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.