A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence

Clostridium difficile is a diarrheagenic pathogen associated with significant mortality and morbidity. While its glucosylating toxins are primary virulence determinants, there is increasing appreciation of important roles for non-toxin factors in C . difficile pathogenesis. Cell wall glycopolymers (CWGs) influence the virulence of various pathogens. Five C . difficile CWGs, including PSII, have been structurally characterized, but their biosynthesis and significance in C . difficile infection is unknown. We explored the contribution of a conserved CWG locus to C . difficile cell-surface integrity and virulence. Attempts at disrupting multiple genes in the locus, including one encoding a predicted CWG exporter mviN , were unsuccessful, suggesting essentiality of the respective gene products. However, antisense RNA-mediated mviN downregulation resulted in slight morphology defects, retarded growth, and decreased surface PSII deposition. Two other genes, lcpA and lcpB , with putative roles in CWG anchoring, could be disrupted by insertional inactivation. lcpA - and lcpB - mutants had distinct phenotypes, implying non-redundant roles for the respective proteins. The lcpB - mutant was defective in surface PSII deposition and shedding, and exhibited a remodeled cell surface characterized by elongated and helical morphology, aberrantly-localized cell septae, and an altered surface-anchored protein profile. Both lcpA - and lcpB - strains also displayed heightened virulence in a hamster model of C . difficile disease. We propose that gene products of the C . difficile CWG locus are essential, that they direct the production/assembly of key antigenic surface polysaccharides, and thereby have complex roles in virulence.