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Influence de l'environnement sur le protéome de surface de Clostridium difficile : analyse globale et caractérisation de la cystéine protéase Cwp84

Abstract : Clostridium difficile, a gram-positive spore-forming, anaerobic bacterium, is the etiological agent of pseudomembranous colitis and of many cases of nosocomial diarrhea. The main risk factor is the use of antibiotics that alters the intestinal microbiota, predisposing to C. difficile intestinal colonization. C. difficile pathogenicity is mediated mainly by its A and B toxins, secreted after host colonization that involves various surface proteins, including different adhesins and proteolytic enzymes as the cysteine protease Cwp84.We sought to analyze the localization and the maturation process of the proteaseCwp84. We showed that the recombinant protein Cwp8430-803, purified as zymogen form, presents a particular maturation process including consecutive cleavages, leading to the mature form of 47 kDa. This protease has a proteolytic activity against the fibronectin. Two identifiable forms of the protease were found to be associated in the bacteria: a form of about 80 kDa and a cleaved one of 47 kDa, identified as the mature protease. They were found mainly in the bacterial cell surface fractions, and weakly in the extracellular fraction. The 80 kDa protein was non covalently associated to the S-layer proteins, while the 47 kDa form was found to be tightly associated with the underlying cell wall. Our data supported that the anchoring of the Cwp84 47 kDa form is presumably due to a re-association of the secreted protein.We also studied the regulation of virulence factors depending of environmental conditions that mimic those encountered by the bacterium in the digestive tract. We showed that an acidic pH affects the expression and the proteolytic process of Cwp84. The mature form was only recovered with an acidic pH. Proteomic and transcriptomic analysis of some surface proteins involved in colonization revealed that their expression was increased in media containing glucose. However, this regulation is probably related to the decrease in pH resulting from fermentation of glucose, rather than a direct effect of glucose. The acidic pH could lead in vivo to modulation of virulence factors expression and is probably a favorable feature in the colonisation process. We also identified new surface associated-proteins, that could represent potential virulence factors; they will be characterized later.
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Submitted on : Thursday, February 21, 2013 - 1:02:19 AM
Last modification on : Friday, October 23, 2020 - 4:55:08 PM
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  • HAL Id : tel-00790732, version 1
  • PRODINRA : 316240



Diana Joanne Chapeton Montes. Influence de l'environnement sur le protéome de surface de Clostridium difficile : analyse globale et caractérisation de la cystéine protéase Cwp84. Sciences agricoles. Université Paris Sud - Paris XI, 2012. Français. ⟨NNT : 2012PA114809⟩. ⟨tel-00790732⟩



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