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

Abstract

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.

The molecular structure of the glycoside hydrolase domain of Cwp19 from Clostridium difficile

“…This suggests either that Cwp19 may act on a substrate or substrates not tested in this study, may only be capable of breaking down very specific substrates or that the rate of reaction for the substrates tested was too slow for a reaction to be observed. The presence of cwp19 in the AP locus, which has been implicated in the formation of PSII, indicates a potential role for Cwp19 in the cleavage of a PSII precursor [18,19].…”

“…The gene coding for Cwp19 is located in the anionic polymer locus (AP locus) which is likely to be involved in synthesis of PSII, the polysaccharide that mediates binding of CWB2 domains to the cell wall. The AP locus is itself immediately downstream of the slpA locus, which contains the first twelve cwp genes and six others with apparent roles relating to the S-layer or cell wall [8,9,18,19]. It is therefore possible that Cwp19 will be involved in processing surface exposed polysaccharides such as peptidoglycan or PSII.…”

“…cwp19 has been shown to be present with more than 95% amino acid sequence identity in a wide variety of C. difficile strains [18,20]. Although expression is yet to be thoroughly analysed, Cwp19 is known to be present in the S-layer under at least some conditions as it copurified with the cysteine protease Cwp84 in a pulldown assay using probes based on E-64, a cysteine protease inhibitor [21].…”

The molecular mechanism of N-acetylglucosamine side-chain attachment to the Lancefield group A Carbohydrate inStreptococcus pyogenes

“…In almost all Gram-positive bacteria, cell wallattached glycopolymers are critical for cell envelope integrity and their depletion is lethal (30). Most streptococcal species including two human pathogens GAS and GBS do not synthesize WTA and instead produce Rhacontaining glycopolymers as functional homologs of WTA (1).…”

Adaptive immune constraints on C. difficile vaccination

“…There are three major surface polysaccharides known as PSI, PSII, and PSIII [12]. PSII may exert several influences on C. difficile including virulence and data thus far indicates that it is expressed by all pathogenic strains [13]. PSII is a phosphate-containing poly-hexa-saccharide molecule that stimulates B cell responses.…”