, and GMP on the ability of Lon to degrade α-casein. Inhibition of in vitro Lon activity occurred for polyP, cAMP, ppGpp, and c-di-GMP. We also demonstrated by HPLC that Lon is able to bind c-di-GMP. Therefore, four cell signals were found to regulate the activity of Lon protease. PMID:24874800

  17. 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.

  18. Characterization of the Lactobacillus plantarum plasmid pCD033 and generation of the plasmid free strain L. plantarum 3NSH.

    Science.gov (United States)

    Heiss, Silvia; Grabherr, Reingard; Heinl, Stefan

    2015-09-01

    Lactobacillus plantarum CD033, a strain isolated from grass silage in Austria, harbors a 7.9 kb plasmid designated pCD033. Sequence analysis identified 14 open reading frames and 8 of these were supposed to be putative coding sequences. Gene annotation revealed no putative essential genes being plasmid encoded, but a plasmid addiction system based on a PemI/PemK-like toxin-antitoxin system, able to stabilize plasmid maintenance. Absence of a replication initiation protein, a double strand origin as well as a single strand origin on plasmid pCD033 suggests replication via a new type of theta mechanism, whereby plasmid replication is potentially initiated and regulated by non-coding RNA. Detailed examination of segregational stability of plasmid vectors consisting of pCD033-fragments, combined with a selection marker, resulted in definition of a stably maintained minimal replicon. A gene encoding a RepB/OrfX-like protein was found to be not essential for plasmid replication. Alignment of the amino acid sequence of this protein with related proteins unveiled a highly conserved amino acid motif (LLDQQQ). L. plantarum CD033 was cured of pCD033 resulting in the novel plasmid free strain L. plantarum 3NSH. Plasmid curing demonstrated that no essential features are provided by pCD033 under laboratory conditions.

  19. Metabolic aspects of bacterial persisters

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    Marcel ePrax

    2014-10-01

    Full Text Available Persister cells form a multi-drug tolerant subpopulation within an isogenic culture of bacteria that are genetically susceptible to antibiotics. Studies with different Gram negative and Gram positive bacteria have identified a large number of genes associated with the persister state. In contrast, the revelation of persister metabolism has only been addressed recently. We here summarize metabolic aspects of persisters, which includes an overview about the bifunctional role of selected carbohydrates as both triggers for the exit from the drug tolerant state and metabolites which persisters feed on. Also alarmones as indicators for starvation have been shown to influence persister levels via different signaling cascades involving the activation of toxin-antitoxin systems and other regulatory factors. Finally, recent data obtained by 13C-isotopologue profiling demonstrated an active amino acid anabolism in Staphylococcus aureus cultures challenged with high drug concentrations. Understanding the metabolism of persister cells poses challenges but also paves the way for the development of anti-persister compounds.

  20. Persistence Increases in the Absence of the Alarmone Guanosine Tetraphosphate by Reducing Cell Growth.

    Science.gov (United States)

    Chowdhury, Nityananda; Kwan, Brian W; Wood, Thomas K

    2016-02-03

    Most bacterial cells are stressed, and as a result, some become tolerant to antibiotics by entering a dormant state known as persistence. The key intracellular metabolite that has been linked to this persister state is guanosine tetraphosphate (ppGpp), the alarmone that was first linked to nutrient stress. In Escherichia coli, ppGpp redirects protein production during nutrient stress by interacting with RNA polymerase directly and by inhibiting several proteins. Consistently, increased levels of ppGpp lead to increased persistence; but, the mechanism by which elevated ppGpp translates into persistence has not been determined. Hence, we explored persistence in the absence of ppGpp so that the underlying mechanism of persister cell formation could be explored. We found that persister cells still form, although at lower levels, in the absence of ppGpp. Additionally, the toxin/antitoxin systems that we investigated (MqsR, MazF, GhoT, and YafQ) remain able to increase persistence dramatically in the absence of ppGpp. By overproducing each E. coli protein from the 4287 plasmid vectors of the ASKA library and selecting for increased persistence in the absence of ppGpp (via a relA spoT mutant), we identified five new proteins, YihS, PntA, YqjE, FocA, and Zur, that increase persistence simply by reducing cell growth.

  1. Polyphosphate, cyclic AMP, guanosine tetraphosphate, and c-di-GMP reduce in vitro Lon activity.

    Science.gov (United States)

    Osbourne, Devon O; Soo, Valerie W C; Konieczny, Igor; Wood, Thomas K

    2014-01-01

    Lon protease is conserved from bacteria to humans and regulates cellular processes by degrading different classes of proteins including antitoxins, transcriptional activators, unfolded proteins, and free ribosomal proteins. Since we found that Lon has several putative cyclic diguanylate (c-di-GMP) binding sites and since Lon binds polyphosphate (polyP) and lipid polysaccharide, we hypothesized that Lon has an affinity for phosphate-based molecules that might regulate its activity. Hence we tested the effect of polyP, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), guanosine tetraphosphate (ppGpp), c-di-GMP, and GMP on the ability of Lon to degrade α-casein. Inhibition of in vitro Lon activity occurred for polyP, cAMP, ppGpp, and c-di-GMP. We also demonstrated by HPLC that Lon is able to bind c-di-GMP. Therefore, four cell signals were found to regulate the activity of Lon protease.

  2. Clinical features of botulism%肉毒杆菌中毒的临床特点

    Institute of Scientific and Technical Information of China (English)

    买买提依明; 马卫英; 热西旦; 颜静; 杨宛霖; 傅毅; 陈生弟

    2013-01-01

    Objective To explore the clinical features of botulism.Methods The clinical data of 5 patients with botulism were retrospectively analyzed.Results All the five cases were onset due to take homemade stinky tofu,and among them three cases were in a collectivity food poisoning.The mean latency was 3.4 days.Initial symptoms were slurred speech 3 cases,dysphagia 3 cases,dizziness 3 cases,fatigue and diarrhea 2 cases respectively,while dyspnea and poor appetite 1 case.As the disease progressed,all 5 cases experienced dizziness and blurred vision,and occurred ptosis,ophthalmoplegia and generalized weakness 4 cases,chest distress,dyspnea and diplopia 3 cases.Botulinum antitoxins A and B were given after confirmation of the diagnosis,symptomatic therapies including oxygen inhalation,anti-infection,maintenance of hydroelectrolytic equilibrium etc.were applied as well.Finally,4 cases were significantly improved,however,two of them remained mild dysphagia,and the rest 1 case was no effective (the treatment was late).Conclusions The symptoms of nervous system damage are occurred in patients with botulism.Dizziness,cerebral nerves and muscle palsy are its main manifestations.Early application of botulinum antitoxins treatment is effectual.%目的 探讨肉毒杆菌中毒的临床特点.方法 回顾性分析5例肉毒杆菌中毒患者的临床资料.结果 本组5例患者均因食用自制臭豆腐发病,其中3例为群体发病.发病潜伏期平均3.4d.首发症状分别为言语不清3例、吞咽困难3例、头晕3例、无力和腹泻2例,呼吸困难1例;随着病程进展,5例患者均出现头晕、视物模糊,出现眼睑下垂、睁眼困难、全身无力和肌力下降4例,胸闷、呼吸困难和复视3例.给予A型和B型肉毒抗毒素治疗,同时给予吸氧、抗感染和维持水电解质等对症治疗.经治疗病情好转4例,其中2例遗留轻度吞咽困难,无效1例(治疗较晚).结论 肉毒杆菌中毒多出现神经系统损害的症状,

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

    Directory of Open Access Journals (Sweden)

    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

  4. Review of the Inhibition of Biological Activities of Food-Related Selected Toxins by Natural Compounds

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    Reuven Rasooly

    2013-04-01

    Full Text Available There is a need to develop food-compatible conditions to alter the structures of fungal, bacterial, and plant toxins, thus transforming toxins to nontoxic molecules. The term ‘chemical genetics’ has been used to describe this approach. This overview attempts to survey and consolidate the widely scattered literature on the inhibition by natural compounds and plant extracts of the biological (toxicological activity of the following food-related toxins: aflatoxin B1, fumonisins, and ochratoxin A produced by fungi; cholera toxin produced by Vibrio cholerae bacteria; Shiga toxins produced by E. coli bacteria; staphylococcal enterotoxins produced by Staphylococcus aureus bacteria; ricin produced by seeds of the castor plant Ricinus communis; and the glycoalkaloid α-chaconine synthesized in potato tubers and leaves. The reduction of biological activity has been achieved by one or more of the following approaches: inhibition of the release of the toxin into the environment, especially food; an alteration of the structural integrity of the toxin molecules; changes in the optimum microenvironment, especially pH, for toxin activity; and protection against adverse effects of the toxins in cells, animals, and humans (chemoprevention. The results show that food-compatible and safe compounds with anti-toxin properties can be used to reduce the toxic potential of these toxins. Practical applications and research needs are suggested that may further facilitate reducing the toxic burden of the diet. Researchers are challenged to (a apply the available methods without adversely affecting the nutritional quality, safety, and sensory attributes of animal feed and human food and (b educate food producers and processors and the public about available approaches to mitigating the undesirable effects of natural toxins that may present in the diet.

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

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    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.

  6. Extraction and inhibition of enzymatic activity of botulinum neurotoxins/A1, /A2, and /A3 by a panel of monoclonal anti-BoNT/A antibodies.

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    Suzanne R Kalb

    Full Text Available Botulinum neurotoxins (BoNTs are extremely potent toxins that are capable of causing death or respiratory failure leading to long-term intensive care. Treatment includes serotype-specific antitoxins, which must be administered early in the course of the intoxication. Rapidly determining human exposure to BoNT is an important public health goal. In previous work, our laboratory focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT/A-G serotypes in buffer and BoNT/A, /B, /E, and /F in clinical samples. We have previously reported the effectiveness of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. Because some antibodies inhibit or neutralize the activity of BoNT, the choice of antibody with which to extract the toxin is critical. In this work, we evaluated a panel of 16 anti-BoNT/A monoclonal antibodies (mAbs for their ability to inhibit the in vitro activity of BoNT/A1, /A2, and /A3 complex as well as the recombinant LC of A1. We also evaluated the same antibody panel for the ability to extract BoNT/A1, /A2, and /A3. Among the mAbs, there were significant differences in extraction efficiency, ability to extract BoNT/A subtypes, and inhibitory effect on BoNT catalytic activity. The mAbs binding the C-terminal portion of the BoNT/A heavy chain had optimal properties for use in the Endopep-MS assay.

  7. Transcriptome mapping of pAR060302, a blaCMY-2-positive broad-host-range IncA/C plasmid.

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    Lang, Kevin S; Danzeisen, Jessica L; Xu, Wayne; Johnson, Timothy J

    2012-05-01

    The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among Escherichia coli and Salmonella enterica strains in the United States. These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and a propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions. Using E. coli strain DH5α harboring the prototype IncA/C plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C plasmids under laboratory growth and in the face of selective pressure. The effects of ampicillin, florfenicol, or streptomycin exposure were compared to those on cells left untreated at logarithmic phase using Illumina platform-based RNA sequencing (RNA-Seq). Under growth in Luria-Bertani broth lacking antibiotics, much of the backbone of pAR060302 was transcriptionally inactive, including its putative transfer regions. A few plasmid backbone genes of interest were highly transcribed, including genes of a putative toxin-antitoxin system and an H-NS-like transcriptional regulator. In contrast, numerous genes within the accessory regions of pAR060302 were highly transcribed, including the resistance genes floR, bla(CMY-2), aadA, and aacA. Treatment with ampicillin or streptomycin resulted in no genes being differentially expressed compared to controls lacking antibiotics, suggesting that many of the resistance-associated genes are not differentially expressed due to exposure to these antibiotics. In contrast, florfenicol treatment resulted in the upregulation of floR and numerous chromosomal genes. Overall, the transcriptome mapping of pAR060302 suggests that it mitigates the fitness costs of carrying resistance-associated genes through global regulation with its transcriptional regulators.

  8. Comparative genomics of the IncA/C multidrug resistance plasmid family.

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    Fricke, W Florian; Welch, Timothy J; McDermott, Patrick F; Mammel, Mark K; LeClerc, J Eugene; White, David G; Cebula, Thomas A; Ravel, Jacques

    2009-08-01

    Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.

  9. Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme.

    Science.gov (United States)

    Hancock, Steven J; Phan, Minh-Duy; Peters, Kate M; Forde, Brian M; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Paterson, David L; Walsh, Timothy R; Beatson, Scott A; Schembri, Mark A

    2017-02-01

    Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including blaCMY and blaNDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a blaNDM-1-positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of blaNDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this blaNDM-containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies.

  10. Regulatory RNAs in the Less Studied Streptococcal Species: from Nomenclature to Identification

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    Mohamed Amine Zorgani

    2016-07-01

    Full Text Available 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.

  11. Genomic and functional characterization of the modular broad-host-range RA3 plasmid, the archetype of the IncU group.

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    Kulinska, Anna; Czeredys, Magdalena; Hayes, Finbarr; Jagura-Burdzy, Grazyna

    2008-07-01

    IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and gammaproteobacteria. RA3 contains 50 open reading frames clustered in distinct functional modules. The replication module encompasses the repA and repB genes embedded in long repetitive sequences. RepA, which is homologous to antitoxin proteins from alpha- and gammaproteobacteria, contains a Cro/cI-type DNA-binding domain present in the XRE family of transcriptional regulators. The repA promoter is repressed by RepA and RepB. The minireplicon encompasses repB and the downstream repetitive sequence r1/r2. RepB shows up to 80% similarity to putative replication initiation proteins from environmental plasmids of beta- and gammaproteobacteria, as well as similarity to replication proteins from alphaproteobacteria and Firmicutes. Stable maintenance functions of RA3 are most like those of IncP-1 broad-host-range plasmids and comprise the active partitioning apparatus formed by IncC (ParA) and KorB (ParB), the antirestriction protein KlcA, and accessory stability components KfrA and KfrC. The RA3 origin of transfer was localized experimentally between the maintenance and conjugative-transfer operons. The putative conjugative-transfer module is highly similar in organization and in its products to transfer regions of certain broad-host-range environmental plasmids.

  12. [Food-borne botulism: review of five cases].

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    Cardoso, Teresa; Costa, Manuela; Almeida, H Cristina; Guimarães, Mário

    2004-01-01

    Food-borne botulism is a disease caused by the ingestion of food contaminated with botulinum toxin, often present in smoked meat, canned food and preserved food; it can occur as sporadic case or as an outbreak. In the last decades there has been an increasing incidence of food-borne botulism in Portugal. The authors do a review of five cases of food-borne botulism, three isolated cases and 2 familiar. Four were associated with the ingestion of smoked ham and one of canned tunafish. The incubation period was 48 hours in one patient and 4 days in another, in the remaining patients it was not possible to determine this period. The clinical picture was dominated in all patients by diplopy, dysphagia, dizziness, blurred vision, dry mouth and constipation, and in two patients there were gastrointestinal complains. In one patient the electromyography findings were compatible with pre-synaptic neuromuscular blockage. A toxin type B was found in the serum of one patient and in the food involved in the two familiar cases. All patients experienced complete recovery with only symptomatic treatment. With this article the authors intend to call attention to this diagnosis, which is not rare, but difficult for someone not familiar with its presentation, being of notice that the diagnosis is essentially clinic with a strong epidemiological history, confirmed by typical electromyography findings and by the identification of the toxin involved. In Portugal there is only descriptions of clinical cases associated with the type B and the type E toxins, not being necessary the resource to the antitoxin therapy.

  13. 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

    2016-01-26

    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.

  14. Analysis of virus genomes from glacial environments reveals novel virus groups with unusual host interactions

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    Bellas, Christopher M.; Anesio, Alexandre M.; Barker, Gary

    2015-01-01

    Microbial communities in glacial ecosystems are diverse, active, and subjected to strong viral pressures and infection rates. In this study we analyse putative virus genomes assembled from three dsDNA viromes from cryoconite hole ecosystems of Svalbard and the Greenland Ice Sheet to assess the potential hosts and functional role viruses play in these habitats. We assembled 208 million reads from the virus-size fraction and developed a procedure to select genuine virus scaffolds from cellular contamination. Our curated virus library contained 546 scaffolds up to 230 Kb in length, 54 of which were circular virus consensus genomes. Analysis of virus marker genes revealed a wide range of viruses had been assembled, including bacteriophages, cyanophages, nucleocytoplasmic large DNA viruses and a virophage, with putative hosts identified as Cyanobacteria, Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes, eukaryotic algae and amoebae. Whole genome comparisons revealed the majority of circular genome scaffolds (CGS) formed 12 novel groups, two of which contained multiple phage members with plasmid-like properties, including a group of phage-plasmids possessing plasmid-like partition genes and toxin-antitoxin addiction modules to ensure their replication and a satellite phage-plasmid group. Surprisingly we also assembled a phage that not only encoded plasmid partition genes, but a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas adaptive bacterial immune system. One of the spacers was an exact match for another phage in our virome, indicating that in a novel use of the system, the lysogen was potentially capable of conferring immunity on its bacterial host against other phage. Together these results suggest that highly novel and diverse groups of viruses are present in glacial environments, some of which utilize very unusual life strategies and genes to control their replication and maintain a long-term relationship with their hosts

  15. Rapid detection of foodborne botulism outbreaks facilitated by epidemiological linking of cases: implications for food defense and public health response.

    Science.gov (United States)

    Newkirk, Ryan W; Hedberg, Craig W

    2012-02-01

    The main objective of this study was to develop an understanding of the descriptive epidemiology of foodborne botulism in the context of outbreak detection and food defense. This study used 1993-2008 data from the Centers for Disease Control and Prevention (CDC) Annual Summaries of Notifiable Diseases, 2003-2006 data from the Bacterial Foodborne and Diarrheal Disease National Case Surveillance Annual Reports, and 1993-2008 data from the Annual Listing of Foodborne Disease Outbreaks. Published outbreak investigation reports were identified through a PubMed search of MEDLINE citations for botulism outbreaks. Fifty-eight foodborne botulism outbreaks were reported to CDC between 1993 and 2008. Four hundred sixteen foodborne botulism cases were documented; 205 (49%) were associated with outbreaks. Familial connections and co-hospitalization of initial presenting cases were common in large outbreaks (>5 cases). In these outbreaks, the time from earliest exposure to outbreak recognition varied dramatically (range, 48-216 h). The identification of epidemiologic linkages between foodborne botulism cases is a critical part of diagnostic evaluation and outbreak detection. Investigation of an intentionally contaminated food item with a long shelf life and widespread distribution may be delayed until an astute physician suspects foodborne botulism; suspicion of foodborne botulism occurs more frequently when more than one case is hospitalized concurrently. In an effort to augment national botulism surveillance and antitoxin release systems and to improve food defense and public health preparedness efforts, medical organizations and Homeland Security officials should emphasize the education and training of medical personnel to improve foodborne botulism diagnostic capabilities to recognize single foodborne botulism cases and to look for epidemiologic linkages between suspected cases.

  16. Transition State Charge Stabilization and Acid-Base Catalysis of mRNA Cleavage by the Endoribonuclease RelE.

    Science.gov (United States)

    Dunican, Brian F; Hiller, David A; Strobel, Scott A

    2015-12-01

    The bacterial toxin RelE is a ribosome-dependent endoribonuclease. It is part of a type II toxin-antitoxin system that contributes to antibiotic resistance and biofilm formation. During amino acid starvation, RelE cleaves mRNA in the ribosomal A-site, globally inhibiting protein translation. RelE is structurally similar to microbial RNases that employ general acid-base catalysis to facilitate RNA cleavage. The RelE active site is atypical for acid-base catalysis, in that it is enriched with positively charged residues and lacks the prototypical histidine-glutamate catalytic pair, making the mechanism of mRNA cleavage unclear. In this study, we use a single-turnover kinetic analysis to measure the effect of pH and phosphorothioate substitution on the rate constant for cleavage of mRNA by wild-type RelE and seven active-site mutants. Mutation and thio effects indicate a major role for stabilization of increased negative change in the transition state by arginine 61. The wild-type RelE cleavage rate constant is pH-independent, but the reaction catalyzed by many of the mutants is strongly dependent on pH, suggestive of general acid-base catalysis. pH-rate curves indicate that wild-type RelE operates with the pK(a) of at least one catalytic residue significantly downshifted by the local environment. Mutation of any single active-site residue is sufficient to disrupt this microenvironment and revert the shifted pK(a) back above neutrality. pH-rate curves are consistent with K54 functioning as a general base and R81 as a general acid. The capacity of RelE to effect a large pK(a) shift and facilitate a common catalytic mechanism by uncommon means furthers our understanding of other atypical enzymatic active sites.

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

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    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.

  18. Efficacy and safety of AVP-21D9, an anthrax monoclonal antibody, in animal models and humans.

    Science.gov (United States)

    Malkevich, Nina V; Hopkins, Robert J; Bernton, Edward; Meister, Gabriel T; Vela, Eric M; Atiee, George; Johnson, Virginia; Nabors, Gary S; Aimes, Ronald T; Ionin, Boris; Skiadopoulos, Mario H

    2014-07-01

    Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis. Timely administration of antibiotics approved for the treatment of anthrax disease may prevent associated morbidity and mortality. However, any delay in initiating antimicrobial therapy may result in increased mortality, as inhalational anthrax progresses rapidly to the toxemic phase of disease. An anthrax antitoxin, AVP-21D9, also known as Thravixa (fully human anthrax monoclonal antibody), is being developed as a therapeutic agent against anthrax toxemia. The efficacy of AVP-21D9 in B. anthracis-infected New Zealand White rabbits and in cynomolgus macaques was evaluated, and its safety and pharmacokinetics were assessed in healthy human volunteers. The estimated mean elimination half-life values of AVP-21D9 in surviving anthrax-challenged rabbits and nonhuman primates (NHPs) ranged from approximately 2 to 4 days and 6 to 11 days, respectively. In healthy humans, the mean elimination half-life was in the range of 20 to 27 days. Dose proportionality was observed for the maximum serum concentration (Cmax) of AVP-21D9 and the area under the concentration-time curve (AUC). In therapeutic efficacy animal models, treatment with AVP-21D9 resulted in survival of up to 92% of the rabbits and up to 67% of the macaques. Single infusions of AVP-21D9 were well tolerated in healthy adult volunteers across all doses evaluated, and no serious adverse events were reported. (This study has been registered at ClinicalTrials.gov under registration no. NCT01202695.).

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

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    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.

  20. Comparison of alternative buffers for use with a new live oral cholera vaccine, Peru-15, in outpatient volunteers.

    Science.gov (United States)

    Sack, D A; Shimko, J; Sack, R B; Gomes, J G; MacLeod, K; O'Sullivan, D; Spriggs, D

    1997-06-01

    During development of Peru-15, a new live oral vaccine for cholera, the role of buffer needed to be evaluated. Generally, oral bacterial vaccines are acid labile and need to be administered by using a formulation which protects them from gastric acid. We compared three different buffers for use with Peru-15, including a standard bicarbonate-ascorbic acid buffer, Alka-Seltzer, and a new electrolyte-rice buffer, CeraVacx. Saline served as the control. Thirty-nine healthy adult volunteers received Peru-15 (10(8) CFU) with one of the three buffers or saline in a double-masked study. The volunteers were monitored for symptoms for 7 days after the dose, serum was tested for antibody responses by vibriocidal antibody and immunoglobulin G antitoxin enzyme-linked immunosorbent assays, and stool samples were tested for excretion of the vaccine strain. Side effects were minimal in all groups. All 30 volunteers who took Peru-15 with a buffer showed significant rises in vibriocidal antibody titer. The magnitude of the rises was higher in the CeraVacx group than in the other two buffer groups. Four of nine volunteers who took the vaccine with saline also showed increased titers, but they were lower than those in any of the three buffer groups. Excretion of the vaccine strain was similar in the buffer groups, but excretion was not associated with the magnitude of the vibriocidal responses. Excretion of Peru-15 was not detected in the saline group. We conclude that buffer does amplify the serological response to Peru-15 and that CeraVacx may provide benefits not provided by other buffers.

  1. [Infant botulism in France, 1991-2009].

    Science.gov (United States)

    King, L-A; Popoff, M-R; Mazuet, C; Espié, E; Vaillant, V; de Valk, H

    2010-09-01

    Infant botulism is caused by the ingestion of spores of Clostridium botulinum and affects newborns and infants under 12 months of age. Ingested spores multiply and produce botulinum toxin in the digestive tract, which then induces clinical symptoms. A single French case was described in the literature prior to 1991. We describe the cases of infant botulism identified in France between 1991 and 2009. All clinical suspicions of botulism must be declared in France. Biological confirmation of the disease is provided by the National reference laboratory for anaerobic bacteria and botulism at the Pasteur Institute. During this period, 7 cases of infant botulism were identified, 1 per year from 2004 to 2008 and 2 in 2009. The median age of affected infants was 119 days and all were female. All infants presented with constipation and oculomotor symptoms. All were hospitalized and required mechanical ventilation. The infants recovered from their botulism. The diagnosis of infant botulism was biologically confirmed for all patients. One 4-month-old infant was treated with a single dose of the human-derived botulism antitoxin specific for infant botulism types A and B (BabyBIG®). The infants all had different feeding habits ranging from exclusive breast feeding to a mix of formula feeding and solid food consumption. The consumption of honey, the only documented risk food for this disease, was reported for 3 of the infants. The honey had been placed on the pacifier of 2 infants and directly in the mouth of the 3rd by the mother. Infant botulism, a form of botulism that was previously rarely recognized in France, has been reported more frequently during the last 6 years. This disease remains rare but nonetheless severe. In light of recent epidemiological data, efforts to raise awareness among parents of infants and health professionals on the danger of infant botulism and particularly, its association with honey consumption seems necessary.

  2. Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

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    Guorui Yao

    2017-03-01

    Full Text Available Botulinum neurotoxins (BoNTs, which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A–G, a new mosaic toxin type termed BoNT/HA (aka type FA or H was reported recently. Sequence analyses indicate that the receptor-binding domain (HC of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2 that target the HC. Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-HC at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG and synaptic vesicle glycoprotein 2 (SV2. Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures.

  3. Twentieth century toxinology and antivenom development in Australia.

    Science.gov (United States)

    Winkel, Kenneth D; Mirtschin, Peter; Pearn, John

    2006-12-01

    It was not until the last decade of the 19th century that an experimental approach (led by Bancroft in Queensland and Martin in Sydney and Melbourne) brought a higher plane of scientific objectivity to usher in the modern era of Australian toxinology. This Australia era, 1895-1905, coincided with and in some respects was the result of the new knowledge emerging from Europe and the Americas of the therapeutic effects of antitoxins. The subsequent systematic study of Australian venoms and toxins through to the 1930s and beyond, by Tidswell, Fairley, Ross, Kellaway and Cleland, set the foundation for Australia's leading reputation in venom research. As elsewhere, this development was to revolutionise the medical management of those victims who in the past had died in Australia from our venomous and toxic fauna. Morgan, Graydon, Weiner, Lane and Baxter at the Commonwealth Serum Laboratories emphasised the importance of cooperation between those expert at catching and milking the venomous creatures and those developing the antivenoms. Commercial antivenom manufacture began in Australia in 1930 with the tiger snake antivenom. This was followed by other antivenoms for the other important species (1955: taipan; 1956: brown snake; 1958: death adder; 1959: Papuan black snake; 1961: sea snake; 1962: polyvalent) including the first marine antivenoms in the world (1956: stonefish antivenom; 1970: box jellyfish) culminating, in 1980, with the release of the funnel web spider antivenom. More recent activity has focused on veterinary antivenoms and production of new generation human antivenoms for export (CroFab and ViperaTAB). This paper reviews some of the milestones of Australian toxinology, and antivenom development in particular, during the 20th century.

  4. Production and evaluation of a recombinant chimeric vaccine against clostridium botulinum neurotoxin types C and D.

    Science.gov (United States)

    Gil, Luciana A F; da Cunha, Carlos Eduardo P; Moreira, Gustavo M S G; Salvarani, Felipe M; Assis, Ronnie A; Lobato, Francisco Carlos F; Mendonça, Marcelo; Dellagostin, Odir A; Conceição, Fabricio R

    2013-01-01

    Bovine botulism is a fatal disease that is caused by botulinum neurotoxins (BoNTs) produced by Clostridium botulinum serotypes C and D and that causes great economic losses, with nearly 100% lethality during outbreaks. It has also been considered a potential source of human food-borne illness in many countries. Vaccination has been reported to be the most effective way to control bovine botulism. However, the commercially available toxoid-based vaccines are difficult and hazardous to produce. Neutralizing antibodies targeted against the C-terminal fragment of the BoNT heavy chain (HC) are known to confer efficient protection against lethal doses of BoNTs. In this study, a novel recombinant chimera, consisting of Escherichia coli heat-labile enterotoxin B subunit (LTB), a strong adjuvant of the humoral immune response, fused to the HC of BoNT serotypes C and D, was produced in E. coli. Mice vaccinated with the chimera containing LTB and an equivalent molar ratio of the chimera without LTB plus aluminum hydroxide (Al(OH)3) developed 2 IU/mL of antitoxins for both serotypes. Guinea pigs immunized with the recombinant chimera with LTB plus Al(OH)3 developed a protective immune response against both BoNT/C (5 IU/mL) and BoNT/D (10 IU/mL), as determined by a mouse neutralization bioassay with pooled sera. The results achieved with guinea pig sera fulfilled the requirements of commercial vaccines for prevention of botulism, as determined by the Brazilian Ministry of Agriculture, Livestock and Food, Supply. The presence of LTB was essential for the development of a strong humoral immune response, as it acted in synergism with Al(OH)3. Thus, the vaccine described in this study is a strong candidate for the control of botulism in cattle.

  5. Production and evaluation of a recombinant chimeric vaccine against clostridium botulinum neurotoxin types C and D.

    Directory of Open Access Journals (Sweden)

    Luciana A F Gil

    Full Text Available Bovine botulism is a fatal disease that is caused by botulinum neurotoxins (BoNTs produced by Clostridium botulinum serotypes C and D and that causes great economic losses, with nearly 100% lethality during outbreaks. It has also been considered a potential source of human food-borne illness in many countries. Vaccination has been reported to be the most effective way to control bovine botulism. However, the commercially available toxoid-based vaccines are difficult and hazardous to produce. Neutralizing antibodies targeted against the C-terminal fragment of the BoNT heavy chain (HC are known to confer efficient protection against lethal doses of BoNTs. In this study, a novel recombinant chimera, consisting of Escherichia coli heat-labile enterotoxin B subunit (LTB, a strong adjuvant of the humoral immune response, fused to the HC of BoNT serotypes C and D, was produced in E. coli. Mice vaccinated with the chimera containing LTB and an equivalent molar ratio of the chimera without LTB plus aluminum hydroxide (Al(OH3 developed 2 IU/mL of antitoxins for both serotypes. Guinea pigs immunized with the recombinant chimera with LTB plus Al(OH3 developed a protective immune response against both BoNT/C (5 IU/mL and BoNT/D (10 IU/mL, as determined by a mouse neutralization bioassay with pooled sera. The results achieved with guinea pig sera fulfilled the requirements of commercial vaccines for prevention of botulism, as determined by the Brazilian Ministry of Agriculture, Livestock and Food, Supply. The presence of LTB was essential for the development of a strong humoral immune response, as it acted in synergism with Al(OH3. Thus, the vaccine described in this study is a strong candidate for the control of botulism in cattle.

  6. Nostocyclopeptide-M1: a potent, nontoxic inhibitor of the hepatocyte drug transporters OATP1B3 and OATP1B1.

    Science.gov (United States)

    Herfindal, Lars; Myhren, Lene; Kleppe, Rune; Krakstad, Camilla; Selheim, Frode; Jokela, Jouni; Sivonen, Kaarina; Døskeland, Stein O

    2011-04-01

    We have isolated a novel cyanobacterial cyclic peptide (nostocyclopeptide M1; Ncp-M1) that blocks the hepatotoxic action of microcystin (MC) and nodularin (Nod). We show here that Ncp-M1 is nontoxic to primary hepatocytes in long-term culture. Ncp-M1 does not affect any known intracellular targets or pathways involved in MC action, like protein phosphatases, CaM-KII, or ROS-dependent cell death effectors. In support of this conclusion Ncp-M1 had no protective effect when microinjected into cells. Rather, the antitoxin effect was solely due to blocked hepatocyte uptake of MC and Nod. The hepatic uptake of MC and Nod is mainly via the closely related organic anion transporters OATP1B1 and OATP1B3, which also mediate hepatic transport of endogenous metabolites and hormones as well as drugs. OATP1B3 is also expressed in some aggressive cancers, where it confers apoptosis resistance. We show that Ncp-M1 inhibits transport through OATP1B3 and OATP1B1 expressed in HEK293 cells. The Ncp-M1 molecule has several nonproteinogenic amino acids and an imino bond, which hamper its synthesis. Moreover, a cyclic all L-amino acid heptapeptide analogue of Ncp-M1 also inhibits the OATP1B1/1B3 transporters, and with higher OATP1B3 preference than Ncp-M1 itself. The nontoxic Ncp-M1 and its synthetic cyclic peptide analogues thus provide new tools to probe the role of OATB1B1/1B3 mediated drug and metabolite transport in liver and cancer cells. They can also serve as scaffolds to design new, exopeptidase resistant OATP1B3-specific modulators.

  7. ``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.

  8. The third replicon of members of the Burkholderia cepacia Complex, plasmid pC3, plays a role in stress tolerance.

    Science.gov (United States)

    Agnoli, Kirsty; Frauenknecht, Carmen; Freitag, Roman; Schwager, Stephan; Jenul, Christian; Vergunst, Annette; Carlier, Aurelien; Eberl, Leo

    2014-02-01

    The metabolically versatile Burkholderia cepacia complex (Bcc) occupies a variety of niches, including the plant rhizosphere and the cystic fibrosis lung (where it is often fatal to the patient). Bcc members have multipartite genomes, of which the third replicon, pC3 (previously chromosome 3), has been shown to be a nonessential megaplasmid which confers virulence and both antifungal and proteolytic activity on several strains. In this study, pC3 curing was extended to cover strains of 16 of the 17 members of the Bcc, and the phenotypes conferred by pC3 were determined. B. cenocepacia strains H111, MCO-3, and HI2424 were previously cured of pC3; however, this had not proved possible in the epidemic strain K56-2. Here, we investigated the mechanism of this unexpected stability and found that efficient toxin-antitoxin systems are responsible for maintaining pC3 of strain K56-2. Identification of these systems allowed neutralization of the toxins and the subsequent deletion of K56-2pC3. The cured strain was found to exhibit reduced antifungal activity and was attenuated in both the zebrafish and the Caenorhabditis elegans model of infection. We used a PCR screening method to examine the prevalence of pC3 within 110 Bcc isolates and found that this replicon was absent in only four cases, suggesting evolutionary fixation. It is shown that plasmid pC3 increases the resistance of B. cenocepacia H111 to various stresses (oxidative, osmotic, high-temperature, and chlorhexidine-induced stresses), explaining the prevalence of this replicon within the Bcc.

  9. Conserved inhibitory mechanism and competent ATP binding mode for adenylyltransferases with Fic fold.

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    Arnaud Goepfert

    Full Text Available The ubiquitous FIC domain is evolutionarily conserved from bacteria to human and has been shown to catalyze AMP transfer onto protein side-chain hydroxyl groups. Recently, it was predicted that most catalytically competent Fic proteins are inhibited by the presence of an inhibitory helix αinh that is provided by a cognate anti-toxin (class I, or is part of the N- or C-terminal part of the Fic protein itself (classes II and III. In vitro, inhibition is relieved by mutation of a conserved glutamate of αinh to glycine. For the class III bacterial Fic protein NmFic from Neisseria meningitidis, the inhibitory mechanism has been elucidated. Here, we extend above study by including bacterial class I and II Fic proteins VbhT from Bartonella schoenbuchensis and SoFic from Shewanella oneidensis, respectively, and the respective E->G mutants. Comparative enzymatic and crystallographic analyses show that, in all three classes, the ATP substrate binds to the wild-type FIC domains, but with the α-phosphate in disparate and non-competent orientations. In the E->G mutants, however, the tri-phosphate moiety is found reorganized to the same tightly bound structure through a unique set of hydrogen bonds with Fic signature motif residues. The γ-phosphate adopts the location that is taken by the inhibitory glutamate in wild-type resulting in an α-phosphate orientation that can be attacked in-line by a target side-chain hydroxyl group. The latter is properly registered to the Fic active center by main-chain β-interactions with the β-hairpin flap. These data indicate that the active site motif and the exposed edge of the flap are both required to form an adenylylation-competent Fic protein.

  10. ON THE PRESENCE OF CERTAIN BODIES IN THE SKIN AND BLISTER FLUID FROM SCARLET-FEVER AND MEASLES.

    Science.gov (United States)

    Field, C W

    1905-07-15

    In sections of control and normal skin, the nuclei of the epithelial cells were often indented by the cell protoplasm, giving them an appearance similar to those indented by Mallory's bodies. It would seem that if these bodies of Mallory's were protozoa they would have been found in the sections from both the living and the dead skin of scarlet-fever and measles, as they were present in the blister fluid. Their absence is certainly more suggestive of a degeneration than of a protozoon. This view is also borne out by the fact that they were not found immediately after death, but were present in another specimen from the same case removed twenty-four hours later. It would seem probable also that the bodies found in the blister fluid were the products of degeneration and cytolytic activity, because they were found in the antitoxin rashes as well as in the cases of scarlet-fever and measles. The histological changes in the skin of these two diseases leads us to expect the presence of cytolytic products both in the blister fluid and in the sections. It certainly cannot be stated that none of these bodies is a protozoön, but it can be positively stated that a great majority of them arise from degenerating cells; and in many cases, I think, it is not possible to differentiate a degeneration from a protozoön by the study of its morphology and staining reactions. The bodies present in blister fluid resemble very closely those granular bodies found in blood under certain conditions, and seen in vaccine lymph and in emulsions of tissues and in exudates. I think, therefore, that they are for the most part, if not wholly, products of degenerating tissue cells and of leucocytes, and within certain limits specific to scarlet-fever and measles.

  11. Staphylococcus aureus Small Colony Variants (SCVs: A Road Map for the Metabolic Pathways Involved in Persistent Infections

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    Richard Allan Proctor

    2014-07-01

    Full Text Available Persistent and relapsing infections, despite apparently adequate antibiotic therapy, occur frequently with many pathogens, but it is an especially prominent problem with Staphylococcus aureus infections. For the purposes of this review, persistence will encompass both of the concepts of long term survival within the host, including colonization, and the concept of resisting antibiotic therapy even when susceptible in the clinical microbiology laboratory. Over the past two decades, the mechanisms whereby bacteria achieve persistence are slowly being unraveled. S. aureus small colony variants (SCVs are linked to chronic, recurrent, and antibiotic-resistant infections, and the study of SCVs has contributed significantly to understanding of persistence. In our earlier work, defects in electron transport and thymidylate biosynthesis were linked to the development of the SCV phenotype (reviewed in 2006, thus this work will be discussed only briefly. Since 2006, it has been found that persistent organisms including SCVs are part of the normal life cycle of bacteria, and often they arise in response to harsh conditions, e.g., antibiotics, starvation, host cationic peptides. Many of the changes found in these early SCVs have provided a map for the discovery mechanisms (pathways for the development of persistent organisms. For example, changes in RNA processing, stringent response, toxin-antitoxin, ribosome protein L6 (RplF, and cold shock protein B (CspB found in SCVs are also found in other persisters. In addition, many classic persister organisms also show slow growth, hence SCVs. Recent work on S. aureus USA300 has elucidated the impact of aerobic expression of arginine deiminase genes on its ability to chronically colonize the skin and survive in abscesses. S. aureus SCVs also express arginine deiminase genes aerobically as well. Thus, many pathways found activated in electron transport type of SCVs are also increased in persisters that have intact

  12. Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity.

    Science.gov (United States)

    Critchley, D R; Streuli, C H; Kellie, S; Ansell, S; Patel, B

    1982-04-15

    Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].

  13. Plasmids of Carotenoid-Producing Paracoccus spp. (Alphaproteobacteria) - Structure, Diversity and Evolution

    Science.gov (United States)

    Maj, Anna; Dziewit, Lukasz; Czarnecki, Jakub; Wlodarczyk, Miroslawa; Baj, Jadwiga; Skrzypczyk, Grazyna; Giersz, Dorota; Bartosik, Dariusz

    2013-01-01

    Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria. PMID:24260361

  14. The Structural Diversity of Carbohydrate Antigens of Selected Gram-Negative Marine Bacteria

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    Elena P. Ivanova

    2011-10-01

    Full Text Available Marine microorganisms have evolved for millions of years to survive in the environments characterized by one or more extreme physical or chemical parameters, e.g., high pressure, low temperature or high salinity. Marine bacteria have the ability to produce a range of biologically active molecules, such as antibiotics, toxins and antitoxins, antitumor and antimicrobial agents, and as a result, they have been a topic of research interest for many years. Among these biologically active molecules, the carbohydrate antigens, lipopolysaccharides (LPSs, O-antigens found in cell walls of Gram-negative marine bacteria, show great potential as candidates in the development of drugs to prevent septic shock due to their low virulence. The structural diversity of LPSs is thought to be a reflection of the ability for these bacteria to adapt to an array of habitats, protecting the cell from being compromised by exposure to harsh environmental stress factors. Over the last few years, the variety of structures of core oligosaccharides and O-specific polysaccharides from LPSs of marine microrganisms has been discovered. In this review, we discuss the most recently encountered structures that have been identified from bacteria belonging to the genera Aeromonas, Alteromonas, Idiomarina, Microbulbifer, Pseudoalteromonas, Plesiomonas and Shewanella of the Gammaproteobacteria phylum; Sulfitobacter and Loktanella of the Alphaproteobactera phylum and to the genera Arenibacter, Cellulophaga, Chryseobacterium, Flavobacterium, Flexibacter of the Cytophaga-Flavobacterium-Bacteroides phylum. Particular attention is paid to the particular chemical features of the LPSs, such as the monosaccharide type, non-sugar substituents and phosphate groups, together with some of the typifying traits of LPSs obtained from marine bacteria. A possible correlation is then made between such features and the environmental adaptations undertaken by marine bacteria.

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

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    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.

  16. 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.

  17. Comparison of seven commercial enzyme-linked immunosorbent assays for the detection of anti-diphtheria toxin antibodies.

    Science.gov (United States)

    Zasada, A A; Rastawicki, W; Śmietańska, K; Rokosz, N; Jagielski, M

    2013-07-01

    Determination of immune status of patients to diphtheria toxin is based mainly on the results of commercially available ELISA kits. The aim of the present study was to compare the results obtained by ELISAs from seven different manufacturers: Mikrogen, Immunolab, Sekisui Virotech, NovaTec, Virion\\Serion, IBL International and Euroimmun. All assays were performed according to the manufacturers' instructions. The concentrations of the anti-diphtheria toxin antibodies in 72 serum samples were calculated on the basis of curves constructed from standards supplied by manufacturers and the new reference material-International Standard for Diphtheria Antitoxin (10/262). The repeatability and reproducibility of all the ELISA kits tested were good. Number of sera with concentrations of the anti-diphtheria toxin antibodies below the WHO-recommended level of protection (0.1 IU/ml) were dependent on the ELISA used: Mikrogen, 20/72 samples (27.7%); Immunolab, 11/72 samples (15.3%); Sekisui Virotech, 0/72 samples (0%); NovaTec 18/72 samples (25.0%); Serion 12/72 samples (16.7%); IBL International, 7/72 samples (9.7 %); and Euroimmun, 17/72 samples (23.6%). However, the results obtained in particular ELISAs, with the exception of Sekisui Virotech, were much more consistent when the concentrations of the anti-diphtheria toxin antibodies in 72 sera measured by using curves constructed from the International Standard 10/262. The data obtained clearly demonstrated that manufacturer-dependent differences between anti-diphtheria IgG ELISA kits exist. The differences in recommendations accepted by the individual manufacturers together with differences shown in our studies in sensitivity greatly affect the clinical interpretation of results.

  18. A Nonsynonymous SNP Catalog of Mycobacterium tuberculosis Virulence Genes and Its Use for Detecting New Potentially Virulent Sublineages

    Science.gov (United States)

    Mikheecheva, Natalya E.; Zaychikova, Marina V.; Melerzanov, Alexander V.

    2017-01-01

    Mycobacterium tuberculosis is divided into several distinct lineages, and various genetic markers such as IS-elements, VNTR, and SNPs are used for lineage identification. We propose an M. tuberculosis classification approach based on functional polymorphisms in virulence genes. An M. tuberculosis virulence genes catalog has been established, including 319 genes from various protein groups, such as proteases, cell wall proteins, fatty acid and lipid metabolism proteins, sigma factors, toxin–antitoxin systems. Another catalog of 1,573 M. tuberculosis isolates of different lineages has been developed. The developed SNP-calling program has identified 3,563 nonsynonymous SNPs. The constructed SNP-based phylogeny reflected the evolutionary relationship between lineages and detected new sublineages. SNP analysis of sublineage F15/LAM4/KZN revealed four lineage-specific mutations in cyp125, mce3B, vapC25, and vapB34. The Ural lineage has been divided into two geographical clusters based on different SNPs in virulence genes. A new sublineage, B0/N-90, was detected inside the Beijing-B0/W-148 by SNPs in irtB, mce3F and vapC46. We have found 27 members of B0/N-90 among the 227 available genomes of the Beijing-B0/W-148 sublineage. Whole-genome sequencing of strain B9741, isolated from an HIV-positive patient, was demonstrated to belong to the new B0/N-90 group. A primer set for PCR detection of B0/N-90 lineage-specific mutations has been developed. The prospective use of mce3 mutant genes as genetically engineered vaccine is discussed. PMID:28338924

  19. 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.

  20. Case of skin injuries due to stings by crown-of-thorns starfish (Acanthaster planci).

    Science.gov (United States)

    Sato, Hironobu; Tsuruta, Yuichiro; Yamamoto, Yu-ichi; Asato, Yutaka; Taira, Kiyohito; Hagiwara, Keisuke; Kayo, Susumu; Iwanaga, Setsuko; Uezato, Hiroshi

    2008-03-01

    A case of skin injuries due to stings by crown-of-thorns starfish, Acanthaster planci, in a 53-year-old Okinawan woman is reported. She went to a beach to gather shellfish on 8 April 2001 and fell to the ground with her left palm on a crown-of-thorns starfish that happened to be close to her. She hurried to the emergency section of our hospital. An emergency doctor sterilized the wound and administered an antibiotic, an analgesic agent and an injection of a tetanus antitoxin. He tried to remove the remaining spines from the palm with great difficulty. Because swelling and subcutaneous indurations of the left palm had persisted thereafter, oral and topical administration of corticosteroid started on 13 April. Physical examination at the dermatology section revealed approximately 10 stab wounds of the left palm with pus, subcutaneous bleeding and many abrasions around them. X-rays of the left hand showed foreign bodies, 2-10 mm in size, located on the lesions. The patient was treated with a topical injection of 2 mg triamcinolone acetonide (Kenacort-A), diluted fivefold with 1% Xylocaine, once a week. Some of the foreign body granulomatous lesions improved but pain and subcutaneous indurations persisted in most of the lesions. Because the X-ray photographs showed many remaining spines, surgical excision to remove them was performed under local anesthesia 3 months after the injury. All the symptoms improved after the operation. Scanning electron microscopic examination of the spines revealed that their tips had fragile lattice-like structures.

  1. 大肠埃希菌TA系统mazEF的研究进展%Recent advancement in study on mazEF, a TA system of E.coli

    Institute of Scientific and Technical Information of China (English)

    叶露; 韦艳霞

    2012-01-01

    大部分细菌的遗传物质中含有毒素-抗毒素系统(TA)的遗传基因.mazEF是大肠埃希菌染色体上的一对毒素抗毒素基因,由毒素基因mazF和抗毒素基因mazE组成.其在细菌的生长调控和细胞程序性死亡中发挥了重要的作用.环境压力激活mazEF后,MazF可以通过对mRNA的剪切作用造成翻译停止.mazEF的存在可以增加细菌对环境压力的耐受性、保持细菌遗传物质的稳定、参与抗生素引起的细胞死亡、也在细菌的耐药性中发挥重要作用.%Most bacterial genomes harbor toxin-anti toxin (TA) systems, MazEF is a chromosome encoded E. coli TA system consisted of the toxin gene mazF and anti-toxin gene mazE. It plays a significant role in the growth modulation and PCD. MazEF can be triggered by various stresses. MazF can cleave mRNA and then inhibit its transcription and translation. The existence of mazEF can improve the tolerance of environmental stresses, the living ability of the groups, and the stability of the genome. MazEF also plays an important role in cell death caused by antibiotics and the resistance to antibiotics.

  2. 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.

  3. Study on the relationship between sub-cluster statistics parameters and biological effects of thymus protease of certain kinds of live scorpion%若干种活蝎胸腺蛋白酶的群子统计参数与生物功效关系的研究

    Institute of Scientific and Technical Information of China (English)

    周游; 牟雪雁; 李琴; 金日光

    2011-01-01

    分析了若干不同产地蛋白酶中的生命动力源相关元素,并根据第四统计力学群子统计参数理论,证明了其无毒性,且有极高的药用价值.结果表明,太行山脉系的活蝎蛋白酶的阴阳性属为(-)(+)(+)(+)中药类;故具有很高的抗毒及杀菌功能,有强烈的清热解毒功效,兼有很好的免疫功能,临床试验表明确实具有良好的抗癌、抗糖尿病、抗艾滋病及抗中风的功能.%Although there have been many studies of the use of the toxins from scorpions to treat disease, the effect of thymus protease from live scorpions has not been previously reported. In this paper, we have analysed the essential elements in the protease of live scorpions from different habitats, and demonstrated its non-toxic and high medicinal value on the basis of statistical parameters of the Fourth Statistics-JRG Sub-cluster Statistics Theory. The results show that the Yin and Yang of live scorpion protease from Taihang Mountain is ( - ) ( + ) ( + ) ( + ); this thymus protease has particularly high anti-toxin and antibacterial effects, an extremely strong heat clearing-detoxication efficacy and a good effect on the immune function. It has good anti-cancer, anti-diabetes, anti-AIDS and antiapoplexy functions which have been confirmed in clinical trials.

  4. 破伤风感染的救治分析%Clinic Study on Tetanic Infection

    Institute of Scientific and Technical Information of China (English)

    李小石; 吕晶

    2014-01-01

    目的:总结破伤风感染的救治经验,探索降低破伤风患者死亡率的有效方法。方法收集2004年~2013年收治的破伤风病例24例,分析其临床特点、救治经过,总结救治破伤风感染的经验。结果24例破伤风病例中,Ablett分级Ⅰ级9例,Ⅱ级7例,Ⅲ级8例,发病平均潜伏期为11 d,平均住院时间26 d,存活率100%。结论创面彻底反复处理;常规注射破伤风抗毒素(tetanus antitoxin, TAT);对于重症破伤风患者强调早期气管切开及镇静、肌松是破伤风救治的最主要有效措施。%Objective To summarize the therapeutic effects of tetanus. Methods A total of 24 patients with tetanus from 2004 to 2013 were collected, to analyze the clinical characteristics and summarize the experience in the treatment of tetanus infection. Results Among all 24 patients, AblettⅠwere 9 cases,ⅡandⅢwere 7 and 8 cases respectively. The average incubation period was 11 days. The average hospitalization time was 26 days, and the survival rate was 100%. Conclusion Early effective wound treatment, injection of TAT, tracheotomy and composure are the effective measures to treat tetanus, and the early tracheotomy, calming and muscle relaxation in severe tetanus patients.

  5. 成人难治性破伤风的诊断与治疗分析%Diagnosis and treatment of adult patients with refractory tetanus

    Institute of Scientific and Technical Information of China (English)

    段劲峰; 唐宇凤; 张芸

    2013-01-01

    目的 探讨成人难治性破伤风的临床表现与治疗方法.方法 回顾性分析5例成人难治性破伤风患者的病历资料.结果 5例患者综合治疗效果不佳,再次对患者所有伤口进行反复清创,并加用头孢三代抗生素,同时延长破伤风抗毒素使用疗程,4例痊愈,1例死亡.结论 应积极控制成人难治性破伤风患者的肺部感染,反复搜寻患者的感染灶并进行彻底清创,必要时可多次使用破伤风抗毒素进行治疗.%Objective To explore the clinical manifestations and therapeutic methods of adults with refractory tetanus. Methods We retrospectively analyzed medical records of the 5 adult patients with refractory tetanus. Results At the beginning,no satisfactory curative effects were obtained after we adopted a variety of treatment methods,including intravenous injection TIG or TAT,nutrition supporting,ventilator, sedative and muscle loose agent. Afterwards, we performed repeated debridement and added cephalosporins of three generation antibiotics. Meanwhile, we extended use of treatment of tetanus antitoxin. As a result ,4 patients recovered and one case died. Conclusions The doctor should actively control pulmonary infection for adult patients with refractory tetanus, repeatedly search all possible focus of infection and thoroughly debride. It should carry out the multiple use of TAT if it is necessary.

  6. Three classes of plasmid (47-63 kb) carry the type B neurotoxin gene cluster of group II Clostridium botulinum.

    Science.gov (United States)

    Carter, Andrew T; Austin, John W; Weedmark, Kelly A; Corbett, Cindi; Peck, Michael W

    2014-08-01

    Pulsed-field gel electrophoresis and DNA sequence analysis of 26 strains of Group II (nonproteolytic) Clostridium botulinum type B4 showed that 23 strains carried their neurotoxin gene cluster on a 47-63 kb plasmid (three strains lacked any hybridization signal for the neurotoxin gene, presumably having lost their plasmid). Unexpectedly, no neurotoxin genes were found on the chromosome. This apparent constraint on neurotoxin gene transfer to the chromosome stands in marked contrast to Group I C. botulinum, in which neurotoxin gene clusters are routinely found in both locations. The three main classes of type B4 plasmid identified in this study shared different regions of homology, but were unrelated to any Group I or Group III plasmid. An important evolutionary aspect firmly links plasmid class to geographical origin, with one class apparently dominant in marine environments, whereas a second class is dominant in European terrestrial environments. A third class of plasmid is a hybrid between the other two other classes, providing evidence for contact between these seemingly geographically separated populations. Mobility via conjugation has been previously demonstrated for the type B4 plasmid of strain Eklund 17B, and similar genes associated with conjugation are present in all type B4 plasmids now described. A plasmid toxin-antitoxin system pemI gene located close to the neurotoxin gene cluster and conserved in each type B4 plasmid class may be important in understanding the mechanism which regulates this unique and unexpected bias toward plasmid-borne neurotoxin genes in Group II C. botulinum type B4.

  7. 小儿皮下感染局部免疫的临床观察%A Clinical Observation on Local Immunity in Subcutaneous Infection among Children

    Institute of Scientific and Technical Information of China (English)

    张金哲

    1987-01-01

    本文利用大量的临床资料分析了不同年龄组之间,同一年龄组不同患儿之间,同一机体不同部位,以及同一感染的不同阶段对感染的免疫反应的不同表现,从病理和免疫发生等方面提出了新的假说,对临康治疗有一定指导意义.%Based on a 30-year clinical observation on the local pathological changes in the subcutaneous infection among children of different ages, the author raised a hypothesis as the following: 1.. The development of the local immunity to staphylococci is in stages similar to that of ana-phylaxis and desensitization reaction to T.A.T. (tetanus anti-toxin) injection.Different stage likes to appear in different age group. The new-borns may have a very low antigen-antibody reaction, give rise a nypoergic local pathology, like that of the neonatae phlegmon of the back of a newborn .Infants react too extensively to staphylococci, presenting as a wide spreading induration of the neck, anaphylactic or hyperergic reaction, like in the infantile sub-maxillary cellulitis. As the children grown up, they will react normally to a usual-dosage sta-phylococcal contamination forming a localized abscess or showing mild or even no clinical inflammation i.e., desensitization and effective immunity formation.2. The natural course of the development of local, immunity includes: no immunity reaction, hypersensitivity, stable and effective immunity, and gradual fading off of the immunity unless subsequent provocation. 3. Among children of the same age group, different local pathology under different special immunological condition could be observed.

  8. Tn5253 family integrative and conjugative elements carrying mef(I) and catQ determinants in Streptococcus pneumoniae and Streptococcus pyogenes.

    Science.gov (United States)

    Mingoia, Marina; Morici, Eleonora; Morroni, Gianluca; Giovanetti, Eleonora; Del Grosso, Maria; Pantosti, Annalisa; Varaldo, Pietro E

    2014-10-01

    The linkage between the macrolide efflux gene mef(I) and the chloramphenicol inactivation gene catQ was first described in Streptococcus pneumoniae (strain Spn529), where the two genes are located in a module designated IQ element. Subsequently, two different defective IQ elements were detected in Streptococcus pyogenes (strains Spy029 and Spy005). The genetic elements carrying the three IQ elements were characterized, and all were found to be Tn5253 family integrative and conjugative elements (ICEs). The ICE from S. pneumoniae (ICESpn529IQ) was sequenced, whereas the ICEs from S. pyogenes (ICESpy029IQ and ICESpy005IQ, the first Tn5253-like ICEs reported in this species) were characterized by PCR mapping, partial sequencing, and restriction analysis. ICESpn529IQ and ICESpy029IQ were found to share the intSp 23FST81 integrase gene and an identical Tn916 fragment, whereas ICESpy005IQ has int5252 and lacks Tn916. All three ICEs were found to lack the linearized pC194 plasmid that is usually associated with Tn5253-like ICEs, and all displayed a single copy of a toxin-antitoxin operon that is typically contained in the direct repeats flanking the excisable pC194 region when this region is present. Two different insertion sites of the IQ elements were detected, one in ICESpn529IQ and ICESpy029IQ, and another in ICESpy005IQ. The chromosomal integration of the three ICEs was site specific, depending on the integrase (intSp 23FST81 or int5252). Only ICESpy005IQ was excised in circular form and transferred by conjugation. By transformation, mef(I) and catQ were cotransferred at a high frequency from S. pyogenes Spy005 and at very low frequencies from S. pneumoniae Spn529 and S. pyogenes Spy029.

  9. Mobility and generation of mosaic non-autonomous transposons by Tn3-derived inverted-repeat miniature elements (TIMEs.

    Directory of Open Access Journals (Sweden)

    Magdalena Szuplewska

    Full Text Available Functional transposable elements (TEs of several Pseudomonas spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i 13 insertion sequences from 5 IS families (IS3, IS5, ISL3, IS30 and IS1380, (ii isoforms of two Tn3-family transposons--Tn5563a and Tn4662a (the latter contains a toxin-antitoxin system, as well as (iii non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs, highly conserved in Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements (262 bp, were identified within two natural plasmids (pZM1P1 and pLM8P2 of Pseudomonas spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element "captured" with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a res site typical for many Tn3-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and

  10. Screening of bifidobacteria and lactobacilli able to antagonise the cytotoxic effect of Clostridium difficile upon intestinal epithelial HT29 monolayer

    Directory of Open Access Journals (Sweden)

    Lorena eValdés-Varela

    2016-04-01

    toxigenic supernatant. Image analysis 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.

  11. Designing Inhibitors of Anthrax Toxin

    Science.gov (United States)

    Nestorovich, Ekaterina M.; Bezrukov, Sergey M.

    2014-01-01

    with anti-PA therapeutics can be time-dependent, requiring coordinated use of membrane permeable small-molecule inhibitors, which block the LF and EF enzymatic activity intracellularly. The desperate search for an ideal anthrax antitoxin allowed researchers to gain important knowledge of the basic principles of small-molecule interactions with their protein targets that could be easily transferred to other systems. At the same time, better identification and validation of anthrax toxin therapeutic targets at the molecular level, which include understanding of the physical forces underlying the target/drug interaction, as well as elucidation of the parameters determining the corresponding therapeutic windows, require further examination. PMID:24447197

  12. Adding to Yersinia enterocolitica Gene Pool Diversity: Two Cryptic Plasmids from a Biotype 1A Isolate

    Directory of Open Access Journals (Sweden)

    Daniela Lepka

    2009-01-01

    Full Text Available We report the nucleotide sequence of two novel cryptic plasmids (4357 and 14 662 base pairs carried by a Yersinia enterocolitica biotype 1A strain isolated from pork. As distinguished from most biotype 1A strains, this isolate, designated 07-04449, exhibited adherence to eukaryotic cells. The smaller plasmid pYe4449-1 carries five attributable open reading frames (ORFs encoding the first CcdA/CcdB-like antitoxin/toxin system described for a Yersinia plasmid, a RepA-like replication initiation protein, and mobilizing factors MobA and MobC. The deduced amino acid sequences showed highest similarity to proteins described in Salmonella (CcdA/B, Klebsiella (RepA, and Plesiomonas (MobA/C indicating genomic fluidity among members of the Enterobacteriaceae. One additional ORF with unknown function, termed ORF5, was identified with an ancestry distinct from the rest of the plasmid. While the C+G content of ORF5 is 38.3%, the rest of pYe4449-1 shows a C+G content of 55.7%. The C+G content of the larger plasmid pYe4449-2 (54.9% was similar to that of pYe4449-1 (53.7% and differed from that of the Y. enterocolitica genome (47.3%. Of the 14 ORFs identified on pYe4449-2, only six ORFs showed significant similarity to database entries. For three of these ORFs likely functions could be ascribed: a TnpR-like resolvase and a phage replication protein, localized each on a low C+G island, and DNA primase TraC. Two ORFs of pYe4449-2, ORF3 and ORF7, seem to encode secretable proteins. Epitope-tagging of ORF3 revealed protein expression at 4°C but not at or above 27°C suggesting adaptation to a habitat outside swine. The hypothetical protein encoded by ORF7 is the member of a novel repeat protein family sharing the DxxGN(xnDxxGN motif. Our findings illustrate the exceptional gene pool diversity within the species Y. enterocolitica driven by horizontal gene transfer events.

  13. Extraction and inhibition of enzymatic activity of botulinum neurotoxins /B1, /B2, /B3, /B4, and /B5 by a panel of monoclonal anti-BoNT/B antibodies

    Directory of Open Access Journals (Sweden)

    Kalb Suzanne R

    2011-11-01

    Full Text Available Abstract Background Botulism is caused by botulinum neurotoxins (BoNTs, extremely toxic proteins which can induce respiratory failure leading to long-term intensive care or death. Treatment for botulism includes administration of antitoxins, which must be administered early in the course of the intoxication; therefore, rapid determination of human exposure to BoNT is an important public health goal. In previous work, our laboratory reported on Endopep-MS, a mass spectrometry-based activity method for detecting and differentiating BoNT/A, /B, /E, and /F in clinical samples. We also demonstrated that antibody-capture is effective for purification and concentration of BoNTs from complex matrices such as clinical samples. However, some antibodies inhibit or neutralize the enzymatic activity of BoNT, so the choice of antibody for toxin extraction is critical. Results In this work, we evaluated 24 anti-BoNT/B monoclonal antibodies (mAbs for their ability to inhibit the in vitro activity of BoNT/B1, /B2, /B3, /B4, and /B5 and to extract those toxins. Among the mAbs, there were significant differences in ability to extract BoNT/B subtypes and inhibitory effect on BoNT catalytic activity. Some of the mAbs tested enhanced the in vitro light chain activity of BoNT/B, suggesting that BoNT/B may undergo conformational change upon binding some mAbs. Conclusions In addition to determining in vitro inhibition abilities of a panel of mAbs against BoNT/B1-/B5, this work has determined B12.2 and 2B18.2 to be the best mAbs for sample preparation before Endopep-MS. These mAb characterizations also have the potential to assist with mechanistic studies of BoNT/B protection and treatment, which is important for studying alternative therapeutics for botulism.

  14. Staphylococcus aureus Small Colony Variants (SCVs): a road map for the metabolic pathways involved in persistent infections.

    Science.gov (United States)

    Proctor, Richard A; Kriegeskorte, André; Kahl, Barbara C; Becker, Karsten; Löffler, Bettina; Peters, Georg

    2014-01-01

    Persistent and relapsing infections, despite apparently adequate antibiotic therapy, occur frequently with many pathogens, but it is an especially prominent problem with Staphylococcus aureus infections. For the purposes of this review, persistence will encompass both of the concepts of long term survival within the host, including colonization, and the concept of resisting antibiotic therapy even when susceptible in the clinical microbiology laboratory. Over the past two decades, the mechanisms whereby bacteria achieve persistence are slowly being unraveled. S. aureus small colony variants (SCVs) are linked to chronic, recurrent, and antibiotic-resistant infections, and the study of SCVs has contributed significantly to understanding of persistence. In our earlier work, defects in electron transport and thymidylate biosynthesis were linked to the development of the SCV phenotype (reviewed in 2006), thus this work will be discussed only briefly. Since 2006, it has been found that persistent organisms including SCVs are part of the normal life cycle of bacteria, and often they arise in response to harsh conditions, e.g., antibiotics, starvation, host cationic peptides. Many of the changes found in these early SCVs have provided a map for the discovery mechanisms (pathways) for the development of persistent organisms. For example, changes in RNA processing, stringent response, toxin-antitoxin, ribosome protein L6 (RplF), and cold shock protein B (CspB) found in SCVs are also found in other persisters. In addition, many classic persister organisms also show slow growth, hence SCVs. Recent work on S. aureus USA300 has elucidated the impact of aerobic expression of arginine deiminase genes on its ability to chronically colonize the skin and survive in abscesses. S. aureus SCVs also express arginine deiminase genes aerobically as well. Thus, many pathways found activated in electron transport type of SCVs are also increased in persisters that have intact electron

  15. The molecular physiological and genetic mechanisms underlying the superb efficacy of quinolones%喹诺酮类药物作用的生理和遗传的分子机制

    Institute of Scientific and Technical Information of China (English)

    龙泉鑫; 何颖; 谢建平

    2012-01-01

    氟喹诺酮类药物是目前使用最广泛的广谱抗生素,占据了全球抗生素类药物市场约18%的份额.其杀菌快速,而且具备大量的衍生物库,为药物更新换代提供了良好的基础.但这些衍生物在杀菌的速度、代谢过程中对氧需求、对蛋白合成的依赖性等方面的差异很大.从药物作用后细菌分子遗传层面的应答特征揭示各类衍生物的深层次作用机制,可以为寻找更有效的新喹诺酮类药物提供坚实基础.SOS应答、毒素-抗毒素系统,细菌程序性死亡、染色体片段化以及活性氧簇等新机制可能不同程度参与了喹诺酮类药物的杀菌过程.本文重点介绍喹诺酮作用的“二步特征”.该特征的引入有助于理解不同喹诺酮类药物的杀菌特征,也有助于开发新的喹诺酮类药物.%The fluoroquinolones are the most widely used broad-spectrum antibiotics, accounting for 18% of global antibacterial market share. They can kill bacteria rapidly with variety of derivatives available. Different quinolones vary significantly in rate and spectrum of killing, oxygen requirement for metabolism and reliance upon protein synthesis. Further understanding the sophisticated mechanisms of action of this important antibiotic family based on the molecular genetic response of bacteria can facilitate the discovery of better quinolone derivatives. Factors such as SOS response, bacterial toxin-antitoxin system, programmed death, chromosome fragmentation and reactive oxygen have been implicated in the action to some extent. "Two steps characteristic" of quinolones killing is also emphasized, which might inspire future better quinolones modification.

  16. Microevolutionary events involving narrow host plasmids influences local fixation of vancomycin-resistance in Enterococcus populations.

    Directory of Open Access Journals (Sweden)

    Ana R Freitas

    Full Text Available Vancomycin-resistance in enterococci (VRE is associated with isolates within ST18, ST17, ST78 Enterococcus faecium (Efm and ST6 Enterococcus faecalis (Efs human adapted lineages. Despite of its global spread, vancomycin resistance rates in enterococcal populations greatly vary temporally and geographically. Portugal is one of the European countries where Tn1546 (vanA is consistently found in a variety of environments. A comprehensive multi-hierarchical analysis of VRE isolates (75 Efm and 29 Efs from Portuguese hospitals and aquatic surroundings (1996-2008 was performed to clarify the local dynamics of VRE. Clonal relatedness was established by PFGE and MLST while plasmid characterization comprised the analysis of known relaxases, rep initiator proteins and toxin-antitoxin systems (TA by PCR-based typing schemes, RFLP comparison, hybridization and sequencing. Tn1546 variants were characterized by PCR overlapping/sequencing. Intra- and inter-hospital dissemination of Efm ST18, ST132 and ST280 and Efs ST6 clones, carrying rolling-circle (pEFNP1/pRI1 and theta-replicating (pCIZ2-like, Inc18, pHTβ-like, two pRUM-variants, pLG1-like, and pheromone-responsive plasmids was documented. Tn1546 variants, mostly containing ISEf1 or IS1216, were located on plasmids (30-150 kb with a high degree of mosaicism and heterogeneous RFLP patterns that seem to have resulted from the interplay between broad host Inc18 plasmids (pIP501, pRE25, pEF1, and narrow host RepA_N plasmids (pRUM, pAD1-like. TAs of Inc18 (ω-ε-ζ and pRUM (Axe-Txe plasmids were infrequently detected. Some plasmid chimeras were persistently recovered over years from different clonal lineages. This work represents the first multi-hierarchical analysis of VRE, revealing a frequent recombinatorial diversification of a limited number of interacting clonal backgrounds, plasmids and transposons at local scale. These interactions provide a continuous process of parapatric clonalization driving a full

  17. Biochemical and Immunological Characterization of Truncated Fragments of the Receptor-Binding Domains of C. difficile Toxin A.

    Directory of Open Access Journals (Sweden)

    Jui-Hsin Huang

    of hemagglutinin (HA activity, with the exception of the F1 fragment, which demonstrates no HA activity; and (iii in the presence of alum, all fragments elicit various levels of anti-toxin A-neutralizing antibody responses, but those neutralizing antibodies elicited by F2 did not protect mice against a TcdA challenge. Because TcdA rRBD, F1 and F3 formulated with alum can elicit immune protective responses against the cytotoxicity of TcdA, they represent potential components of future candidate vaccines against C. difficile-associated diseases.

  18. A Single-Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic [alpha]-Exosite Binding Region

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Jianbo; Thompson, Aaron A.; Fan, Yongfeng; Lou, Jianlong; Conrad, Fraser; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Stevens, Raymond C.; Marks, James D. (UIUC); (Scripps); (UCSF)

    2010-08-13

    Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K{sub d}) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K{sub d} for BoNT/A Lc of 1.47 x 10{sup -10} M and an IC{sub 50} (50% inhibitory concentration) of 4.7 x 10{sup -10} M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 {angstrom} resolution. The structure reveals that the Aa1 VHH binds in the {alpha}-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc {alpha}-exosite as a target for inhibitor development.

  19. The orphan gene ybjN conveys pleiotropic effects on multicellular behavior and survival of Escherichia coli.

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    Dongping Wang

    Full Text Available YbjN, encoding an enterobacteria-specific protein, is a multicopy suppressor of temperature sensitivity in the ts9 mutant strain of Escherichia coli. In this study, we further explored the role(s of ybjN. First, we demonstrated that the ybjN transcript was about 10-fold lower in the ts9 strain compared to that of E. coli strain BW25113 (BW. Introduction of multiple copies of ybjN in the ts9 strain resulted in over-expression of ybjN by about 10-fold as compared to that of BW. These results suggested that temperature sensitivity of the ts9 mutant of E. coli may be related to expression levels of ybjN. Characterization of E. coli ybjN mutant revealed that ybjN mutation resulted in pleiotropic phenotypes, including increased motility, fimbriation (auto-aggregation, exopolysaccharide production, and biofilm formation. In contrast, over-expression of ybjN (in terms of multiple copies resulted in reduced motility, fimbriation, exopolysaccharide production, biofilm formation and acid resistance. In addition, our results indicate that a ybjN-homolog gene from Erwinia amylovora, a plant enterobacterial pathogen, is functionally conserved with that of E. coli, suggesting similar evolution of the YbjN family proteins in enterobacteria. A microarray study revealed that the expression level of ybjN was inversely correlated with the expression of flagellar, fimbrial and acid resistance genes. Over-expression of ybjN significantly down-regulated genes involved in citric acid cycle, glycolysis, the glyoxylate shunt, oxidative phosphorylation, amino acid and nucleotide metabolism. Furthermore, over-expression of ybjN up-regulated toxin-antitoxin modules, the SOS response pathway, cold shock and starvation induced transporter genes. Collectively, these results suggest that YbjN may play important roles in regulating bacterial multicellular behavior, metabolism, and survival under stress conditions in E. coli. These results also suggest that ybjN over

  20. Isolation and functional characterization of the novel Clostridium botulinum neurotoxin A8 subtype.

    Science.gov (United States)

    Kull, Skadi; Schulz, K Melanie; Weisemann, Jasmin; Kirchner, Sebastian; Schreiber, Tanja; Bollenbach, Alexander; Dabrowski, P Wojtek; Nitsche, Andreas; Kalb, Suzanne R; Dorner, Martin B; Barr, John R; Rummel, Andreas; Dorner, Brigitte G

    2015-01-01

    pave the way for the development of novel therapeutics and tailor-made antitoxins.

  1. Isolation and functional characterization of the novel Clostridium botulinum neurotoxin A8 subtype.

    Directory of Open Access Journals (Sweden)

    Skadi Kull

    toxin function might pave the way for the development of novel therapeutics and tailor-made antitoxins.

  2. Developing Universal Genetic Tools for Rapid and Efficient Deletion Mutation in Vibrio Species Based on Suicide T-Vectors Carrying a Novel Counterselectable Marker, vmi480.

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    Peng Luo

    Full Text Available Despite that Vibrio spp. have a significant impact on the health of humans and aquatic animals, the molecular basis of their pathogenesis is little known, mainly due to the limited genetic tools for the functional research of genes in Vibrio. In some cases, deletion of target DNAs in Vibrio can be achieved through the use of suicide vectors. However, these strategies are time-consuming and lack universality, and the widely used counterselectable gene sacB does not work well in Vibrio cells. In this study, we developed universal genetic tools for rapid and efficient deletion mutations in Vibrio species based on suicide T-Vectors carrying a novel counterselectable marker, vmi480. We explored two uncharacterized genes, vmi480 and vmi470, in a genomic island from Vibrio mimicus VM573 and confirmed that vmi480 and vmi470 constitute a two-component toxin-antitoxin system through deletion and expression of vmi480 and vmi470. The product of vmi480 exhibited strong toxicity to Escherichia coli cells. Based on vmi480 and the PBAD or PTAC promoter system, we constructed two suicide T-vectors, pLP11 and pLP12, and each of these vectors contained a multiple cloning region with two AhdI sites. Both vectors linearized by AhdI digestion could be stored and directly ligated with purified PCR products without a digestion step. By using pLP11 and pLP12 coupled with a highly efficient conjugation system provided by E. coli β2163, six genes from four representative Vibrio species were easily deleted. By using the counterselective marker vmi480, we obtained 3-12 positive colonies (deletion mutants among no more than 20 colonies randomly selected on counterselection plates. The strategy does not require the digestion of PCR products and suicide vectors every time, and it avoids large-scale screening colonies on counterselective plates. These results demonstrate that we successfully developed universal genetic tools for rapid and efficient gene deletion in Vibrio

  3. Ranking of persister genes in the same Escherichia coli genetic background demonstrates varying importance of individual persister genes in tolerance to different antibiotics

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    Nan eWu

    2015-09-01

    Full Text Available Despite the identification of many genes and pathways involved in the persistence phenomenon of bacteria, the relative importance of these genes in a single organism remains unclear. Here, using Escherichia coli as a model, we generated mutants of 21 known candidate persister genes and compared the relative importance of these mutants in persistence to various antibiotics (ampicillin, gentamicin, norfloxacin, and trimethoprim at different times. We found that oxyR, dnaK, sucB, relA, rpoS, clpB, mqsR, and recA were prominent persister genes involved in persistence to multiple antibiotics. These genes map to the following pathways: antioxidative defense pathway (oxyR, global regulators (dnaK, clpB, and rpoS, energy production (sucB, stringent response (relA, toxin–antitoxin (TA module (mqsR, and SOS response (recA. Among the TA modules, the ranking order was mqsR, lon, relE, tisAB, hipA, and dinJ. Intriguingly, rpoS deletion caused a defect in persistence to gentamicin but increased persistence to ampicillin and norfloxacin. Mutants demonstrated dramatic differences in persistence to different antibiotics at different time points: some mutants (oxyR, dnaK, phoU, lon, recA, mqsR, and tisAB displayed defect in persistence from early time points, while other mutants (relE, smpB, glpD, umuD, and tnaA showed defect only at later time points. These results indicate that varying hierarchy and importance of persister genes exist and that persister genes can be divided into those involved in shallow persistence and those involved in deep persistence. Our findings suggest that the persistence phenomenon is a dynamic process with different persister genes playing roles of variable significance at different times. These findings have implications for improved understanding of persistence phenomenon and developing new drugs targeting persisters for more effective cure of persistent infections.

  4. Institutos de pesquisa em saúde Health research institutes

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    Erney Plessmann Camargo

    2004-06-01

    Full Text Available Ao fim do século 19, em seguida aos avanços na assepsia cirúrgica e no esclarecimento do papel de microorganismos como causadores de moléstias infecciosas, foram criados em escala mundial "institutos de pesquisa com os objetivos de conduzir pesquisa sobre as causas, prevenção e tratamento de homens e animais e de desenvolver produtos preventivos e curativos como vacinas e antitoxinas". Para atingir esses objetivos os institutos recrutaram os melhores cientistas disponíveis em todos os lugares. Este foi um período fulgurante para a pesquisa sublinhado por inúmeras descobertas sobre as doenças infecciosas. Passada essa fase, os institutos entraram em depressão e até mesmo insolvência. Muitos se recuperaram, alguns não. Muitos ainda sofrem de uma "crise de identidade". Neste ensaio analisamos o destino dos institutos de pesquisa em saúde no Brasil, as causas de seu sucesso e eventuais fracassos e as medidas possíveis de ajudá-los a contornar suas presentes dificuldades.By the end of the 19th century, after the advances in surgical asepsis and the understanding of the role of microorganisms in the etiology of many infectious diseases, research institutes in the public health area were created worldwide with the objectives of undertaking "scientific research into the causes, prevention and treatment of disease in man and animals and to prepare and supply protective and curative materials such as vaccines and antitoxins". To pursue these objectives the institutes hired the best scientists from everywhere. This was an exhilarating period for research in which discoveries proliferated. After that, institutes got into depression and even insolvency. Most recovered, many not. Many are still suffering from an "identity crisis". In this paper we analyze the fate of the health research institutes in Brazil, the causes of their success and eventual failures and the possible ways to get out of their current difficulties.

  5. 霍乱口服疫苗的研究与应用%Development of oral cholera vaccine and its vaccination

    Institute of Scientific and Technical Information of China (English)

    阚飙

    2015-01-01

    应用疫苗是人群霍乱防控的重要策略之一.霍乱疫苗研制强调黏膜免疫策略,使之发挥诱导抗菌抗毒素免疫的作用.疫苗配方中如果带有毒素B亚单位(包括在灭活疫苗中添加以及在活疫苗中进行表达),能通过交叉免疫发挥预防其他一些病原导致腹泻病的作用.口服灭活疫苗已成熟,活疫苗的研制与应用也在积极探索之中.霍乱疫苗的研发还不仅仅在于技术层面,根据流行地区和人群的特点,也要求低成本、储运条件简单、接种次数少.对危险人群以及出现疫情的地区如何使用霍乱疫苗,虽然以往存在争论,但通过评估和在疫情中的应用,已逐步形成了共识.%The application of the cholera vaccine is one of the cholera prevention and control strategies.Cholera vaccines stimulate mucosal immune to play the role of antibacteria and antitoxin.When the cholera toxin B subunit is added in the cholera vaccine,it could also defend against some diarrhea associated pathogens by cross-protection.Oral inactivated cholera vaccines are commercially available now.The oral live vaccine candidates are under development.The development of cholera vaccine is not only on the technical aspect,based on the situations of epidemic areas and population,cost,storage and transportation condition should also be considered.Though the argument on the use of cholera vaccine in epidemic areas and population in high risk existed previously,its vaccination has reached agreement now based on the clinical trials and evaluations during epidemic period.

  6. Development and Preliminary Application of ELISA Method for Diphtheria Toxoid%白喉类毒素ELISA检测方法的建立及初步应用

    Institute of Scientific and Technical Information of China (English)

    刘朝阳; 景辉; 陈艳红; 张斌; 马相虎; 朱金华

    2011-01-01

    目的 建立白喉类毒素双抗体夹心ELISA检测方法,并进行验证及初步应用.方法 以马抗白喉类毒素血清作为包被抗体,HRP标记的白喉絮状反应抗毒素作为酶标抗体,建立白喉类毒素双抗体夹心ELISA检测方法,并对该方法进行重复性、特异性验证、最佳线性范围和检测限确定及初步应用.结果 经验证,该方法重复性好,特异性强,白喉类毒素含量在0.9765~125.0000ng/ml之间时线性关系良好,检测限为7.812 ng/ml,该方法检测了3批白喉类毒素原液,与动物实验结果基本相符.结论 已建立了白喉类毒素双抗体夹心ELISA检测方法,可用于检测白喉类毒素的含量和抗原性.%Objective To develop, verify and preliminarily apply a double antibody sandwich ELISA method for diphtheria toxoid. Methods A double antibody sandwich ELISA method was developed using horse antiserum against diphtheria toxoid as coating antibody and HRP-labeled diphtheria flocculation antitoxin as enzyme labeled antibody, then verified for reproducibility and specificity, determined for optimal linear range and detection limit, and applied preliminarily. Results The developed method showed high reproducibility and specificity, of which the optimal linear range and detection limit were 0. 976 5 ~ 125. 000 0 ng/ml and 7. 812 ng/ml respectively. Three batches of bulk diphtheria toxoid were detected by the developed method, and the results were basically consistent with thoes by animal test. Conclusion A double antibody sandwich ELISA method was developed, which might be used for determination of content and antigenicity of diphtheria toxoid.

  7. Microevolutionary Events Involving Narrow Host Plasmids Influences Local Fixation of Vancomycin-Resistance in Enterococcus Populations

    Science.gov (United States)

    Freitas, Ana R.; Novais, Carla; Tedim, Ana P.; Francia, María Victoria; Baquero, Fernando; Peixe, Luísa; Coque, Teresa M.

    2013-01-01

    Vancomycin-resistance in enterococci (VRE) is associated with isolates within ST18, ST17, ST78 Enterococcus faecium (Efm) and ST6 Enterococcus faecalis (Efs) human adapted lineages. Despite of its global spread, vancomycin resistance rates in enterococcal populations greatly vary temporally and geographically. Portugal is one of the European countries where Tn1546 (vanA) is consistently found in a variety of environments. A comprehensive multi-hierarchical analysis of VRE isolates (75 Efm and 29 Efs) from Portuguese hospitals and aquatic surroundings (1996–2008) was performed to clarify the local dynamics of VRE. Clonal relatedness was established by PFGE and MLST while plasmid characterization comprised the analysis of known relaxases, rep initiator proteins and toxin-antitoxin systems (TA) by PCR-based typing schemes, RFLP comparison, hybridization and sequencing. Tn1546 variants were characterized by PCR overlapping/sequencing. Intra- and inter-hospital dissemination of Efm ST18, ST132 and ST280 and Efs ST6 clones, carrying rolling-circle (pEFNP1/pRI1) and theta-replicating (pCIZ2-like, Inc18, pHTβ-like, two pRUM-variants, pLG1-like, and pheromone-responsive) plasmids was documented. Tn1546 variants, mostly containing ISEf1 or IS1216, were located on plasmids (30–150 kb) with a high degree of mosaicism and heterogeneous RFLP patterns that seem to have resulted from the interplay between broad host Inc18 plasmids (pIP501, pRE25, pEF1), and narrow host RepA_N plasmids (pRUM, pAD1-like). TAs of Inc18 (ω-ε-ζ) and pRUM (Axe-Txe) plasmids were infrequently detected. Some plasmid chimeras were persistently recovered over years from different clonal lineages. This work represents the first multi-hierarchical analysis of VRE, revealing a frequent recombinatorial diversification of a limited number of interacting clonal backgrounds, plasmids and transposons at local scale. These interactions provide a continuous process of parapatric clonalization driving a full

  8. 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

  9. Severe Tetanus in Adults:Report of Four Cases and Review of Literature%成人重症破伤风4例诊治体会

    Institute of Scientific and Technical Information of China (English)

    张滋凤; 余明主; 曾小明

    2011-01-01

    目的 探讨成人重症破伤风的治疗方法.方法 回顾性研究自2008年1月至2011年3月应用改进后的破伤风治疗方法治疗成人重症破伤风4例,分析治疗方法及疗效.结果 4例患者均予以注射人破伤风免疫球蛋白或破伤风抗毒素、抗感染、营养支持、肌松镇静解痉等综合治疗.4例均行气管切开、呼吸机辅助呼吸.4例患者中1例并发肺部感染,1例合并肺部感染,1例有吸毒史.4例患者均治愈出院.结论 应用改进后的破伤风治疗方法,可明显提高所研究病例的治愈率,改善重症破伤风患者的预后.%Objective To investigate the treatment of adults with severe tetanus. Methods A retrospective analysis of treatment methods and efficacy was conducted on adults with severe tetanus who received the improved treatment of tetanus between Jan. 2008 and Mar. 2011. Results Patients were treated with human tetanus immune globulin or tetanus antitoxin,antibiotics,nutritional support, antispasmodic and sedative muscle relaxants, and other comprehensive treatment approaches. All patients underwent tracheotomy and mechanical ventilation. Among the 4 cases, 1 was complicated by pulmonary infection, 1 was associated with pulmonary infection,and 1 had a history of drug abuse. All the 4 patients were cured and discharged from hospital. Conclusion The improved treatment of tetanus can significantly increase the cure rate and improve the prognosis in patients with severe tetanus.

  10. Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System.

    Science.gov (United States)

    Bradshaw, Marite; Tepp, William H; Whitemarsh, Regina C M; Pellett, Sabine; Johnson, Eric A

    2014-12-01

    Clostridium botulinum subtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producing C. botulinum strain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenic C. botulinum expression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided in trans by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed in Escherichia coli has indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the in vitro, cellular, and in vivo activities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.

  11. Prioritizing drug targets in Clostridium botulinum with a computational systems biology approach.

    Science.gov (United States)

    Muhammad, Syed Aun; Ahmed, Safia; Ali, Amjad; Huang, Hui; Wu, Xiaogang; Yang, X Frank; Naz, Anam; Chen, Jake

    2014-07-01

    A computational and in silico system level framework was developed to identify and prioritize the antibacterial drug targets in Clostridium botulinum (Clb), the causative agent of flaccid paralysis in humans that can be fatal in 5 to 10% of cases. This disease is difficult to control due to the emergence of drug-resistant pathogenic strains and the only available treatment antitoxin which can target the neurotoxin at the extracellular level and cannot reverse the paralysis. This study framework is based on comprehensive systems-scale analysis of genomic sequence homology and phylogenetic relationships among Clostridium, other infectious bacteria, host and human gut flora. First, the entire 2628-annotated genes of this bacterial genome were categorized into essential, non-essential and virulence genes. The results obtained showed that 39% of essential proteins that functionally interact with virulence proteins were identified, which could be a key to new interventions that may kill the bacteria and minimize the host damage caused by the virulence factors. Second, a comprehensive comparative COGs and blast sequence analysis of these proteins and host proteins to minimize the risks of side effects was carried out. This revealed that 47% of a set of C. botulinum proteins were evolutionary related with Homo sapiens proteins to sort out the non-human homologs. Third, orthology analysis with other infectious bacteria to assess broad-spectrum effects was executed and COGs were mostly found in Clostridia, Bacilli (Firmicutes), and in alpha and beta Proteobacteria. Fourth, a comparative phylogenetic analysis was performed with human microbiota to filter out drug targets that may also affect human gut flora. This reduced the list of candidate proteins down to 131. Finally, the role of these putative drug targets in clostridial biological pathways was studied while subcellular localization of these candidate proteins in bacterial cellular system exhibited that 68% of the

  12. 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

  13. Sequences of two related multiple antibiotic resistance virulence plasmids sharing a unique IS26-related molecular signature isolated from different Escherichia coli pathotypes from different hosts.

    Directory of Open Access Journals (Sweden)

    Carola Venturini

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC and atypical enteropathogenic E. coli (aEPEC are important zoonotic pathogens that increasingly are becoming resistant to multiple antibiotics. Here we describe two plasmids, pO26-CRL125 (125 kb from a human O26:H- EHEC, and pO111-CRL115 (115kb from a bovine O111 aEPEC, that impart resistance to ampicillin, kanamycin, neomycin, streptomycin, sulfathiazole, trimethoprim and tetracycline and both contain atypical class 1 integrons with an identical IS26-mediated deletion in their 3´-conserved segment. Complete sequence analysis showed that pO26-CRL125 and pO111-CRL115 are essentially identical except for a 9.7 kb fragment, present in the backbone of pO26-CRL125 but absent in pO111-CRL115, and several indels. The 9.7 kb fragment encodes IncI-associated genes involved in plasmid stability during conjugation, a putative transposase gene and three imperfect repeats. Contiguous sequence identical to regions within these pO26-CRL125 imperfect repeats was identified in pO111-CRL115 precisely where the 9.7 kb fragment is missing, suggesting it may be mobile. Sequences shared between the plasmids include a complete IncZ replicon, a unique toxin/antitoxin system, IncI stability and maintenance genes, a novel putative serine protease autotransporter, and an IncI1 transfer system including a unique shufflon. Both plasmids carry a derivate Tn21 transposon with an atypical class 1 integron comprising a dfrA5 gene cassette encoding resistance to trimethoprim, and 24 bp of the 3´-conserved segment followed by Tn6026, which encodes resistance to ampicillin, kanymycin, neomycin, streptomycin and sulfathiazole. The Tn21-derivative transposon is linked to a truncated Tn1721, encoding resistance to tetracycline, via a region containing the IncP-1α oriV. Absence of the 5 bp direct repeats flanking Tn3-family transposons, indicates that homologous recombination events played a key role in the formation of this complex

  14. 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

  15. 小儿破伤风应用静脉丙种球蛋白的免疫力效果观察%Observation on immune effect of intravenous immune globulin in the treatment of pediatric tetanus

    Institute of Scientific and Technical Information of China (English)

    龚德彰

    2013-01-01

    Objective To preliminarily study and discuss the specific clinical effect of intravenous immune globulin in the treatment of pediatric tetanus, in order to increase the clinical effect of treatment to the pediatric tetanus and guarantee their life safety. Methods 28 pediatric patients with tetanus treated in this hospital from October 2010 to December 2012 were randomly divided into the observation group and control group, including 14 cases in each group. Pediatric patients in the control group received the routine treatment of tetanus antitoxin; while pediatric patients in the observation group received the routine treatment and intravenous immune globulin. Observed and made statistics for the clinical effect of pediatric patients in two groups. Results After receiving the treatment, the clinical effect in the observation group was significantly better than the one in the control group, with the significant difference (P<0.05). Conclusion Tetanus has a great impact on the immune function of pediatric patients. Based upon the routine treatment, the intravenous immune globulin will have a significant clinical effect in the treatment to pediatric tetanus, which will significantly increase the clinical effect, guarantee patients' life safety, reduce the pains of disease to them and thus is worthy to be propularized.%  目的为了进一步提高我院治疗小儿破伤风的临床疗效,保障患儿的生命安全,本资料就小儿破伤风应用静脉丙种球蛋白的免疫力的具体临床效果进行了浅显的研究和探讨。方法将我院在2010年10月~2012年12月期间收治的28例破伤风患儿随机分为观察组和对照组,两组患者均14例、给予对照组患儿采用破伤风抗毒素进行常规治疗;给予观察组患儿在常规治疗的基础上加用静脉丙种球蛋白进行治疗,对两组患儿的治疗效果进行观察和统计。结果两组患儿在接受治疗后,观察组患儿的治疗效果要明显的

  16. Evaluation of tetanus toxoid immunization of childbearing age women in Inner Mongolia Autonomous Region%内蒙古自治区育龄期妇女接种破伤风类毒素免疫效果评价

    Institute of Scientific and Technical Information of China (English)

    闫绍宏; 魏永刚; 武贵森; 夏伟; 金仁杰; 陈永生; 白吉日根

    2013-01-01

    目的 评价内蒙古自治区农村、牧区育龄期妇女开展破伤风类毒素(TT)接种的效果.方法 在开展破伤风类毒素接种的突泉县和未开展接种的扎赉特旗分别采用东西南北中5个方位抽取10个乡级单位,用间接血凝方法检测487名18~35岁育龄期妇女破伤风抗毒素(TAT)抗体水平.结果 开展TT接种的突泉县育龄期妇女达到破伤风保护水平43.1%(>0.01 IU/ml),TAT几何平均滴度为0.034 IU/ml;而未开展TT接种的扎赉特旗育龄期妇女破伤风保护率为8.33%,TAT几何平均滴度为0.005 5 IU/ml.结论 育龄期妇女TT接种是消除新生儿破伤风的策略之一,做好新生儿破伤风高危地区育龄期妇女TT接种,消除新生儿破伤风效果显著,应加强对18~35岁低年龄组育龄期妇女的TT接种.%OBJECTIVE To evaluate the effect of tetanus toxoid immunization of childbearing age women in rural and pasturing areas of Inner Mongolia Autonomous Region.METHODS 10 counties were collected from the east,west,north,south and middle directions in Tuquan County which developed the tetanus toxoid immunization as well as the Zalaiteqi which did not develop the immunization respectively.From these 10 counties,487 childbearing age women were aged 18-35 collected randomly and their antibody level of serum tetanus antitoxin (TAT) were detected by indirect blood coagulation and general situation investigation on.RESULTS In Tuquan County,the protection rate reached 43.1% (> 0.01 IU/ml) and the TAT geometry average titer was 0.034 IU/ml.In Zalaiteqi,the protection rate was 8.33% and the TAT geometry average titer was 0.005 5 IU/ml.CONCLUSION The tetanus toxoid immunization of childbearing age women is one of the strategies to eliminate the neonatal tetanus,so we should strengthen the tetanus toxoid immunization of childbearing age women from 18 to 35 years old.

  17. High-level Expression of the Hc Domain of Clostridium botulinum Neurotoxin Serotype A in Escherichia coli and Its Immunogenicity as an Antigen%A型肉毒毒素Hc结构域在大肠杆菌中的高水平表达及免疫原性

    Institute of Scientific and Technical Information of China (English)

    余云舟; 孙志伟; 王双; 俞炜源

    2007-01-01

    对A型肉毒毒素受体结合区Hc基因的全序列进行优化和人工合成,获得了全长1 287bp,编码429aa的Hc基因.以pTIG-Trx为原核表达载体,实现了Hc在大肠杆菌中的高效可溶性表达及纯化,该表达水平可占可溶性全菌总蛋白的36%~53%,经一步亲和层析纯化可获得电泳级纯度的目的蛋白,在常规培养条件下,产量达到30mg/L以上.然后,纯化的重组蛋白Hc免疫小鼠后能够诱导产生高滴度特异性的抗体,也能诱导产生特异性的细胞免疫应答反应.小鼠体内A型肉毒毒素中和试验结果表明免疫组小鼠血清中含高滴度的体内中和抗体.结果表明,利用本实验的原核表达系统不仅能够高水平可溶性地表达A型肉毒毒素受体结合区Hc,而且重组Hc具有良好的免疫原性,可以用于制备治疗性抗毒素和作为亚单位候选疫苗用于预防A型肉毒毒素中毒.%A completely synthetic gene encoding the Hc domain of Clostridium botulinum neurotoxin serotype A (AHc, 1287bp,429aa, ~ 50kD) was constructed with oligonucleotides. After expressed in Escherichia coli, soluble product AHc was gained and verified by SDS-PAGE and Western blot analysis. The expressive level of recombinant AHc in E. coli was very high (36% ~53% of soluble total proteins) and the purified yield was more than 30mg/L by one-step purification. Then,the purified AHc was used to vaccinate Balb/c mice, which developed a strong and specific immune response as expected following administration of AHc protein via the subcutaneous route. Results from BoNT/A neutralization assay showed that the serum from mice vaccinated with AHc contained high titer protective antibody. These results showed that the soluble, stable and high-levelly expressive AHc not only could be produced by the prokaryotic expression system built in our lab, but also owned strong immunogenicity to prepare antitoxin for treatment and as sub-unit candidate vaccine for prophylaxis against

  18. Quality assurance of C. perfringens epsilon toxoid vaccines--ELISA versus mouse neutralisation test.

    Science.gov (United States)

    Rosskopf-Streicher, Ute; Volkers, Peter; Noeske, Kerstin; Werner, Esther

    2004-01-01

    exact efficacy of the test serum is given in International Units (IU). The tested serum must show a minimum content of 5 IU. This in vivo method requires a very high number of experimental animals. Approximately 400 mice (or 50 guinea pigs) are used per vaccine batch. The monograph for C. perfringens vaccines, which has recently been revised, expressly indicates that a validated serological method may be used for batch testing. In addition, a reference serum known as clostridium multicomponent serum has been available since 2000. The objective is to test vaccine batches against this reference and by means of a competitive ELISA developed in the precursor project, using a monoclonal antibody for direct determination of specific antitoxins in rabbit sera. This ELISA method was subjected to an international validation to verify whether the protocol and the precision can be transferred within and between the participating laboratories.

  19. 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

  20. Nuclease activity and cytotoxicity to host cells of toxic protein VapC produced by Leptospira species%钩端螺旋体毒性蛋白VapC核酸酶活性及其对宿主细胞毒性作用的研究

    Institute of Scientific and Technical Information of China (English)

    辛晓阳; 林旭瑷; 李立伟; 严杰

    2012-01-01

    Objective To determine the function of toxic protein VapC in toxin/antitoxin system of Leptospira species and the cytotoxicity to host cells of the toxic protein.Methods Using genomic DNA of pathogenic L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai as the template,several PCRs were performed to amplify entire vapB,vapC and vapBC genes.Subsequently,the prokaryotic expression systems of vapB,vapC and vapBC genes were constructed.Expression of the target recombinant proteins rVapB and rVapC was detected by SDS-PAGE and the expressed rVapB and rVapC were extracted by NiNTA affinity chromatography.Activity of rVapB and rVapC to lyse the DNAs or RNAs from L.interrogans strain Lai and THP-1 cells were then determined.The changes of transcription and expression of vapB and vapC genes of L.interrogans strain Lai before and after infection of THP-1 cells were detected by real-time fluorescent quantitative RT-PCR and Western blot assay.The eukaryotic expression vectors of the vapB and vapC genes were generated for transfection of host cells and CCK-8 agent was used to detect the effect of leptospiral VapB and VapC proteins on activity of host cells.Results The nucleotide and putative amino acid sequences of the cloned vapB and vapC genes were completely identical with the reported corresponding genes.The constructed prokaryotic expression systems could express rVapB and rVapC,respectively.rVapC displayed RNase avtivity but did not lyse DNA.When L.interrogans strain Lai infected THP-1 cells,the transcription and expression of vapB and vapC genes were upregulated and partial VapC protein was secreted from the leptospiral cells.The mass mortality was observed in HEK293 human renal tubular epithelial ceils containing the vapC gene through transfection.Conclusion VapC protein of L.interrogans strain Lai is a RNase and is secreted during infection of host cells with obvious cytotoxicity.%目的 了解钩端螺旋体毒素-抗毒素系统中毒性蛋白VapC的

  1. 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