Variability of Clostridium difficile surface proteins and specific serum antibody response in patients with Clostridium difficile-associated disease

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 Cwp 66 with a two domain-structure [Cw 66 N-terminal and Cwp 66 C-terminal domains]) and (ii) protease (the Cwp 84 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 cwp 66 genes. Immunoblot analysis showed that FliC and FliD were detected in the majority of isolates. The N-terminal domain of Cwp 66 and Cwp 84 were present in all strains tested, in contrast to the Cwp 66 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, Cwp 84, and the Cwp 66 C-terminal domain, but not to the Cwp 66 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 Cwp 84 proteins appeared to be good potential vaccine candidates.

FIG. 1.

PCR ribotyping profiles of the 17 Clostridium difficile isolates studied (1 to 17). Lane MW, 100-bp molecular size marker.

FIG. 2.

PCR-RFLP profiles of cwp66. (A) The amplified 3′ part of the cwp66 gene product was digested with RsaI, SspI, Sca I, and DraI. The different restriction profiles for the enzymes were designated a, b, c, d, e, and f. Lanes M, 100-bp ladders; lanes a, profile a; lanes b, profile b; lanes c, profile c; lanes d, profile d; lanes e, profile e; lanes f, profile f. The digested amplified products were subjected to electrophoresis in a 1.2% (wt/vol) agarose gel. (B) The amplified 5′ part of the cwp66 gene product was digested with DraI, Hind III, KpnI, and RsaI. The different restriction profiles for the enzymes were designated a, b, c, and d. Lanes M, 100-bp ladders; lanes a, profile a; lanes b, profile b; lanes c, profile c; lanes d, profile d. The digested amplified products were subjected to electrophoresis in a 1.2% (wt/vol) agarose gel.

FIG. 3.

Immunoblot analysis of flagellar preparations for FliC and FliD and cell wall protein extracts for Cwp66 and Cwp84. (A) Flagellar preparation revealed with anti-FliC and anti-FliD rabbit sera. (B) Protein extract revealed with anti-Cwp66 N-terminal rabbit serum (only one band was revealed at the same molecular mass of 50 kDa for all isolates). (C) Protein extract revealed with anti-Cwp66 C-terminal rabbit serum. Several bands at different molecular masses, depending on the strain tested, were revealed. (D) Protein extract revealed with anti-Cwp84 mouse serum. Only one band was revealed at the same molecular mass, 84 kDa, for all isolates. MW, molecular weight low-range standard (Bio-Rad).