Variability of Clostridium difficile Surface Proteins and Specific Serum Antibody Response in Patients with Clostridium difficile -Associated Disease

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Abstract
Pathogen attachment is a crucial early step in mucosal infections. This step is mediated by important virulence factors, such as surface proteins. Clostridium difficile surface proteins have been identified as (i) adhesins (the flagellar cap protein FliD; the flagellin FliC; and the cell wall protein Cwp66 with a two domain-structure [Cw66 N-terminal and Cwp66 C-terminal domains]) and (ii) protease (the Cwp84 protein). To address the roles of these proteins in the pathogenesis of Clostridium difficile and to identify vaccine antigen candidates, we analyzed the variability of the proteins and their immunogenicities in 17 patients with C. difficile -associated disease. PCR-restriction fragment length polymorphism analysis of amplified gene products revealed interstrain homogeneity with fliC and fliD , in contrast to cwp66 genes. Immunoblot analysis showed that FliC and FliD were detected in the majority of isolates. The N-terminal domain of Cwp66 and Cwp84 were present in all strains tested, in contrast to the Cwp66 C-terminal domain, the expression of which was heterogeneous. The 17 sera from the corresponding patients were analyzed by enzyme-linked immunosorbent assay to detect antibodies directed against these 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 of C. difficile during the course of the disease. In addition, the FliC, FliD, and Cwp84 proteins appeared to be good potential vaccine candidates.