== Flagella were isolated from the 17 isolates by the procedure described by Delme et al. proteins. Many patients developed antibodies to FliC, FliD, Cwp84, and the Cwp66 C-terminal domain, but not to the Cwp66 N-terminal domain. In conclusion, this study confirms the expression of these surface proteins ofC. difficileduring the course of the disease. In addition, the FliC, FliD, and Cwp84 proteins appeared to be good potential vaccine candidates. The expression of virulence by bacterial pathogens often requires the production and actions of toxins and adhesins. Whereas toxins are generally released by the pathogens into the extracellular medium and can thus act Lerociclib dihydrochloride at distant sites, surface proteins allow the microorganisms to adhere to host determinants (2,10,20). Clostridium difficileis a gram-positive, spore-forming enteric pathogen. After disruption of the intestinal barrier by antibiotics, spores ofC. difficile, acquired exogenously or endogenously, germinate, and bacteria multiply in the intestine.C. difficilesynthesizes two major toxins, toxin A and toxin B, both of which are responsible for the clinical manifestations of the disease, which include diarrhea or, in the worst case, pseudomembranous colitis (18). The colonization mechanism ofC. difficilehas recently been studied and is supposed to be a two-step process. The bacteria are initially able to interact with the apical microvilli of the intestinal epithelial cells and begin to release toxins A and B, which disrupt epithelial barrier function (16). The basolateral pole of epithelial cells thus becomes accessible, and a large number of bacteria are able to interact with receptors via their surface proteins (5). Lerociclib dihydrochloride In addition to mediating the attachment of bacteria to host tissues, adhesins may have additional functions in the development of the infection. They IL12RB2 may be biological effectors in vivo and thus influence the outcome of the host-pathogen interaction (9). Flagella contribute to the virulence of pathogenic bacteria through chemotaxis, as well as adhesion to and invasion of host surfaces (19) Some of the surface proteins ofC. difficilehave been characterized: the proteins of the S-layer (4), the flagellin FliC, the major structural component of the flagellar filament, the flagellar cap protein FliD, and the cell wall proteins Cwp66 and Cwp84. FliD has been shown to have in vitro and in vivo adhesive properties and, in particular, Lerociclib dihydrochloride to play a role in attachment to mucus (25). Cwp66 is a surface protein with a two-domain structure. The C-terminal domain (Cwp66-Cter) is exposed to the cell surface, displays repeated motifs, and has been described as an adhesin; the N-terminal domain (Cwp66-Nter), which shows homology to the CwlB autolysin ofBacillus subtilis, is supposed to anchor Cwp66 to the cell wall ofC. difficile(26). Cwp84 is a protein with proteolytic activity which could have a role in the physiology of the bacteria (21). The level of host immune response to toxins has been shown to correlate with the severity of the disease (13). Mulligan et al. showed that antibodies were also directed against surface proteins ofC. difficile(15). In addition, it has been shown by Drudy et al. that a high level of immunoglobulin M (IgM) antibody toC. difficileS-layer proteins is associated with a markedly reduced risk of recurrentC. difficile-associated diarrhea (7). In a previous study, we demonstrated that antibody levels against FliC, FliD, and Cwp66-Nter were significantly higher in a control group versus a group of patients withClostridium difficile-associated-disease (CDAD), suggesting that these proteins are able Lerociclib dihydrochloride to induce an immune response that could play a role in the defense mechanism of the host (17). The aim of the present study was to analyze the genotypic and phenotypic variability of these surface proteins and their immunogenicities to confirm their expression in humans in order to identify novel antigens for active immunization. == MATERIALS AND METHODS == == C. difficilestrains and growth Lerociclib dihydrochloride conditions. == SeventeenC. difficilestrains were isolated from patients with CDAD (Microbiology Unit, Pr Delme, Catholic University of Louvain, Brussels, Belgium). The diagnosis ofC. difficiledisease was confirmed by culture and detection of toxin B in fecal samples.C. difficilestrains were grown under anaerobic conditions on Colombia cystein agar plates (Oxoid) supplemented with 5% horse blood (Biomerieux, Marcy l’Etoile, France) or in tryptone-glucose-yeast broth (Difco) for 48 h in aerobiosis. TheC. difficilestrain 79-685, isolated from a patient with pseudomembranous colitis, was a gift from the Department of Microbiology of the University of Strasbourg, Strasbourg, France, and was used as the reference strain. == Serum samples..