A lesson in bacterial variability: The C. difficile cell wall protein CwpV

Abstract

Clostridium difficile is the main cause of antibiotic-associated diarrhea, leading to significant morbidity and mortality, and putting considerable economic pressure on healthcare systems. Current knowledge of the molecular basis of pathogenesis is limited primarily to the activities and regulation of two major toxins. In contrast, little is known of the mechanisms used to colonise the enteric system. C. difficile expresses a proteinaceous array on its cell surface known as the S-layer, consisting primarily of SlpA and a family of homologues, the cell wall protein (CWP) family. CwpV is the largest member of this family. CwpV is expressed in a phase-variable manner controlled by an invertible DNA switch, the cwpV switch. The novel mechanism controlling this phase variation has been charaterised using enzymatic reporter assays. A site-specific recombinase (RecV) catalyzing the inversion of the cwpV switch has been identified. Knocking out this recombinase has enabled isolation of cwpV switch locked ON and locked OFF strains of C. difficile, indicating that cwpV switch orientation is the primary determinant of CwpV expression. CwpV is post-translationally cleaved and expressed on the cell surface as two proteins that form a stable complex, with one subunit responsible for the noncovalent cell wall anchoring of the other large repetitive subunit. Due to the significant heterogeneity of C. difficile strains the characteristics of CwpV across a panel of strains were investigated. The cwpV switch and recV are conserved across diverse strains and all strains tested express CwpV in a phase variable manner. The N-terminus of CwpV is well conserved, however the C-terminal repetitive domain of CwpV varies markedly. Five different types have been identified and shown to be antigenically distinct. All types of CwpV repeats promote aggregation of C. difficile cells, which may be an important function during infection. These findings suggest a complex evolutionary history for CwpV.