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Karlsson, Sture. "Toxin production in Clostridium difficile /." Stockholm : Karolinska institutet, 2004. http://diss.kib.ki.se/2004/91-77349-812-2/.
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Mullan, Nivette K. "Mucosal cell responses to Clostridium difficile toxins." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/13217/.
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Colonic inflammation in C. difficile infection is mediated by released toxins A and B. I have investigated responses to C. difficile toxin A and B by primary human colonic myofibroblasts, which represent a distinct subpopulation of mucosal cells that are normally located below the intestinal epithelium and epithelial cell lines, Caco-2 and HT29. Myofibroblasts, isolated from normal human colonic mucosal specimens, Caco-2 and HT29 cells incubated with purified toxin A or B displayed a dose dependent response. Myofibroblast morphology changed to a stellate shaped cell, with processes that were immunoreactive for alpha smooth muscle actin. Most of the myofibroblasts remained viable, with persistent stellate morphology, despite exposure to high concentrations (up to 10 μ g/ml) of toxin A for 72 h. In contrast, a majority of the toxin B exposed myofibroblasts lost their processes prior to cell death over 24-72 h. Investigating toxin A+B on myofibroblasts, at low concentrations, toxin A provided protection against toxin B-induced cell death. Most of the intestinal epithelial, HT29 cells remained viable despite exposure to high concentrations of either toxin (up to 10 μ g/mi). By contrast, a significant loss in cell viability was observed in Caco-2 cells exposed to either toxin. Within 4 h, myofibroblast and epithelial cell types exposed to either toxin A or B lost expression of the non-glucosylated form of Racl, but total intracellular RhoA remained unchanged in myofibroblasts and Caco-2 cells. A time-dependent reduction in RhoA expression was seen in HT29 in response to toxin A or B. Active RhoA expression was lost within 4h in myofibroblasts exposed to either toxin. Despite pre-exposure to high concentrations of toxin A for 3 h, colonic myofibroblasts were able to recover their morphology and proliferative capacity during prolonged culture in medium. This was also shown when pre-exposure to toxin A was extended to 48 h. However, toxin B-pre-exposed myofibroblasts were not able to recover. In conclusion, primary human colonic mucosal myofibroblasts are resistant to toxin A (but not toxin B)-induced cell death. Responses by colonic myoflbroblasts may play an important role in mucosal protection, repair, and regeneration in colitis due to C. difficile infection. Investigation into the apparent resilience of HT29 cells has highlighted the importance of cell specific substrate specificity by C. difficile toxins.
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Hussack, Greg. "Single-domain Antibody Inhibitors of Clostridium difficile Toxins." Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/20362.
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Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.
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Shilling, Michael P. "Optimizing detection and control of Clostridium difficile and its toxins." Thesis, Kent State University, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=3618927.
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Clostridium difficile infection (CDI) is a bacterial disease affecting the lower gastrointestinal tract of patients whose normal colonic microbiota are altered, generally by administration of antibiotic therapies. C. difficile produces toxins that cause severe diarrhea, with potentially fatal complications in the immunocompromised. CDI has spread unabated despite the best prevention efforts of clinical practitioners. This dissertation is a broad-based study of several factors of importance in prevention and control CDI. In clinical environments, transport media are used to maintain the integrity of clinical samples for later laboratory testing. A transport medium for enteric bacteria was assessed as a preservative in outpatient fecal samples submitted for CDI testing. The transport medium used preserved C. difficile toxin out to five days. The possible effect of fecal pH and trypsin content on toxin stability was investigated. Fecal pH was ruled out as a factor due to CDI selected samples tending toward neutral pH. Trypsin was found to degrade toxin in controlled experiments; however, results from clinical samples were mixed. Oils and fatty acids, including virgin coconut oil (VCO), have antimicrobial effects on a variety of human pathogens. Use of natural products like VCO may reduce or prevent CDI by killing C. difficile while preserving the protective bowel flora. Virgin coconut oil (VCO) and three of its constituent fatty acids were evaluated for their toxic effect on C. difficile and were found to have bactericidal activity, the most potent of which was lauric acid. The outer surface of C. difficile spores is thought to contain proteins that are critical for attachment of the spores to surfaces, including human hands. This strong attachment assists in transmission of infectious spores; however, the source of this strong attachment in the spore remains unknown. Transmission electron micrography shows that C. difficile lacks a true exosporium, rather, they are coated in the remains of the mother cell. Results from subsequent fluorescence microscopy and ELISA with antibodies raised against spore coat proteins confirm that this residue is not part of the spore coat and can be easily removed by chemical treatment, increasing spore binding to anti-coat antibodies.
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Shilling, Michael. "OPTIMIZING DETECTION AND CONTROL OF CLOSTRIDIUM DIFFICILE AND ITS TOXINS." Kent State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=kent1374852321.
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Tsuchiya, Ana Claudia 1987. "Avaliação de métodos e ocorrência de Clostridium difficile em carnes." [s.n.], 2012. http://repositorio.unicamp.br/jspui/handle/REPOSIP/255444.
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Orientador: Arnaldo Yoshiteru KuayeDissertação (mestrado) - Universidade Estadual de Campionas, Faculdade de Engenharia de Alimentos
Made available in DSpace on 2018-08-20T10:25:27Z (GMT). No. of bitstreams: 1 Tsuchiya_AnaClaudia_M.pdf: 449310 bytes, checksum: 3afe68453ac7a3b16148cef90be5c03f (MD5) Previous issue date: 2012
Resumo: Clostridium difficile é um bacilo anaeróbio responsável por doença intestinal associada ao tratamento prévio com antibióticos, manifestando desde uma diarreia leve até casos graves de colite pseudomembranosa causada principalmente pelas toxinas A (TcdA) e B (TcdB). Os casos de infecção estão relacionados à contaminação em hospitais, porém pesquisas recentes sugerem possível associação ao consumo de alimentos contaminados, pois C. difficile já foi isolado de bovinos, suínos e aves e suas carnes sugerindo os animais como reservatórios. Desta forma, os estudos são de suma importância para o entendimento da transmissão da doença causada por C. difficile. Diante de poucas pesquisas de C. difficile e da inexistência de método padronizado para seu isolamento a partir dos alimentos, o trabalho consistiu em três etapas: 1) avaliação de metodologia [utilizando dois procedimentos (tratamento com álcool e plaqueamento direto) e dois meios seletivos (ágar Clostridium difficile moxalactan norfloxacina ¿ CDMNA e ágar cicloserina cefoxitina frutose ¿ CCFA)] de detecção de C. difficile em carnes (bovina moída e peito de frango [Peitoralis profundus e superficialis]); 2) avaliação da ocorrência de C. difficile em amostras de carnes resfriadas (bovina moída, bovina peça [Semimembranosus], suína [Longuisimus dorsi] e frango [Peitoralis profundus e superficialis]), compreendendo detecção, isolamento e identificação dos isolados; 3) avaliação do perfil toxigênico dos isolados através da detecção de genes tcdA e tcdB codificadores de TcdA e TcdB e respectivamente avaliação de produção do toxinas pelos isolados. A partir da comparação de dois procedimentos, observou-se que o plaqueamento direto foi mais eficaz e recuperou uma maior quantidade de C. difficile se comparado com o tratamento com álcool e o ágar Clostridium difficile moxalactan norfloxacina (CDMNA) apresentou maior taxa de recuperação em relação ao ágar cicloserina cefoxitina frutose (CCFA). A ocorrência de C. difficile foi observada em 11,5% (17/147) das amostras analisadas, totalizando 80 isolados, destes 41,2% (33/83) apresentaram positivo para pelo menos um gene de virulência (A-B+), ou para ambos os genes (A+B+). Houve concordância de 70,5% entre os testes fenotípicos e genotípicos utilizados para detecção de toxinas. Desta forma, sugere-se que alimentos de origem animal são uma potencial fonte de transmissão de C. difficile para humanos
Abstract: Clostridium difficile is an anaerobic bacillus responsible for intestinal deseases in individuals previouslly treated with antibiocs, who can manifest from a mild diarrhea to severe cases of pseudomembranous colitis, mainly caused by toxins A (TcdA) and B (TcdB). The infections are related to contamination in hospitals, but recent researches sugests a possible association with the consumiption of contaminated foods as C. difficile has been isolated from bovines, suines and poultries and their meat, sugesting animals as reservatories. Thus, studies are extremelly important to elucidate the transmition of the desease caused by C. difficile. Faced with few researches about this bacteria and the lack of a standard method for its isolation from food, this work is divided in three steps: 1) Evaluation of the methodology for C. difficile detection in meet (commercial bovine mince and chicken breast ¿ [Peitoralis superficialis and Peitoralis profundus] [using two procedures (treatment with alcohol and direct plating) and two selective mediums (agar Clostridium difficile moxalactan norfloxacin ¿ CDMNA and cycloserine cefoxitin fructose agar¿ CCFA)]; 2) Assessing of the occurrence of C. difficile in samples of chilled meat (bovine: commercial mince and the whole Semimembranosus; suine: whole Longuisimus dorsi; chicken: Peitoralis profundus and Peitoralis superficialis) by detection, isolation and identification of the isolated; 3) Evaluation of the toxicogenic profile of the isolated by the detection of the genes tcdA and tcdB, which are encoding of TcdA and TcdB respectivelly, and the capacity of toxine production by the isolated bacterias. From the comparison of the two proceeding above it was observed that the direct plating was more efficient and recovered a larger aumont of C. difficile than the treatment with alcohol. Furthermore, the CDMNA agar presented a higher recovery rate compared to CCFA agar. It was observed the occurrence of C. difficile in 11,5% (17/147) of the analyzed samples, comprising 80 isolates, of which 41,2% (33/83) showed a positive response for at least one virulence gene (A-B+), or for both genes (A+B-). In addition, there was a 70,5% concordance between the phenotypic and genotypic tests used to detect toxins. In this way, it is suggested that foods of animal origin are a potential source of transmission of C. difficile for humans
Mestrado
Tecnologia de Alimentos
Mestre em Tecnologia de Alimentos
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Davies, Abigail. "Structure and biochemical analysis of toxins from the superbug Clostridium difficile." Thesis, University of Bath, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619282.
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Clostridium difficile is a gram positive, anaerobic bacterium that is the leading cause of antibiotic-associated pseudomembranous colitis worldwide. C. difficile is an extremely infectious bacterium that produces spores that are highly resistant to standard disinfectant agents and can survive on surfaces for long periods of time. Both the resistance of the spores combined with multiple patients with low-immune systems has lead to an increase in hospital-acquired C. difficile infection, which has had a severe economic impact on the healthcare system. Due to the emerging antibiotic resistance problems and the common occurrence of patient relapse using the current drugs of choice, alternative therapeutic avenues are being explored. C.difficile produces two potent exotoxins; Toxin A and Toxin B that are the causative agents of infection. These toxins have multi-modular domain organisations, with each domain playing a role in cytotoxicity. Some of these domains have been characterised structurally using X-ray crystallography. In this thesis, the low resolution SAXS structure of Toxin A will be presented along with the advances made towards determining the X-ray crystallographic structure of the full-length Toxin A. In addition to Toxins A and B, some strains of C. difficile produce a binary toxin, CDT, which is made up of two individually produced components, CDTa and CDTb. The CDTa component is the enzymatically active component, whereas CDTb is the transport component, directly involved in translocating CDTa into target cells. The precise role of CDT in pathogenesis is unclear, however there is evidence that CDT ADP-ribosylates monomeric actin in target cells, but the detailed mechanism by which this reaction takes place is unknown. Here site directed mutagenesis of key residues of the active site of CDTa was performed and the effect of these mutations on the enzyme’s cytotoxicity tested. By separately mutating three active site residues, the cytotoxic effect of CDTa can be completely eradicated, details of which will be discussed in this thesis. Additionally, the progress made towards determining the X-ray crystallographic structure of the transport component, CDTb, will be discussed.
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Martinez, Ramon D. "Purification and characterization of Clostridium sordellii toxins HT and LT and comparison to toxins A and B of Clostridium difficile." Diss., Virginia Polytechnic Institute and State University, 1989. http://hdl.handle.net/10919/54238.
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Clostridium sordellii cause gas gangrene in man and animals, and more recently it has been implicated as a causal agent of diarrhea and enterotoxemia in domestic animals. This organism was once believed to cause pseudomembranous colitis (PMC) in humans, however, Clostridium difficile, not C. sordellii, was found to be the causative agent of this disease. It is now known that C. difficile produces two toxins, designated A and B, that are implicated in the pathogenesis of the disease. C. sordellii produces two toxins, designated HT (Hemorrhagic Toxin) and LT (Lethal Toxin), that are similar to toxins A and B of C. difficile. The goal of my research was to purify and characterize the two toxins of C. sordellii, and compare their properties to those of C. difficile. Toxin HT was purified from C. sordellii (VPI strain 9048) culture filtrate by ultrafiltration through an XM-100 membrane filter and immunoaffinity chromatography using a monoclonal antibody to toxin A of C. difficile as the ligand. Toxin LT was purified to 80% homogeneity by ultrafiltration on an XM-100 membrane filter and ion-exchange chromatography. Toxin HT migrated as a major band with molecular weight of 525,000 and a minor band at 450,000 on non-denaturing PAGE. By SDS-PAGE the molecular weight was estimated at 300,000. Isoelectric focusing indicated a pI of 6.1. Like toxin A, toxin HT was cytotoxic to cultured cells, lethal for mice, and elicited an accumulation of hemorrhagic fluid in rabbit ileal loops. Toxin LT exhibited properties similar to toxin B, although LT was about a 1000-fold less cytotoxic than toxin B. By SDS-PAGE the molecular weight was estimated at 260,000. Immunodiffusion analysis revealed a reaction of partial identity between these toxins and their amino-terminal sequences were very similar.
Toxins HT and LT of C. sordellii have retained remarkable immunological similarities as well as physicochemical and biological properties with toxins A and B of Q. difficult however the toxins are not identical.Ph. D.
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Vohra, Prerna. "Clostridium difficile : expression of virulence factors, resistance to disinfectants and interactions with human cells." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/6490.
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Clostridium difficile is the most common cause of nosocomial diarrhoea today. Through the changing epidemiology of C. difficile infection, the emergence and decline of different strains of varying virulence and a broad spectrum of disease from asymptomatic carriage and mild infection to severe pseudomembranous colitis have been observed. The main aim of this three-part thesis was to identify bacterial factors that might explain these variations by comparing five C. difficile strains - strain 630, an historic strain, strain VPI 10463, a reference strain, the hypervirulent ribotype 027 and the current locally endemic ribotypes 001 and 106. The first study focussed on the growth-related phenotypic and genotypic expression of virulence factors in C. difficile. Growth was studied over twenty-four hours, with simultaneous assessment of toxin and spore production. Total toxin production was measured by a commercial ELISA, while a quantitative ELISA for toxin A and a quantitative cytotoxicity assay for toxin B were developed for individual toxin levels, and spores were enumerated by viable counts. Ribotype 027 produced large amounts of toxin A and toxin B and was the second highest spore producer after ribotype 106. Growth may not affect virulence, but the ability to produce more toxins and spores could. To study the transcription of the genes involved in these processes, a real-time RT-PCR was developed. The transcription of the pathogenicity locus (tcdA-E) that regulates toxin production in C. difficile, and of spo0A, the initiator of sporulation, was studied. There were three key observations: firstly, the transcription of tcdC, the negative regulator of toxin production, did not decrease over time, suggesting it has a modulatory rather than repressive effect on the process. Secondly, tcdE expression was highest in ribotype 027, which might explain its hypertoxicity by greater toxin release. Thirdly, there was almost steady state expression of spo0A during the exponential growth phase in ribotypes 106 and 027, the highest spore producers, suggesting prolonged activation of sporulation. Thus, distinct inter-strain differences exist between C. difficile strains in vitro, which could mirror their virulence in vivo, and several traits contribute synergistically to the hypervirulence of ribotype 027. The second study aimed to identify suitable laboratory disinfectants against C. difficile. The efficacy of four commonly-used disinfectants and one decontaminant was tested; one disinfectant was a chlorine-based agent commonly used in hospitals. In conventional susceptibility tests, all five agents were effective against vegetative cells and spores of C. difficile. However, only the chlorine-based disinfectant was effective against spores dried onto surfaces, but this too required more than two minutes of treatment. The presence of organic matter significantly impaired the efficacy of the non-chlorine agents. The spores of epidemic strains were destroyed less effectively and exposure to sub-MIC levels of disinfectant increased sporulation, especially in ribotype 001, a common outbreak strain. Environmental sampling of the laboratory and surrounding areas showed considerable dissemination of C. difficile, highlighting the need for effective decontamination in conjunction with basic hygiene methods like hand-washing. The third study examined the biological activity of C. difficile. Macrophages were challenged in vitro with S-layer proteins, flagella, heat-shock proteins and culture supernatants of the five strains and cytokine production was measured by specially developed ELISAs. No significant inter-strain differences were observed, although the epidemic strains generally elicited a slightly greater cytokine response. Using epithelial cell lines it was observed that epidemic strains showed greater adherence; from inhibition assays, flagella and S-layer proteins were found to contribute equally to this. Through these studies, inter-strain differences between epidemic and historic isolates were identified with respect to virulence factors, survival in the environment and possible behaviour within the host. A sum of these observations suggests increased virulence in contemporary versus historical C. difficile strains. Finally, a supplementary study characterising a collection of ribotype 027 strains isolated in Scotland and the Netherlands by typing schemes, gene sequencing, susceptibility testing and phenotypic studies was performed. In agreement with other studies, the clonality of these hypervirulent strains was observed.
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Jefferson, Kimberly Kay. "Clostridium difficile toxins A and B: exploring the possible mechanism of action." Thesis, Virginia Tech, 1995. http://hdl.handle.net/10919/45056.
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Clostridium difficile is a common cause of antibiotic-associated diarrhea and
occasionally causes the life-threatening disease pseudomembranous colitis. The
pathogenicity of the organism has been attributed to the production of two large
exotoxins, toxin A (308,000 daltons) and toxin B (269,000 daltons). Toxin A is a
powerful enterotoxin and is generally thought to play the more important role in the
pathology of the disease. Toxin B may exert its effect after the initial tissue damage by
toxin A. Both toxins cause rounding of mammalian culture cells by disrupting the
cytoskeletal system. The similar biological activities and high percentage of sequence
homology between the two toxins suggest that they have a similar mechanism of action.
I found that purified preparations of both toxins cleave skeletal muscle actin at a single
site, producing a 38,000 dalton actin fragment, and that the toxins are capable of
autodigestion. The proteolytic activity may be involved in the mechanism of action of
the toxins. I also analyzed an aberrant strain of C. difficile which reportedly lacked the
gene for toxin B. Such a strain would be very useful for the study of the mechanism of
toxin A. I concluded however, that the strain contained the genes for both toxin A and
toxin B. The toxin genes and resulting proteins appear, however, to be slightly different
from those of other strains.
Master of Science
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Lewallen, Daniel M. "Development of synthetic carbohydrates for capturing toxins." University of Cincinnati / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1288969515.
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Barroso, Lisa Ann. "Effect of Autoregulated TxeR on the Expression of Clostridium difficile Toxins." Thesis, Virginia Tech, 1999. http://hdl.handle.net/10919/35988.
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Clostridium difficile is a major nosocomial pathogen responsible for causing pseudomembranous colitis. It is estimated that 25% of antibiotic-associated diarrhea is due to C. difficile. These diseases result from intestinal tissue damage caused by two of the largest known bacterial toxins, A and B. Molecular studies of the C. difficile toxins have identified a 19.6 kb toxigenic element that contains both toxin genes flanked by three small open reading frames (ORFs). The focus of this study is to elucidate the function of the ORF, designated txeR, which is located at the beginning of the toxigenic element. The deduced amino acid sequence of txeR predicts a 22-kDa protein that contains a helix-turn-helix motif characteristic of DNA binding regulatory proteins. To determine if the protein TxeR regulates expression from the toxA, toxB, and txeR promoters, gene fusions were constructed that contained the various promoter regions and a reporter gene. The immunodominant region of toxin A located at the carboxy-terminus, termed the repeating units (ARU), was selected as the reporter gene. Expression studies were performed in Escherichia coli host strains. Levels of ARU expression were measured by enzyme-linked immunosorbent assay using an ARU-specific monoclonal antibody.
Expression levels of ARU from the toxin B promoter region with TxeR supplied on the same plasmid (in cis) or on a different plasmid (in trans) were determined. In cis, ARU levels were 50-fold higher than strains without txeR. In trans, expression of ARU from the toxin B promoter region increased over 800-fold. When TxeR was supplied in trans to a toxin A promoter region-ARU fusion, expression levels of ARU increased over 500-fold. To test for autoregulation, TxeR was supplied in trans to the txeR promoter region fused to ARU. The effect was an increase of ARU expression up to 20-fold over background. These results suggest that TxeR is a trans-acting regulator that stimulates expression of the C. difficile toxins and is subjected to autoregulation.
Master of Science
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Strysio, Oliver Moritz [Verfasser]. "Molecular characterization of single-domain antibodies against Clostridium difficile toxins / Oliver Moritz Strysio." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2020. http://d-nb.info/1223621030/34.
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Monaghan, Tanya Marie. "Circulating antibody and memory B cell responses to Clostridium difficile toxins A and B." Thesis, University of Nottingham, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.595681.
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In C. difficile infection (COl), the antibody-mediated immune response to secreted toxins A and B appears to be important in determining the nature of clinical disease. The aim of this study was to evaluate multiple aspects of the humoral immune response to C. difficife toxins A and B in patients with C. difficile-associated disease (COAO), among which included subjects with inflammatory bowel disease. Humoral immune measurements were also quantified for healthy controls and patients with cystic fibrosis (CF) who have been reported to rarely develop COl. Isolated peripheral blood mononuclear cells (PBMCs) were fluorescently labelled with Alexa Fluor® 488 and other B cell markers to identify transiently circulating toxin A-specific antigen-activated B cells during clinical disease by flow cytometry. PBMCs were also polyclonally stimulated in vitro for 6 days to induce proliferation and differentiation of memory B cells (MBCs) to antibody-secreting cells (ASCs). ELiSPOT assays were used to quantify toxin A- and B-specific IgG ASCs. Toxin A- and B-specific antibody levels in sera and supernatant samples of cultured PBMCS were studied by ELISA. A small proportion of toxin A-specific, antigen-activated B cells were detected in the circulation soon after COl. Differential antibody and MBC responses to C. difficile toxins A and B were detected over many months following CDl and in CF patients. The magnitude of these responses did not significantly differ between patients with single and recurrent CDAD. A greater proportion of toxin B-specific MBCs were present in the peripheral circulation in the CDAD and CF groups. No correlation was seen between the frequency of circulating toxin-specific antigen-activated or MBCs and contemporaneous serum anti-toxin IgG levels in either group. These data suggest that serum anti-toxin antibodies can underestimate the breadth of humoral immunity and strongly support the use of toxin-specific B (memory) cell analyses to complement serological studies.
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Gustafsson, Agneta. "Antibiotic associated diarrhea in horses : with special reference to Clostridium difficile /." Uppsala : Dept. of Large Animal Clinical Sciences, Swedish Univ. of Agricultural Sciences, 2004. http://epsilon.slu.se/v166.pdf.
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Kasendra, Magdalena Julia <1985>. "Clostridium difficile toxins facilitate bacterial colonization by modulating the fence and gate function of colonic epithelium." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6364/.
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The contribution of Clostridium difficile toxin A and B (TcdA and TcdB) to cellular intoxication has been extensively studied, but their impact on bacterial colonization remains unclear. By setting-up two- and three-dimensional in vitro models of polarized gut epithelium, we investigated how C. difficile infection is affected by host cell polarity and whether TcdA and TcdB contribute to such events. Indeed, we observed that C. difficile adhesion and penetration of the epithelial barrier is substantially enhanced in poorly polarized or EGTA-treated cells, indicating that bacteria bind preferentially to the basolateral cell surface. In this context, we demonstrated that sub-lethal concentrations of C. difficile TcdA are able to alter cell polarity by causing redistribution of plasma membrane components between distinct surface domains. Taken together, the data suggest that toxin-mediated modulation of host cell organization may account for the capacity of this opportunistic pathogen to gain access to basolateral receptors leading to a successful colonization of the colonic mucosa.
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Hammond, Georgia Ann. "The toxigenic element of Clostridium difficile strain 10463 and its transcriptional analysis in strains which differ in toxigenicity." Diss., Virginia Tech, 1994. http://hdl.handle.net/10919/37447.
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Clostridium difficile is a Gram positive, anaerobic bacterium which produces two potent protein toxins, A and B. The genes for toxins A and B have been previously cloned and sequenced and lie within 1.4 kb of each other.
Upstream and downstream boundaries between sequences shared by both toxigenic and nontoxigenic strains and those sequences which are unique to toxigenic strains were established.
A toxigenic element was defined in C. difficile strain 10463 which is 19.6 kb in length and is comprised of five open reading frames, including the toxin A and B genes. One of these open reading frames is previously unidentified and is located upstream of toxin B.
Products of Polymerase Chain Reaction (PCR) amplification of three regions in the toxigenic element: the upstream boundary, the downstream boundary, and the region between the toxin A and B genes, were all identical in length in six toxigenic strains, indicating that the toxigenic element is conserved among these strains. A short fragment unique to nontoxigenic strains and occupying the same position on the chromosome as the toxigenic element was identified. peR products of this region were identical in length in three nontoxigenic strains.
Transcriptional analyses were undertaken using probes to each of the five open reading frames in the toxigenic element. Transcripts were detected for four of the open reading frames which are contiguous and transcribed in the same direction. In addition, a very large transcript, corresponding to the length of the four open reading frames and processing intermediates were detected, indicating that the toxin genes are cotranscribed. A promoter region and processing sites were identified. Sizes were determined for each of the individual transcripts which correspond well with the sizes of the open reading frames.
Six toxigenic strains which vary considerably in toxin production were selected for analysis to determine whether DNA sequence variation could account for the observed differences in toxin production. DNA restriction fragment length polymorphisms were examined, toxin-specific transcripts were analyzed, and sequences of regulatory regions were determined and compared. Whereas quantitative differences in toxin-specific transcripts were found among the toxigenic strains, the remaining analyses showed that DNA sequences were conserved among these strains.Ph. D.
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Du, Timothy. "Use of an in-vitro polarized cell culture model to study the translocation of Clostridium difficile toxins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0011/MQ41694.pdf.
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Zhu, Ziyu [Verfasser]. "Bacitracin reduces the cytotoxic effects of Clostridium difficile toxins A and B on mammalian cells and human intestinal organoids / Ziyu Zhu." Ulm : Universität Ulm, 2021. http://d-nb.info/1228439109/34.
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Perelle, Sylvie. "Toxine IOTA de "Clostridium perfringens" et toxines apparentées." Paris 11, 1996. http://www.theses.fr/1996PA114811.
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Ticchi, Laurence. "Clostridium difficile et ses toxines : prévalence de "Clostridium difficile" et de la toxine A asymptomatique." Paris 5, 1990. http://www.theses.fr/1990PA05P183.
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Cosmetatos, Isabelle Cosmetatos Isabelle. "Fecal isolation of "Clostridium difficile" and its toxins in horses with typhlo-colitis : Oral administration of neomycin and phthalylsulfathiazole in horses : effects on clinical, hematological and hematochemical parameters and influence on the isolation rate of "C. difficile" in feces /." [S.l.] : [s.n.], 1995. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Kerzmann, Amy N. "Mechanistic analysis of Clostridium difficile toxin A." [Bloomington, Ind.] : Indiana University, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3378359.
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Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2009.Title from home page (viewed on Jul 12, 2010). Source: Dissertation Abstracts International, Volume: 70-10, Section: B, page: 6182. Advisers: Andrew L. Feig; James T. Drummond.
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Almdni, Sabir M. Shakir. "Recombinant antibodies against Clostridium difficile toxin A." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4727/.
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Clostridium difficile is a major cause of nosocomial intestinal infection. The pathogen possesses two potent toxins, Toxin A and Toxin B, both of which contribute to diarrhoea, intestinal inflammation and tissue damage. Antibiotics are effective against the disease, however around 20 % of patients on treatment relapse after the termination of antibiotic therapy. The binding of Toxin A to a receptor on human intestinal epithelial cells initiates disease: this is considered the starting point from which the toxin elicits its effect. One feature of the carboxy-terminal domain of Toxin A is the presence of repeating units of amino acids that form a series of binding sites able to recognise disaccharides and trisaccharides on glycolipid and glycoprotein receptor molecules. Antibody response against the toxin can protect against C. difficile disease and efforts to generate vaccines have focused upon the carboxy-terminal, receptor binding domain. The aims of this project were to use phage display to isolate recombinant antibodies against those features of the carboxy-terminal domain of Toxin A thought to be responsible for receptor-binding and to assess if the antibodies were capable protecting against the action of Toxin A. Using published crystallographic data that has shown the interaction of Toxin A and trisaccharide, a region of about 113 amino acids from the carboxy-terminal region of Toxin A was expressed as a fusion to maltose-binding protein. The MBP fusion protein was expressed, purified on amylose resin, and characterised. The fusion protein was then used to isolate single chain antibodies from the Tomlinson libraries of scFvs, a synthetically diversified phage display library of single scaffold human antibodies. Conventional bio-panning methods were used in which the MBP fusion protein was bound to a plastic surface and the phage display libraries were pre-mixed with native MBP to inhibit the isolation of anti-MBP antibodies. Progressive enrichment of scFvs through 3 rounds of selection was observed. Those scFvs that showed strongest reaction against the target protein in ELISA but failed to react with native MBP were sequenced, expressed as soluble antibodies and purified on nickel chelating columns. While the resulting panel of scFvs showed similarities of sequence, none were identical. All were reactive with native, full-length Toxin A and appeared to bind to conformational (nonlinear) epitopes. Cross-reaction with Toxin B from C. difficile was also evident. A panel of truncation mutants were generated from the MBP fusion protein and using these in ELISA with the scFvs, reactivity appeared to be directed to features of a long repeat sequence of Toxin A. To assay whether the isolated scFvs possessed biological activity of significance, in vitro and in vivo protection assays were established. For experiments in vitro, the action of Toxin A upon cultured Vero cells was studied. Native Toxin A triggered a conversion of the cells from stellate to rounded morphology. When cells were exposed to 100 ng of toxin, this effect was evident within 60 minutes; at a 10-fold lower dose, the minimum quantity to which a response was detectable, virtually all cells had undergone rounding within 2 h. When individual scFvs were mixed with 10 ng of Toxin A prior to addition to Vero cells, there was a consistent delay in cytopathic activity that extended to 5 h. In this assay, the percentage of cells that had retained their stellate morphology 5 h post-challenge was dependent on the scFv used. To quantify the potency of this neutralising activity, the amount of each scFv required to achieve 50% protection during a 2 h challenge period was established. This revealed 3.5-fold difference between the most and the least effefctive scFv. The most potent scFv was used in an in vivo assay in which Toxin A was administered to the ligated intestinal loops of rats. Again, protective activity was evident. Overall, phage display technology enabled the assembly of a panel of scFv antibodies against the putative receptor binding site in the carboxy-terminal domain of Toxin A from C. difficile. The scFvs were able to protect against the cytopathic activity of Toxin A in vitro and in vivo and proposals are made about how these observations could be taken forward in a model of C. difficile infection that best mimics the human disease.
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Lim, Chien-Sen Jenson. "Functional studies of toxin A from Clostridium difficile." Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.407433.
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Craggs, Joanna K. "Structure-function relationships of Clostridium difficile toxin A." Thesis, University of Nottingham, 1999. http://eprints.nottingham.ac.uk/12047/.
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Ten overlapping fragments covering the entire Clostridium difficile toxin A gene were cloned and expressed in Escherichia coli. Eight fragments (a', a2, b, c, d, e, f and g) represented the first 5.55kb of the gene whereas two fragments (hl and h2) each spanned the entire C-terminal repeat region of the molecule. All activities relating to binding to carbohydrates (i. e. cold haemagglutination of rabbit erythrocytes), binding to bovine thyroglobulin and non-specific binding to a murine monoclonal antibody were restricted solely to peptides H1 (amino acids [aa] 1834-2683) and H2 (aa 1832-2683). Peptide H2 alone also displayed the ability to bind to cells and to be internalised into endosome-like compartments within the cells. Taken together with the observation that peptide H2 caused a cytopathic effect on Vero cells which was atypical of the holotoxin, these results may indicate that the repeat region of toxin A stimulates intracellular signalling pathways prior to Rho glucosylation. Peptide A2 (aa 1-536) glucosylated recombinant RhoA (rRhoA) in vitro, whereas peptides A'(aa 1-205), B (aa 542-859), C (aa 114-859), D (aa 869-1330), E (aa 542- 1161), G (aa 869-1830) and H2 (aa 1832-2683) did not. The results obtained for peptides A', A2 and C indicate that the first 536 as encompass the catalytic domain for this activity, that more than the first 205 as alone are needed for expression of enzymic activity, and that for a peptide to be active it must not lack the first 113 aa. The first 113 as of the holotoxin are probably essential for the correct folding of the catalytic domain and expression of its activity. These studies were also the first to locate the toxin A ATP binding site to a peptide spanning as 542-859 (peptide B) of the holotoxin. Antibody reaction profiles of antiserum to holotoxin A against toxin A peptides and of antiserum to the peptides against holotoxin A indicate that this region is unexposed in the native state. Also of interest was the observation that the only peptides, which contained the nucleotidebinding site (B and E), lacked the ability to glucosylate rRhoA. Further peptide A2, which possessed glucosyltransferase activity, lacked the nucleotide-binding site. These studies therefore, suggest that a nucleotide-binding site is not required for in vitro glucosylation of rRhoA by toxin A, and fail to identify a role for the toxin A nucleotide binding site. An engineered truncated form of toxin A, consisting of the first 539 as of the holotoxin (encompassing glucosyltransferase activity) fused to the 852 as C-terminal peptide H2 (repeat end binding portion) caused a conventional cytopathic effect (CPE), but was 1,400 fold less cytotoxic to Vero cells than the holotoxin. Peptide A2 (aa 1-536) alone had no effect on Vero cells or in rabbit ileal loops suggesting that peptide H2 aided delivery of the glucosyltransferase molecule into cells leading to a CPE. The truncated toxin lacked the nucleotide binding site and the putative membrane-translocating domain (internal hydrophobic region). The reduced activity of the truncated toxin suggests that although not essential for cytotoxic activity, the nucleotide-binding site and the internal hydrophobic region are important for stability and/or efficient translocation of the holotoxin into the cytosol.
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Burger, Silke. "Darstellung und Charakterisierung von rekombinantem Clostridium-difficile-Toxin A." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=974133590.
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Agoumellah, Fatiha. "Pathologie de Clostridium difficile chez le nourrisson de moins d'un an." Paris 5, 1998. http://www.theses.fr/1998PA05P199.
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Frey, Steven M. "The localization of two epitopes recognized by the monoclonal antibody PCG-4 on toxin A of Clostridium difficile." Thesis, This resource online, 1990. http://scholar.lib.vt.edu/theses/available/etd-05022009-040613/.
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Krencker, Diane. "Diarrhees associees a la presence de clostridium difficile et/ou de sa toxine : a propos de 76 cas." Université Louis Pasteur (Strasbourg) (1971-2008), 1991. http://www.theses.fr/1991STR1M188.
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BALDACINI, OLIVIER. "La cytotoxine de clostridium sordellii. Etude comparee a la toxine b de clostridium difficile." Université Louis Pasteur (Strasbourg) (1971-2008), 1992. http://www.theses.fr/1992STR13037.
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La mise au point d'un protocole de purification rapide de la cytotoxine ou de la toxine l de clostridium sordellii nous a permis de comparer ses caracteristiques physicochimiques et biologiques et ses modalites d'action avec celles de la cytotoxine ou toxine b de clostridium difficile, egalement purifiee dans notre laboratoire. La toxine l et la toxine b, bien que presentant des homologies sur le plan immunologique, induisent des modifications morphologiques cellulaires distinctes: les deux toxines provoquent la depolymerisation des microfilaments mais les caracteristiques morphometriques et la distribution de l'actine f residuelle different selon que les cellules sont traitees par la toxine l ou la toxine b. Ces deux cytotoxines entrainent egalement des variations de phosphorylation de proteines cellulaires et en particulier de la tropomyosine et de la vimentine, deux composants du cytosquelette. Enfin, nous avons montre qu'un inhibiteur de phosphatases, l'acide okadaique, et la toxine l utilisent des mecanismes d'intoxication en partie communs. L'ensemble des resultats obtenus permet de proposer de nouvelles hypotheses quant aux mecanismes d'action des cytotoxines l et b, lesquelles agiraient en empruntant les voies de la phosphorylation-dephosphorylation cellulaires
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Cavalcante, Ingrid Chaves. "Study of a new adenosine receptor A2A agonist, ATL313, on Clostridium difficile toxin A-induced enteritis in ileal pouch isolated of mice." Universidade Federal do CearÃ, 2005. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=84.
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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgicoC. difficile toxin A (TxA) plays an important pathogenic role in antibiotic-induced diarrhea and pseudomembranous colitis, a condition characterized by intense mucosal inflammation and secretion. Agonist activity at A2A adenosine receptors (A2A ARs) attenuates inflammation and damage in many tissues. This study evaluated the effect of a new selective A2A AR agonist (4-{3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}piperidine-1-carboxylic acid methyl ester; ATL313) on TxA-induced enteritis in murine ileal loops. ATL313 (0.05-5 nM) and/or the A2A AR antagonist (ZM241385; 5 nM) or PBS were injected inside ileal loops immediately prior to challenge with TxA (1-10 mg/loop) or PBS. Intestinal fluid volume/length and weight/length ratios were calculated 3 h later. Ileal tissue samples were collected for measurement of myeloperoxidase (MPO) content, evaluation of ADA activity, for histopathology and apoptotic immunohistochemistry (ApopTagÃ) and for assessment of TNF-α levels by ELISA. TxA (1-10 Âg/loop) significantly (p<0.05) increased volume/length and weight/length, reaching maximum values at 5Âg/loop dosage. ATL313 (5 nM) treatment significantly (p<0.05) reduced TxA-induced volume/length and weight/length, as well as prevented mucosal disruption and TxA-induced apoptosis. These protective effects were reversed by ZM241385 (5 nM), the A2A AR antagonist. ATL313 (5 nM) also reduced neutrophil infiltration, as measured by MPO content; reduced the toxin A-induced increase in ADA activity. Prior to the challenge with TxA, a systemic injection of fucoidin, but not PBS, also reduced tissue destruction and toxin A-induced increase in ADA activity. In conclusion, the A2A AR agonist ATL313 has a great antiinflammatory effect in TxA-induced mice enteritis, significantly reducing tissue destruction and ADA activity. In addition, our data suggested that TxA-induced increase in ADA activity and tissue damage in murine ileal loops are related to the neutrophil infiltration induced by this toxin.
A toxina A do Clostridium difficile (TxA) desempenha um importante papel na patogÃnese da diarrÃia induzida por antibiÃticos e na colite pseudomembranosa, uma condiÃÃo caracterizada por intensa secreÃÃo e inflamaÃÃo da mucosa. A estimulaÃÃo de receptores A2A da adenosina reduz a inflamaÃÃo e o dano tecidual. Neste estudo, avaliou-se o efeito de um novo agonista seletivo para receptores A2A da adenosina (metil Ãster do Ãcido 4-{3-[6-amino-9-(5-ciclopropilcarbamoil-3,4- dihidroxitetrahidrofuran-2-il)-9H-purin-2-il]prop-2-inil}piperidina-1-carboxÃlico; ATL313) na enterite induzida pela TxA em alÃas ileais de camundongos. O ATL313 (0,05-5 nM) e/ou o antagonista dos receptores A2A da adenosina (ZM241385; 5 nM) ou PBS foram injetados em alÃas ileais imediatamente antes da injeÃÃo de TxA (1-10 Âg/alÃa) ou PBS. As razÃes volume de secreÃÃo/comprimento da alÃa e peso/comprimento da alÃa foram calculadas 3h depois. Amostras de tecido foram coletadas para dosagem de atividade de mieloperoxidade (MPO), atividade de ADA, histopatologia, imunohistoquÃmica para apoptose (ApopTag_) e dosagem de TNF-a_ por ELISA. A injeÃÃo de TxA (1-10 Âg) nas alÃas ileais aumentou significativamente (p<0,05) as razÃes volume de secreÃÃo/comprimento da alÃa e peso/comprimento da alÃa com pico em 5Âg. O tratamento das alÃas com ATL313 (5 nM) reduziu significativamente (p<0,05) a secreÃÃo e o edema, preveniu a destruiÃÃo da mucosa e a apoptose induzidos por TxA. Tais efeitos protetores foram revertidos pelo antagonista dos receptores A2A de adenosina, o ZM241385 (5 nM). O tratamento com ATL313 (5 nM), reduziu ainda a infiltraÃÃo neutrofÃlica, avaliada pela dosagem de MPO, e reduziu o aumento da atividade de ADA induzidos pela TxA, bem como a dosagem de TNF-a no tecido das alÃas ileais. O prÃ-tratamento sistÃmico com fucoidina, mas nÃo com PBS, tambÃm reduziu o dano na mucosa e atividade de ADA no tecido das alÃas ileais tratadas com TxA. Assim, conclui-se que na enterite induzida pela TxA em camundongos, o agonista dos receptores A2A da adenosina (ATL313) possui um potente efeito antiinflamatÃrio, reduzindo consideravelmente a lesÃo tecidual e a atividade de ADA. Nossos resultados tambÃm indicam que o aumento da atividade de ADA e o dano tecidual induzido pela TxA em alÃa ileal de camundongos està relacionado com a infiltraÃÃo neutrofÃlica induzida por esta toxina.
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Santos, Ana Angélica Queiroz Assunção. "Estudo das alterações morfológicas induzidas pela Toxina A, do Clostridium difficile em células epiteliais intestinais e do efeito protetor da Glutamina e Alanil-glutamina." reponame:Repositório Institucional da UFC, 2011. http://www.repositorio.ufc.br/handle/riufc/6909.
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SANTOS, Ana Angélica Queiroz Assunção. Estudo das alterações morfológicas induzidas pela Toxina A, do Clostridium difficile em células epiteliais intestinais e do efeito protetor da Glutamina e Alanil-glutamina. 2011. 120 f. Dissertação (Mestrado em Ciências Médicas) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2010.Submitted by denise santos (denise.santos@ufc.br) on 2013-12-06T13:02:50Z No. of bitstreams: 1 2011_dis_aaqasantos.pdf: 6513486 bytes, checksum: b4873c52295127b43af4843d288a42bf (MD5)
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Clostridium difficile is the major cause of antibiotic-associated colitis, a disease with significant morbidity and mortality. Glutamine (Gln), a non-essential aminoacid, is a major fuel for the dynamic intestinal cell population. Alanyl-glutamine (Ala-Gln) is a dipeptide that is highly soluble and well tolerated. The aim of this study was to analyze the changes induced by Clostridium difficile toxin A (TcdA) in intestinal epithelial cell morphology and cytoskeletal element and the effect of Gln and Ala-Gln treatment, using advanced microscopic techniques. Twelve well cell culture plates, with 13 mm diameter glass coverlids, were seeded with 5x105 IEC-6 cells and grown for 24h in DMEM media. Afterwards, the wells were incubated for 24h as follow: control, TcdA (10 ng/mL), TcdA + Gln (10 mM) and TcdA + Ala-Gln (10 mM). The cells were than fixed in 4% formaldehyde for 14 h and afterwards they were examined by atomic force microscopy (AFM), scanning electronic microscopy (SEM) and Fluorescent microscopy. For the SEM the samples were fixed to samples holders with carbon adhesive tape and covered with a 15 mm gold film for conductivity by sputter. To fluorescent microscopy the cells were permeabilizided with PBS/Triton after that they were marked with stained with FITC-RhoA, Rodhamine-phallodin and DAPI performed using an inverted fluorescence microscope. An immunoblotting was realized with the same groups. The PVDF membrane was incubated with RhoA antibody overnight and afterwards activated by Amershan kit. The proteic control was made by α- tubulin. Also performed experiments of cellular proliferation and oxidative stress. As observed by AFM, SEM and Fluorescent microscopy TcdA caused intense cell shrinkage with multiple extensions. This change in shape was associated with collapse of the F-actin cytoskeleton demonstrated by fluorescent microscopy. An increase of RhoA production was detected in the groups treated with Gln e Ala-Gln. We demonstrated that TcdA dramatically altered the intestinal cell morphology and cytoskeleton organization and that Ala-Gln and Gln supplementation consistently prevented the intestinal epithelial cell damage induced by TcdA probably by increasing RhoA expression. The TcdA induced a reduction of 8.4% in cell proliferation while the Ala-Gln and Gln increased by 13.2% and 12.7%, respectively. The TcdA induced cells to oxidative damage, which was reversed by the use of Gln and Ala-Gln.. Our findings provide rationale for the potential use of Ala-Gln and Gln as adjuvant therapy in Clostridium difficile disease. Investigation of morphological and cytoskeleton changes using advanced microscopic techniques may aid in the evaluation of the protective or therapeutic activity of drugs against TcdA effects.
Clostridium difficile é a maior causa de colite associada ao uso de antibióticos, com significante morbidade e mortalidade. Glutamina (Gln), um aminoácido não-essencial, é a maior fonte combustível para a dinâmica das células intestinais. Alanil-glutamina (Ala-Gln) é um dipeptídeo, altamente solúvel e bem tolerado. O objetivo deste estudo foi analisar as alterações induzidas pela toxina A (TcdA) do Clostridium difficile na morfologia e elementos do citoesqueleto das células epiteliais intestinais e o efeito do tratamento com Gln e Ala-Gln, utilizando técnicas avançadas de microscopia. Placas de cultura de células com doze poços, previamente acrescidas de lamínulas de vidro, com diâmetro de 13mm, IEC-6, 5x105 foram semeadas e cultivadas por 24 horas em meio DMEM. Depois, as células foram incubadas por 24h de acordo com os seguintes grupos: Controle, TcdA (10 ng / mL), TcdA + Gln (10 mM) e TcdA + Ala-Gln (10 mM). As células foram fixados em formol a 4% por 14h, depois foram examinados na microscopia de força atômica (AFM), microscopia eletrônica de varredura (SEM), microscopia confocal e fluorescente. Para o SEM as amostras foram fixadas em porta amostras fita adesiva de carbono e cobertos com uma película de ouro 15 mm para adquirir condutividade por pulverização catódica. Para microscopia de fluorescência e o confocal, as células foram permeabilizadas com PBS/Triton depois foram marcados com RhoA- FITC, Faloidina –Rodamina e DAPI e as imagens capturadas através de um microscópio invertido de fluorescência ou confocal. Um immunoblotting foi realizado com os mesmos grupos. A membrana PVDF foi incubada com o anticorpo RhoA overnight, em seguida ativado pelo kit Amershan. O controle protéico foi feito por α-tubulina. Também realizarmos experimentos de proliferação celular e estresse oxidativo. Observou-se que a TcdA causa intenso encolhimento celular restando múltiplas extensões filamentosas. Esta alteração na forma foi associada ao colapso do citoesqueleto de F-actina demonstrada na microscopia de fluorescência. Um aumento da produção RhoA foi detectada no grupo tratado com Gln e Ala-Gln. Demonstramos que a morfologia das células intestinais e organização do citoesqueleto foram dramaticamente alteradas pela TcdA e que a suplementação com Ala-Gln e Gln impediu o dano celular epitelial intestinal induzida pela TcdA provavelmente por aumentar a expressão RhoA. A TcdA induziu uma redução de 8,4% na proliferação celular, enquanto o Ala-Gln e Gln aumentou 13,2% e 12,7%, respectivamente. A TcdA induziu as células ao dano oxidativo, que foi revertido com o uso de Gln e Ala-Gln. Nossos resultados fornecem justificativa para o uso potencial de Ala-Gln e Gln como terapia adjuvante na doença causada pelo Clostridium difficile. Investigação de alterações morfológicas e citoesqueleto usando avançadas técnicas de microscopia pode auxiliar na avaliação da atividade de proteção ou terapêutica de drogas contra os efeitos TcdA.
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El, Meouche Imane. "Etude du régulateur transcriptionnel SigmaD chez Clostridium difficile." Rouen, 2014. http://www.theses.fr/2014ROUES008.
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La principale partie de ce travail porte sur l'étude du facteur SigD de C. Difficile et de son implication dans la régulation de l'autolyse, de la mobilité et de la production de ses deux facteurs majeurs de virulence, les toxines A et B. Nous avons pu montrer, par inactivation du gène sigD, que SigD régule positivement la mobilité de C. Difficile mais n'affecte pas, ou peu son autolyse. Le régulon global de SigD a été ensuite déterminé par une analyse transcriptonique en microarrays. Parmi les gènes sous-exprimés chez le mutant du gène sigD, nous retrouvons les gènes flagellaires tardifs, les gènes des toxines A et B et le gène de leur régulateur TcdR. Nous avons pu identifier des promoteurs SigD-dépendants notamment en amont des gènes de la flagelline FliC, de l'anti-SigD FlgM et deTcdR. De plus, nous avons prouvé que SigD se lie avec l'ARN polymérase pour démarrer la transcription de tcdR, dont il est un régulateur direct. Par ailleurs, nous avons identifié une séquence consensus propre au régulateur SigD chez C. Difficile. Enfin, nous avons déterminé le rôle antagoniste de FlgM, l'anti-SigD, dans la répression des gènes SigD dépendants. SigD s'avère être un régulateur positif et direct de la mobilité et de la synthèse des toxines chez C. Difficile. Une partie complémentaire du travail s'intéresse aux autolysines Acd et Cwp19 de C. Difficile. Des mutants simples des gènes acd et cwp19 ont permis de montrer que seule Cwp19 intervient dans l'autolyse de C. Difficile en présence du TritonX-100. Cette autolysine est impliquée dans la lyse à long terme chez C. Difficile. Un double mutant acd-cwp19 semble avoir le même profil autolytique que le simple mutant cwp19. La glucosaminidase Acd ne semble donc pas avoir une implication majeure dans l'autolyse de C. Difficile. Des analyses complémentaires en cours de réalisation permettront de déterminer l'activité enzymatique de l'autolysine Cwp19The main part of this work focuses on the characterization of the C. Difficile SigD factor and its role in the regulation of autolysis, motility and production of the two major virulence factors, toxins A and B. After the inactivation of sigD, we show that SigD factor positively controls C. Difficile motility whereas its contribution to the autolysis, if any, is modest. The global regulon of SigD was then determined by transcriptonic analysis using microarrays. Among the down-regulated genes in the sigD mutant strain, we find the late flagellar genes, the genes encoding toxins A and B and the gene encoding their regulator TcdR. We could identify SigD-dependent promoters upstream many genes including those encoding the flagellin FliC, the anti-SigD FlgM, and TcdR. In addition, we proved that SigD binds with RNA polymerase to initiate the transcription of tcdR. Furthermore, we identified a specific SigD-dependent consensus sequence in C. Difficile. Finally, we determined the antagonistic role of FlgM, the anti-SigD, in the repression of SigD-dependent genes. We prove that SigD is a positive and direct regulator of motility and toxin synthesis in C. Difficile. Another part of the work focuses on the autolysins Cwp19 and Acd of C. Difficile. Single mutants for acd and cwp19 genes showed that only Cwp-19 is involved in the long-term lysis of C. Difficile. A double-mutant acd-cwp19 seems to have the same autolytic profile as cwp19 single mutant. The glucosaminidase Acd does not seem to have a major involvement in autolysis of C. Difficile. Additional analyzes are in progress to determine the enzymatic activity of the Cwp19 autolysin
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Sekulovic, Ognjen. "Étude de l'impact des prophages sur la biologie de Clostridium difficile." Mémoire, Université de Sherbrooke, 2010. http://savoirs.usherbrooke.ca/handle/11143/4049.
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La bactérie Clostridium difficile est maintenant considérée comme un pathogène majeur responsable d'infections nosocomiales en Amérique du Nord et en Europe. De plus, l'émergence de souches hypervirulentes, telle la souche NAP1/027 responsable de récentes épidémies, est un phénomène inquiétant. Un enjeu crucial au cours des prochaines années sera de mieux comprendre les mécanismes de virulence et d'évolution de C. difficile. Les bactériophages (c.-à-d. des virus bactériens, ou phages) sont des joueurs clés dans l'évolution de la plupart des bactéries, pathogènes ou non. Les données concernant l'impact des phages sur C. difficile sont très limitées. Par contre, deux études récentes démontrent que les phages semblent influencer la virulence de C. difficile en altérant la production de toxines TcdA et TcdB. L'objectif de mes travaux de recherche est donc d'étudier au niveau microbiologique et moléculaire les phages de C. difficile et de démontrer leur impact sur l'évolution et la virulence de ce pathogène. Dans ce contexte, plusieurs phages ont été induits à partir d'isolats cliniques de C. difficile. Dans cette collection, un phage en particulier, le (pCD38-2, a été choisi pour la caractérisation subséquente dû à sa divergence génomique par rapport aux autres phages et à sa capacité d'infecter la grande majorité des souches ayant le ribotype hypervirulent (027). Ces caractéristiques uniques ont justifié le séquençage complet de son génome. Par contre, aucun facteur de virulence évident n'a été identifié. À l'opposé, une analyse bio-informatique a permis l'identification d'une région spécifique comportant plusieurs gènes de conversion lysogénique potentiels. L'impact de ces gènes sur la virulence bactérienne reste à être déterminé. De plus, lorsqu'on introduit le phage cpCD38-2 dans la souche sensible CD274, on observe une accumulation plus rapide et plus grande des toxines après 48h dans le surnageant de la culture. Ce phénomène a été confirmé avec des tests ELISA sur des réplicas biologiques indépendants ainsi que par un immunodosage avec anticorps spécifiques aux deux toxines. Par ailleurs, une étude transcriptionelle par PCR en temps réel a permis de constater que le phage (pCD38-2 influence également l'expression des gènes tcdA et tcdB dans le temps. Par contre, l'effet du phage cpCD38-2 est variable lorsqu'on l'introduit dans d'autres souches de C. difficile. Donc, les résultats de nos travaux indiquent que certains phages auraient un impact sur la virulence de C. difficile en altérant la production et la transcription des gènes de toxines. Nos données laissent toutefois sous-entendre que cet effet peut varier selon les souches de C. difficile. [Symboles non conformes]
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SIFFERT, JEAN-CLAUDE. "Le cytotoxine ou toxine b de clostridium difficile : actions sur le systeme des phagocytes mononuclees : implication physiopathologique." Besançon, 1991. http://www.theses.fr/1991BESA3090.
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Foschetti, Danielle Abreu. "Toxinas A e B do Clostridium difficille induzem a expressão diferencial de receptor de Adenosina em células epiteliais intestinais: papel do receptor A2B." reponame:Repositório Institucional da UFC, 2014. http://www.repositorio.ufc.br/handle/riufc/14909.
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FOSCHETTI, D. A. Toxinas A e B do Clostridium difficille induzem a expressão diferencial de receptor de Adenosina em células epiteliais intestinais: papel do receptor A2B. 2014. 160 f. Tese(Doutorado em Farmacologia) – Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2014.Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-01-25T11:59:35Z No. of bitstreams: 1 2014_tese_dafoschetti.pdf: 5271291 bytes, checksum: 110e5955cf8edb636759d629ae0d7b60 (MD5)
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Clostridium difficile is recognized to be a nosocomial pathogen that causes intense intestinal inflammation, epithelial barrier disruption and diarrhea. Adenosine production is increased under inflammatory situations. The adenosine receptor A2B is the most expressed receptor in the human and mice intestine. We investigated the effect of short- and long-term exposure to TcdA and TcdB in HCT-8 cells and isolated cecum epithelial cells. HCT-8 cells were exposed to TcdA or TcdB (10 ng/ml) for 2, 6 and 24h. We used a murine cecal loop model and murine infection model to evaluate the effects of TcdA and C. difficile infection, respectively. We demonstrated that HCT-8 and isolated intestinal cecum epithelial cells naturally express high levels of A2BR receptors. TcdA or TcdB alters the cell morphology, viability and proliferation pattern and caused gene expression increase of all AR subtypes in HCT-8. In isolated cecum epithelial cells, TcdA significantly (p<0.05) increased volume/length, weight/length, histopathology scores, neutrophil infiltration, as measured by MPO content, and induced an altered gene expression increase of all AR subtypes. PSB603 (10 nM) treatment significantly (p<0.05) reduced TcdA-induced tissue damage. Our findings support the hypothesis that Clostridium difficile toxins affect adenosine receptor expression and this action may be related to their severe inflammatory effect. We concluded that adenosine receptors may play a crucial role in regulating the inflammatory system in intestinal epithelium during C. difficile infection.
O Clostridium difficile é reconhecido por ser uma bactéria nosocomial, levando a intensa inflamação intestinal, quebra da barreira epitelial e diarreia. A produção de adenosina está aumenta durante eventos inflamatórios. O receptor de adenosina A2B (A2BR) é o mais expresso no intestino de humanos e camundongos. Nós investigamos o efeito de exposição às TcdA e TcdB, a curto e longo prazo, em células HCT-8 e células epiteliais intestinais isoladas do ceco. Células HCT-8 foram expostas a TcdA ou TcdB (10 ng/ml) por 2, 6 e 24h. Foi usado o modelo de alça cecal e de infecção pelo bacilo em murinos para avaliar os efeitos da TcdA e da infecção pelo C. difficile, respectivamente. Foi demonstrado que HCT-8 e células epiteliais intestinais isoladas do ceco naturalmente expressam altos níveis do receptor A2BR. TcdA e TcdB alteraram a morfologia celular, viabilidade e proliferação e causaram aumento da expressão gênica de todos os subtipos de receptores de adenosina e das citocinas IL-6 e IL-8 em HCT-8. Em células epiteliais intestinais isoladas do ceco, a TcdA significativamente causou um aumento do peso e volume/comprimento da alça cecal, escores histológicos e infiltrado neutrofílico, medido por MPO, e também causou alterações da expressão gênicas dos receptores de adenosina, tanto no modelo de alça cecal quanto na infecção pelo bacilo. O tratamento com PSB603, um antagonista do receptor A2BR, foi capaz de reverter os efeitos causados pelas toxinas do C. difficile. Nossos dados suportam a hipótese que as toxinas do C. difficile alteram a expressão dos receptores de adenosina e isso pode estar relacionado com os severos efeitos inflamatórios. Nós concluimos que os receptores de adenosina tenham um papel importante na regulação da inflamação em células epiteliais intestinais na infecção pelo C. difficile.
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Lister, Michelle M. "Understanding the genetic mechanisms of Clostridium difficile toxin regulation and clinical relapse." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/53216/.
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Clostridium difficile is the leading cause of health care associated diarrhoea and remains a burden for the NHS. Disease symptoms can range from mild diarrhoea through to fulminant pseudomembranous colitis, resulting in mortality for some patients. Recurrence is a major problem and estimates are that 20% of all patients with disease will either relapse (with the same strain) or have a re-infection (with a different strain). Arguably, the main virulence factors are toxins A (TcdA) and toxin B (TcdB) which cause disease symptoms. The genes encoding TcdA and TcdB are located within the pathogenicity locus (PaLoc) along with three accessory genes; tcdR, tcdE and tcdC. The regulatory network has been studied but we aimed to add to this knowledge by using two under investigated strains R20291 a so-called hypervirulent strain and VPI 10463 a strain known to produce higher levels of toxin. Two different methods of investigation were employed during this study to improve our understanding of both the regulation of TcdA / TcdB but also the genetic mechanisms behind clinical relapse. These methods were; using forward and reverse genetic analysis to assess phenotypic differences and using bioinformatics to identify genes and / or single nucleotide variants (SNP) that may play a role. Using a combination these methods we have identified potential regulators of toxin production in both strains. We have also identified unique genes and SNPs that might provide a fitness benefit to strains of C. difficile that were isolated from patients who had suffered relapse episodes.
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Trombert, Sabine. "Acquisition de "Clostridium difficile" chez le nouveau-né et cinétique d'implantation." Paris 5, 1995. http://www.theses.fr/1995PA05P087.
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Beaudoin, Axelle. "Développement et application d'un test ELISA pour l'étude des anticorps dirigés contre clostridium difficile." Mémoire, Université de Sherbrooke, 2009. http://savoirs.usherbrooke.ca/handle/11143/3958.
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Clostridium difficile est un pathogène entérique pouvant causer des diarrhées allant de modérées à sévères, des colites pseudomembraneuses et même la mort. Les traitements actuels contre la bactérie ont des taux de rechutes élevés. De plus, il n'existe pas à ce jour de vaccin permettant de prévenir l'infection. Cette étude épidémiologique porte sur la protection contre la colonisation et/ou la maladie conférée par la présence d'anticorps sériques naturels spécifiques aux antigènes de la bactérie. Nous avons développé un test ELISA (Enzyme-Linked ImmunoSorbent Assay) pour la détection des anticorps contre les toxines A et B de C. difficile et contre les protéines du flagelle (flagellines) dans des échantillons de sérum. Notre test ELISA servant à détecter les anticorps dirigés contre la toxine B a été calibré de façon à obtenir la meilleure corrélation possible avec le test de neutralisation de la cytotoxicité de la toxine B. Les paramètres du test ELISA ainsi mis au point ont été appliqués aux autres antigènes (toxine A et flagellines), pour lesquels un test de référence n'existe pas. Par la suite, nous avons tenté de définir le rôle de la réponse immunitaire humorale de l'hôte en corrélant les résultats de l'analyse des sérums de deux groupes de patients hospitalisés avec les informations sur le suivi des symptômes et la recherche de la présence de la bactérie dans les selles. Le premier groupe de patients nous a permis d'étudier la relation entre la survenue de la colonisation ou de l'infection par C. difficile et la production d'anticorps. Nos résultats montrent que les anticorps sériques ne semblent pas protéger le patient de l'implantation de la bactérie au tube digestif mais que suite à l'infection, une réponse humorale est mise en place contre les toxines du pathogène. Les patients du deuxième groupe, des patients hospitalisés infectés par C. difficile, ont été surveillés pour la survenue de symptômes sévères, du décès ou de rechutes suite à un épisode de maladie. Nous avons observé une plus grande prévalence d'anticorps sériques dirigés contre la toxine B et contre certaines flagellines chez les patients ayant eu une infection simple, sans complications ni rechutes. Les résultats de nos travaux indiquent donc que certains patients développent une réponse humorale contre les antigènes de C. difficile et que les anticorps produits, particulièrement ceux dirigés contre la toxine B, semblent impliqués dans la défense du patient contre la survenue de complications et de rechutes. Nos données laissent toutefois sous-entendre que d'autres caractéristiques de l'hôte contribuent de façon importante à la défense contre la colonisation et l'infection par C. difficile. Des travaux supplémentaires sont nécessaires afin de définir les paramètres qui permettront d'élaborer un protocole de vaccination optimal contre C. difficile.
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Meessen-Pinard, Mathieu. "Caractérisation de phages tempérés et évaluation de leurs impacts sur le phénotype bactérien de clostridium difficile." Mémoire, Université de Sherbrooke, 2010. http://savoirs.usherbrooke.ca/handle/11143/4053.
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Clostridium difficile est un pathogène entérique qui cause d'importantes infections nosocomiales dont le traitement est parfois problématique. Il n'existe, à l'heure actuelle, que deux antibiotiques approuvés pour traiter les infections à C. difficile et le taux de rechute est assez important. Ce projet a initialement visé à isoler et caractériser des phages virulents contre C. difficile en vue de les utiliser en phagothérapies comme outils thérapeutiques alternatifs. Les eaux usées et les selles de patients infectés par C. difficile ont été utilisées pour isoler et détecter les phages virulents. Or, quatre phages différents (9MMPOI-O4) ont été isolés mais aucun de ces phages ne s'est révélé être virulent. Les quatre phages tempérés ont donc été caractérisés et leur impact a été évalué sur quelques phénotypes bactériens chez C. difficile dont la motilité et la production des toxines A et B. La caractérisation morphologique des phages (pMMPOl-04 a permis de déterminer que ceux-ci appartiennent à la familles des Myoviridae alors que la caractérisation génomique a permis de démontrer que certains de ces phages sont assez différents entre eux mais également par rapport aux autres phages tempérés, isolés et caractérisés dans la littérature. De façon générale, les phages (pMMPOl-04 ne semblent pas s'induire spontanément de manière plus importante mais suggère que la présence de certains antibiotiques pourrait augmenter l'induction de certains de ces phages. L'impact des phages (pMMPOl-04 sur la motilité chez C. difficile n'a pas démontré que ceux-ci avaient un rôle à jouer sur ce phénotype. Par contre, certains des phages (pMMP semblent augmenter ou diminuer la production en toxines A et B. Les résultats de nos travaux indiquent donc que certains des phages caractérisés présentent des différences importantes qui suggèrent une grande diversité parmi les phages tempérés chez C. difficile. De plus, certains des phages cpMMP auraient la capacité de participer aux transferts horizontaux de matériels génétiques et d'affecter la régulation de certains facteurs de virulence chez C. difficile tel que la production en toxines A et B. Évidemment, des travaux supplémentaires seront nécessaires pour confirmer la modification du phénotype de production en toxines par ces phages mais également sur d'autres phénotypes associés aux autres facteurs de virulence de cette bactérie. [Symboles non conformes]
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Solomon, Katie. "An investigation into the effects of purified Clostridium difficile toxin A on monocytes." Thesis, University of Nottingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.401583.
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Junqueira, Ana FlÃvia Torquato de AraÃjo. "Estudo do efeito do inibidor da enzima adenosina desaminase, EHNA, sobre a enterite induzida pela toxina a do Clostridium difficile em alÃa ileal isolada de camundongos." Universidade Federal do CearÃ, 2008. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=1305.
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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgicoO Clostridium difficile tem como principal fator de virulÃncia a toxina A (TxA), a qual provoca inflamaÃÃo e destruiÃÃo tecidual aguda em intestinos de animais experimentais e de pacientes com a doenÃa induzida por esta bactÃria. Em locais de injÃria tecidual, adenosina à produzida em altas concentraÃÃes, onde exerce uma sÃrie de efeitos antiinflamatÃrios, limitados por sua rÃpida degradaÃÃo pela enzima adenosina desaminase. O objetivo deste trabalho foi investigar o efeito da inibiÃÃo da enzima adenosina desaminase pelo EHNA (eritro-9-(2-hidrÃxi-3-nonil)-adenina) sobre a enterite induzida pela TxA do C. difficile em alÃa ileal de camundongos. Para isto, injetamos EHNA (90 μmol/kg) ou PBS i.p. 30 minutos antes da administraÃÃo de TxA (10 a 100 μg) ou PBS na alÃa ileal isolada. Os animais foram sacrificados 3 horas depois da induÃÃo da enterite e as alÃas foram retiradas para estudo. As razÃes peso/comprimento da alÃa e volume de secreÃÃo/comprimento da alÃa foram calculadas e amostras de tecido foram coletadas para histopatologia, dosagem de atividade de mieloperoxidase (MPO), dosagem de TNF-α, IL-1β e IL-10 por ELISA, imunohistoquÃmica para TNF-α, IL-1β, NOS induzÃvel e PTX3, e PCR para TNF-α, IL-1β e PTX3. A injeÃÃo de TxA (10 a 100 μg) nas alÃas ileais aumentou significativamente (p<0,05) as razÃes peso/comprimento da alÃa e volume de secreÃÃo/comprimento da alÃa com resultados consistentes a partir de 50 μg. A TxA promoveu significativa (p<0,05) destruiÃÃo tecidual, edema, infiltraÃÃo de cÃlulas inflamatÃrias, aumento das citocinas TNF-α e IL-1β, e elevaÃÃo de iNOS e PTX3. Todos esses parÃmetros foram significativamente revertidos com o uso do EHNA (p<0,05). Em adiÃÃo, a TxA nÃo alterou os nÃveis de IL-10 em relaÃÃo ao controle, mas o prÃ-tratamento com EHNA promoveu uma elevaÃÃo nos nÃveis desta citocina. Assim, concluÃmos que na enterite induzida pela TxA em camundongos o EHNA demonstrou um potente efeito antiinflamatÃrio, reduzindo consideravelmente a lesÃo tecidual, a migraÃÃo neutrofÃlica, a expressÃo e os nÃveis de citocinas prÃinflamatÃrias (TNF-α, IL-1β) e produzindo um aumento nos nÃveis de IL-10. AlÃm disso, a administraÃÃo de TxA induziu um aumento na expressÃo da proteÃna PTX3 e no nÃmero de cÃlulas imunomarcadas para iNOS no tecido ileal, ambos revertidos pelo EHNA
The main factor of virulence in Clostridium difficile is toxin A (TxA), which can induce inflammation and acute tissue injury in the bowels of animals and humans affected by this organism. The high concentration of adenosine generated upon injury produces a number of antiinflammatory effects limited by rapid degradation by adenosine deaminase. The objective of this study was to determine the effect of EHNA (erythro-9-(2-hydroxy-3-nonyl)-adenine) inhibition of adenosine deaminase upon TxA-induced ileal loop enteritis in mice. EHNA (90 μmol/kg) or PBS was injected i.p. 30 minutes prior to TxA (10-100 μg) or PBS instillation into the ligated ileal loop. The animals were euthanized 3 hours after enteritis induction and the ileal loops were retrieved for analysis. The weight/length ratio and the secretion volume/length ratio were calculated and tissue samples were submitted to histopathological study, myeloperoxidase assay (MPO), measurement of TNF-α, IL-1β and IL-10 levels with ELISA, immunohistochemical tests for TNF-α, IL-1β, inducible NOS and PTX3, and PCR assay for TNF-α, IL-1β and PTX3. The instillation of TxA (10-100 μg) into the ileal loop significantly increased (p<0.05) the weight/length ratio and the secretion volume/length ratio with consistent results above 50 μg. TxA induced a significant amount (p<0.05) of histological damage, edema and inflammatory cell infiltration and increased the production of TNF-α, IL-1β, iNOS and PTX3. All changes were significantly reverted by treatment with EHNA (p<0.05). Moreover, IL-10 levels remained unchanged in animals treated with TxA, but increased in animals receiving EHNA. In conclusion, in mice with TxA-induced enteritis EHNA produced considerable antiinflammatory effects, reducing tissue injury, neutrophil migration, the expression and levels of proinflammatory cytokines (TNF-α and IL-1β) and producing an increase in IL-10 levels. In addition, TxA instillation increased PTX3 expression and the number of cells immunolabeled for iNOS in the ileal tissue, both of which were reverted by EHNA
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Sirard, Stéphanie. "Analyse génotypique et phénotypique d'isolats cliniques de Clostridium difficile et comparaison en fonction de la sévérité des symptômes." Mémoire, Université de Sherbrooke, 2011. http://hdl.handle.net/11143/5547.
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Clostridium difficile est la principale cause de diarrhées nosocomiales liées à la prise d'antibiotiques. La souche hypervirulente NAP1/027 est apparue récemment et a causé de nombreuses épidémies en Amérique du Nord et en Europe. On considère généralement que cette souche produit plus de toxines, sporule davantage, provoque des infections plus sévères menant à des complications et est souvent associée aux cas de récurrence. Toutefois, des études récentes ont montré des données contradictoires à ce sujet. L'objectif de mes travaux de recherche est donc de déterminer si l'issue clinique des infections à C. difficile (ICD) peut être prédite en fonction du génotype et de certains phénotypes bactériens associés à la virulence, comme la production de toxines et la sporulation. Pour ce faire, 21 isolats cliniques associés à des ICD de sévérité différente (légère à modérée, sévère, compliquée) ont été caractérisés par des méthodes de typage courantes, incluant le ribotypage par PCR, le typage des répétitions en tandem, l'analyse de loci multiples de répétitions en tandem polymorphe, la détection des toxines A, B et CDT, ainsi que le séquençage du gène tcdC. Les taux de sporulation et la production des toxines A et B ont aussi été évalués in vitro, de même que la résistance des isolats à certains antibiotiques. La mobilité, la sensibilité des isolats à certains bactériophages et leur contenu en prophages ont aussi été étudiés. Les résultats de mes travaux démontrent que les méthodes de typage utilisées ne permettent pas de prévoir avec certitude le phénotype bactérien ni de prédire la sévérité des ICD. En effet, les souches NAP1/027 peuvent autant provoquer des ICD non-sévères que mener à des complications. Le phénotype n'est pas non plus nécessairement un indice de la sévérité. Les souches NAP1/027 produisent généralement plus de toxines, mais ne possèdent pas forcément la capacité de sporulation qu'on leur attribue généralement. Par conséquent, les généralisations à propos des souches NAP1/027 devraient être évitées.
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Lima, Bruno Bezerra. "Efeitos das toxinas A e B do Clostridium difficile sobre a via de WNT/Beta-catenina em células epiteliais intestinais." reponame:Repositório Institucional da UFC, 2014. http://www.repositorio.ufc.br/handle/riufc/12264.
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LIMA, Bruno Bezerra. Efeitos das toxinas A e B do Clostridium difficile sobre a via de WNT/Beta-catenina em células epiteliais intestinais. 2014. 79 f. Tese (Doutorado em Farmacologia) – Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2014.Submitted by denise santos (denise.santos@ufc.br) on 2015-05-19T12:12:30Z No. of bitstreams: 1 2014_tese_bblima.pdf: 2558018 bytes, checksum: 9b5ba19b7dbb3fd33020f7fcd72564f7 (MD5)
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Clostridium difficile toxins A and B (TcdA and TcdB) are homologous glycosyltransferases that inhibit a group of small GTPases within host cells, but several mechanisms underlying their pathogenic activity remain unclear. Here, we evaluated the effects of TcdA and TcdB on the Wnt/Beta-catenin pathway, the major driving force behind the proliferation of epithelial cells in colonic crypts. IEC-6 and RKO cells stimulated with Wnt3a-conditioned medium were incubated with 10, 50 and 100 ng/mL of TcdA or TcdB for 24h, resulting in a dose-dependent inhibition of the Wnt signaling, as demonstrated by a T-cell factor (TCF) reporter assay. This was further confirmed by immunofluorescence staining for nuclear localization of Beta-catenin and western blotting for Beta-catenin and c-Myc (encoded by a Wnt target gene). Moreover, our western blot analysis showed a decrease in the Beta-catenin protein levels, which was reversed by z-VAD-fmk, a pan-caspase inhibitor. Nonetheless, TcdA was still able to inhibit the Wnt/Beta-catenin pathway even in the presence of z-VAD-fmk, lithium chloride (a GSK3B inhibitor), or constitutively active Beta-catenin, as determined by a TCF reporter assay. Furthermore, pre-incubation of RKO cells with TcdA for 12h also attenuated Wnt3a-mediated activation of Wnt signaling, suggesting that inactivation of Rho GTPases plays a significant role in that inhibition. Taken together, these findings suggest that attenuation of the Wnt signaling by TcdA and TcdB is important for their anti-proliferative effects.
As toxinas A e B do Clostridium difficile (TcdA e TcdB) são glicosiltransferases homólogas que inibem um grupo de pequenas GTPases dentro da célula hospedeira, contudo, vários mecanismos envolvidos na atividade patogênica dessas toxinas permanecem desconhecidos. No presente estudo, avaliamos os efeitos das TcdA e TcdB na via de Wnt/Beta-catenina que representa a força motora principal responsável pela proliferação das células epiteliais nas criptas colônicas. Foram utilizadas células IEC-6 (células epiteliais de criptas de Rattus novergicus) e RKO (células de adenocarcinoma de cólon humano). Estas células foram estimuladas com meio condicionado contendo Wnt3a e incubadas com 10, 50 ou 100 ng/mL de TcdA ou 1, 5 ou 10 ng/mL de TcdB por 24h, resultando em uma inibição dose-dependente da via de sinalização canônica de Wnt, como demonstrado pelo ensaio de repórter de fator de célula T (TCF). Esse resultado foi corroborado pelos dados da imunofluorescência com marcação para a localização nuclear de Beta-catenina e por western blotting para Beta-catenina e c-MYC (gene-alvo da via de Wnt). Além disso, os dados do experimento de western blot evidenciaram uma diminuição dos níveis da proteína Beta-catenina, o qual foi prontamente revertido por z-VAD-fmk, um pan-inibidor de caspase. Entretanto, a TcdA ainda foi capaz de inibir a via de Wnt/Beta-catenina mesmo na presença do z-VAD-fmk, cloreto de lítio (um inibidor de GSK3Beta) ou plasmídeo de Beta-catenina constitutivamente ativo, como determinado pelo ensaio do TCF (TOPFlash/Luciferase). O estudo evidenciou ainda que a pré-incubação de células RKO com TcdA por 12h também atenuou a ativação da via de Wnt mediada por Wnt3a, o que sugere que a inativação de RhoGTPases, particularmente Rac1, possui um papel nessa inibição. Em conclusão, esses achados sugerem que a inibição da via canônica de Wnt pelas TcdA e TcdB representa um mecanismo importante da sua patogênese e explica seus efeitos anti-proliferativos.
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Gauckler, Philipp Alexander [Verfasser]. "Identifizierung inhibitorischer Peptide gegen die Toxine von Clostridium difficile aus humanem Hämofiltrat / Philipp Alexander Gauckler." Ulm : Universität Ulm, 2018. http://d-nb.info/1162193549/34.
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Maikova, Anna. "The CRISPR-Cas system of human pathogen Clostridium difficile : function and regulation." Thesis, Université de Paris (2019-....), 2019. http://www.theses.fr/2019UNIP7091.
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Clostridium difficile (nouveau nom Clostridioides difficile) est une bactérie à Gram-positif, sporulante, anaérobie stricte, présente dans le sol et les environnements aquatiques, ainsi que dans le tractus intestinal des mammifères. C. difficile est l’un des principaux clostridies pathogènes. Cette bactérie est devenue un vrai problème de santé publique associé à l'antibiothérapie dans les pays industrialisés. La diarrhée associée à C. difficile est actuellement la diarrhée nosocomiale la plus fréquente en Europe et dans le monde. Depuis la dernière décennie, la proportion de formes d’infections graves a augmentée en raison de l’émergence des souches hypervirulantes et épidémiques comme la souche R20291 de ribotype 027. L’infection à C. difficile provoque la diarrhée, la colite et même la mort. De nombreux aspects de la pathogenèse de C. difficile restent mal compris. En particulier, les mécanismes moléculaires de son adaptation aux conditions changeantes de l'hôte doivent être examinés.Durant le cycle d'infection, C. difficile survit dans des communautés intestinales riches en bactériophages, en utilisant des systèmes qui contrôlent les échanges génétiques favorisés dans ces environnements complexes. Au cours de la dernière décennie, les systèmes CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (associés aux CRISPR) d'immunité adaptative chez les procaryotes contre des éléments génétiques exogènes sont devenus le centre d'intérêt scientifique parmi les divers systèmes de défense bactérienne.Des études antérieures ont révélé la présence d'ARN CRISPR abondants chez C. difficile. Cette bactérie possède un système CRISPR original, caractérisé par la présence d'un grand nombre de cassettes CRISPR (12 dans la souche 630 et 9 dans la souche hypervirulante R20291), de deux ou trois opérons cas conservés dans la majorité des génomes séquencés de C. difficile et la localisation au sein des prophages de plusieurs cassettes CRISPR. Cependant, le rôle de CRISPR-Cas dans la physiologie et le cycle infectieux de cet important pathogène reste obscur.Les objectifs de ce travail sont les suivants:1) étudier le rôle et la fonctionnalité du système CRISPR-Cas de C. difficile dans les interactions avec des éléments d'ADN étrangers (tels que les plasmides), 2) révéler la manière dont le système CRISPR-Cas de C. difficile est régulé et fonctionne dans des conditions de culture bactérienne différentes, incluant la réponse aux stress.Dans la présente thèse, la fonctionnalité du système CRISPR-Cas de C. difficile a été étudiée (chapitre 2). Grâce à des tests d'efficacité de conjugaison, la fonction défensive (en interférence) du système CRISPR-Cas a été démontrée. La corrélation entre les niveaux d'expression des ARN CRISPR et les niveaux d'interférence observés a également été montrée. De plus, grâce à la série d’expériences d’interférence, la fonctionnalité des motifs PAM (protospacer adjacent motifs) a été confirmée en accord avec des prédictions in silico. Le consensus fonctionnel de PAM a été déterminé expérimentalement avec les bibliothèques des plasmides. La fonction adaptative du système CRISPR-Cas de C. difficile a été également démontrée pour la souche de laboratoire. Le rôle de plusieurs opérons cas dans la fonctionnalité du système CRISPR de C. difficile est démontré aussi dans ce chapitre.Le chapitre 3 montre le lien entre le système CRISPR-Cas et un nouveau système toxine-antitoxine de type I, ainsi que leur possible co-régulation dans des conditions de biofilm et de stress. Ce chapitre définit également le rôle possible du c-di-GMP dans la régulation du système CRISPR-Cas de C. difficile. De plus, le chapitre 4 décrit l'utilisation du système CRISPR-Cas endogène comme nouvel outil pour la rédaction du génome de C. difficile.En conclusion, les données obtenues mettent en évidence les caractéristiques originales du système CRISPR-Cas actif de C. difficile et démontrent son potentiel biotechnologiqueClostridium difficile (the novel name – Clostridioides difficile) is a Gram-positive, strictly anaerobic spore forming bacterium, found in soil and aquatic environments as well as in mammalian intestinal tracts. C. difficile is one of the major pathogenic clostridia. This bacterium has become a key public health issue associated with antibiotic therapy in industrialized countries. C. difficile-associated diarrhoea is currently the most frequently occurring nosocomial diarrhoea in Europe and worldwide. Since the last decade the number of severe infection forms has been rising due to emergence of the hypervirulent and epidemic strains as ribotype 027 R20291 strain. C. difficile infection causes diarrhoea, colitis and even death. Many aspects of C. difficile pathogenesis remain poorly understood. Particularly, the molecular mechanisms of its adaptation to changing conditions inside the host are to be scrutinized. During the infection cycle C. difficile survives in bacteriophage-rich gut communities possibly by relying on some special systems that control the genetic exchanges favored within these complex environments. During the last decade, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems of adaptive prokaryotic immunity against exogenic genetic elements has become the center of interest among various anti-invader bacterial defense systems.Previous studies revealed the presence of abundant and diverse CRISPR RNAs in C. difficile. C. difficile has an original CRISPR system, which is characterized by the presence of an unusually large set of CRISPR arrays (12 arrays in the laboratory 630 strain and 9 ones in the hypervirulent R20291 strain), of two or three sets of cas genes conserved in the majority of sequenced C. difficile genomes and the prophage location of several CRISPR arrays. However, the role CRISPR-Cas plays in the physiology and infectious cycle of this important pathogen remains obscure.The general aims of this work run as follows: 1) to investigate the role and the functionality of C. difficile CRISPR-Cas system in the interactions with foreign DNA elements (such as plasmids), 2) to reveal the way C. difficile CRISPR-Cas system expression is regulated and functions in different states of bacterial culture, including its response to stresses. In the present PhD thesis the functionality of C. difficile CRISPR-Cas system was investigated (Chapter 2). Through conjugation efficiency assays defensive function (in interference) of C. difficile CRISPR-Cas system was demonstrated. The correlation between the previously known levels of expression of CRISPR RNAs and the observed levels of interference has also been shown. Moreover, through the series of interference experiments the functionality of PAMs (protospacer adjacent motifs) was confirmed, which have already been predicted in silico. Additionally, the general functional PAM consensus was determined using PAM libraries experiments. Furthermore, an adaptive function of C. difficile CRISPR-Cas system was shown for laboratory strain. The role of multiple cas operons in C. difficile CRISPR functionality is also demonstrated in this Chapter.In Chapter 3 the link between C. difficile CRISPR-Cas system and a new type I toxin-antitoxin system is demonstrated, as well as a possible co-regulation under biofilm and stress conditions of CRISPR-Cas system and these toxin-antitoxin modules. This Chapter also defines a possible role of c-di-GMP in regulation of C. difficile CRISPR-Cas system. Additionally, Chapter 4 describes the utilization of endogenous C. difficile CRISPR-Cas system as a novel tool for genome editing in C. difficile. Altogether, the obtained data highlight the original features of active C. difficile CRISPR-Cas system and demonstrate its biotechnological potential
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RAMALHETE, Sara de Castro Gonçalves. "Exploring the relationship between toxin and spore prodution in the human enteric pathogen Clostridium difficile." Master's thesis, Instituto de Higiene e Medicina Tropical, 2015. http://hdl.handle.net/10362/19069.
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Clostridium difficile é presentemente a principal causa de doença gastrointestinal associada à utilização de antibióticos em adultos. C. difficile é uma bactéria Gram-positiva, obrigatoriamente anaeróbica, capaz de formar endósporos. Tem-se verificado um aumento dos casos de doença associada a C. difficile com sintomas mais severos, elevadas taxas de morbilidade, mortalidade e recorrência, em parte, devido à emergência de estirpes mais virulentas, mas também devido à má gestão do uso de antibióticos. C. difficile produz duas toxinas, TcdA e TcdB, que são os principais fatores de virulência e responsáveis pelos sintomas da doença. Estas são codificadas a partir do Locus de Patogenicidade (PaLoc) que codifica ainda para um regulador positivo, TcdR, uma holina, TcdE, e um regulador negativo, TcdC. Os esporos resistentes ao oxigénio são essenciais para a transmissão do organismo e recorrência da doença.
A expressão dos genes do PaLoc ocorre em células vegetativas, no final da fase de crescimento exponencial, e em células em esporulação. Neste trabalho construímos dois mutantes de eliminação em fase dos genes tcdR e tcdE. Mostrámos que a auto-regulação do gene tcdR não é significativa. No entanto, tcdR é sempre necessário para a expressão dos genes presentes no PaLoc.
Trabalho anterior mostrou que, com a exceção de tcdC, os demais genes do PaLoc são expressos no pré-esporo. Mostrámos aqui que TcdA é detectada à superfície do esporo maduro e que a eliminação do tcdE não influencia a acumulação de TcdA no meio de cultura ou em associação às células ou ao esporo. Estas observações têm consequências para o nosso entendimento do processo infecioso: sugeremque o esporo possa ser também um veículo para a entrega da toxina nos estágios iniciais da infecção, que TcdA possa ser libertada durante a germinação do esporo, e que o esporo possa utilizar o mesmo receptor reconhecido por TcdA para a ligação à mucosa do cólon.Clostridium difficileis currently the major cause of antibiotic-associated gastrointestinal diseases in adults. This is a Gram-positive bacterium, endospore-forming and an obligate anaerobe that colonizes the gastrointestinal tract.Recent years have seen a rise in C. difficile associated disease (CDAD) cases, associated with more severe disease symptoms, higher rates of morbidity, mortality and recurrence, which were mostly caused due to the emergence of “hypervirulent” strains but also due to changing patterns of antibiotics use. C. difficile produces two potent toxins, TcdA and TcdB, which are the main virulence factors and the responsible for the disease symptoms. These are codified from a Pathogenicity Locus (PaLoc), composed also by the positive regulator, TcdR, the holin-like protein, TcdE, and a negative regulator, TcdC. Besides the toxins, the oxygen-resistant spores are also essential for transmission of the organism through diarrhea; moreover, spores can accumulate in the environment or in the host, which will cause disease recurrence.The expression of the PaLoc genes occurs in vegetative cells, at the end of the exponential growth phase, and in sporulating cells. In this work, we constructed two in-frame deletion mutants of tcdR and tcdE. We showed that the positive auto regulation oftcdR is not significant. However, tcdR is always necessary for the expression of the PaLoc genes.A previous work showed that, except tcdC, all the PaLoc genes are expressed in the forespore. Here, we detected TcdA at the spore surface. Furthermore, we showed that the in-frame deletion of tcdE does not affect the accumulation of TcdA in the culture medium or in association with cells or spores. This data was important for us to conclude about the infeccious process: it suggests that the spore may be the vehicle for the delivery of TcdA in early stages of infection, that TcdA may be released during spores germination and that this spore may use the same receptor recognized by TcdA to bind to the colonic mucosa.
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Clark, Andrew Elton, and Andrew Elton Clark. "The Clostridium difficile Flagellar System Mediates Toxin Synthesis, Pathogenicity, and Activation of Innate Immune Responses." Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/623153.
Full textAbstract:
Clostridium difficile has emerged as the most common healthcare-associated infection responsible for an estimated annual cost of $6 billion to the US healthcare system. Disease is mediated through the action of two glucosylating toxins which manifests as a fulminant diarrhea. Despite the severity of C. difficile infections, large gaps in knowledge, particularly pertaining to the molecular mechanisms of host colonization and interaction with the innate immune response, have hampered the development of novel targeted therapies. This work explores the molecular mechanisms used by C. difficile, in the context of the flagellar system, to colonize the host and interact with the resident immune system. The flagellin (FliC) protein was shown to not only be a critical mediator of C. difficile motility, but also participated in regulating sporulation, biofilm formation, and the expression of toxin genes. The FliC protein also robustly activated host inflammatory signaling through interaction with TLR5, demonstrating this molecule’s importance as a mediator of both bacterial and host responses to C. difficile infection. The flagellar capping protein (FliD) by contrast was found to have an important role mediating attachment to host tissues as well as to regulate and prevent the secretion of FliC into the extracellular environment. Finally, a novel FliC-specific glycosylation system was characterized. Through the addition of glycans, the reactivity of FliC with TLR5 was dampened, resulting in a weaker inflammatory response to infection. Flagellin glycans were also found to be necessary for C. difficile flagellar assembly, and different strains exhibited significant variation in both the number and structure of glycan moieties attached. The C. difficile flagellar system is a key mediator of bacterial motility, virulence, and host-pathogen interactions during infection with this enigmatic organism.
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GIRY, MURIELLE. "Les gtpases de la famille rho : cibles intracellulaires des toxines a et b de clostridium difficile." Paris 6, 1995. http://www.theses.fr/1995PA066607.
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Les proteines qui appartiennent a la superfamille ras presentent une organisation fonctionnelle particulierement bien conservee au cours de l'evolution. Elles sont caracterisees par un poids moleculaire d'environ 21 kd, une capacite a lier les nucleotides guanyliques, une activite gtpasique intrinseque et des modifications post-traductionnelles qui leur permettent une localisation membranaire. Ces gtpases controlent de nombreux aspects de la croissance et de la differenciation cellulaire, de l'organisation du cytosquelette et du transport des vesicules entre les compartiments membranaires. Nous nous sommes interesses aux proteines de la famille rho qui controlent le cytosquelette microfilamentaire. Nous avons utilise l'exoenzyme c3 de c. Botulinum qui a pour substrats differents membres de la famille rho. Nous avons realise une toxine chimerique en fusionnant le fragment b de la toxine diphterique qui permet l'entree de la molecule dans la cellule, avec c3 qui apporte donc l'activite catalytique. Cette chimere entraine la depolymerisation de l'actine-f et une redistribution des elements de la plaque d'adhesion des fibroblastes en culture. Rho semble donc exercer un role dans l'organisation des proteines de la plaque d'adhesion qui permettrait ensuite la formation des cables de tension d'actine. La structure des points focaux d'adhesion est comparable a celles des jonctions intercellulaires qui se creent au sein d'un epithelium polarise. Nous avons donc utilise cette chimere dans un modele d'epithelium intestinal (cellules t84). Nous avons montre que rho controle l'organisation du cytosquelette du pole apical de ce type de cellules, en particulier l'anneau perijonctionnel. Rho regule la distribution de zo-1 a ce niveau, sans influencer la localisation de la cadherine-e specifique de la jonction intermediaire. Ainsi, rho controle la jonction serree, donc la permeabilite de l'epithelium intestinal. Clostridium difficile, bacterie anaerobie presente dans la flore intestinale, est l'agent enteropathogene responsable de la colite pseudomembraneuse et de diarrhees associees aux antibiotiques. Elle produit deux toxines puissantes: les toxines a et b, dont les actions intracellulaires entrainent la destruction du cytosquelette d'actine. Clostridium sordellii est un microorganisme anaerobie qui produit deux toxines immunologiquement et structurellement apparentees aux toxines a et b de c. Difficile: les toxines ht et lt. Les toxines de c. Difficile induisent dans les cellules intestinales in vivo des effets similaires a ceux provoques par l'exoenzyme c3. Nous avons montre que rho est bien la cible intracellulaire directe de ces toxines. Par contre, la toxine lt de c. Sordellii qui provoque aussi une perte des cables de tension d'actine, reorganise cependant l'actine corticale en nombreuses microvillosites qui contiennent de la fimbrine. Le mecanisme d'action de cette toxine est independant de rho, et est probablement controle par une gtpase appartenant a la meme famille. Les proteines apparentees a rho sont des elements cles des phenomenes moleculaires qui ont lieu a l'interface membrane-cytosol. Elles sont les cibles specifiques de nombreuses toxines bacteriennes dont l'utilisation permettra l'etude des mecanismes biologiques qui dependent du cytosquelette filamentaire