Cwp84, a Surface-associated Cysteine Protease, Plays a Role in the Maturation of the Surface Layer of Clostridium difficile

Abstract

Clostridium difficile is a major and growing problem as a hospital-associated infection that can cause severe, recurrent diarrhea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood but undoubtedly involves protein components within the surface layer (S-layer), which play a role in adhesion. In C. difficile, the S-layer is composed of two principal components, the high and low molecular weight S-layer proteins, which are formed from the post-translational cleavage of a single precursor, SlpA. In the present study, we demonstrate that a recently characterized cysteine protease, Cwp84 plays a role in maturation of SlpA. Using a gene knockout approach, we show that inactivation of the Cwp84 gene in C. difficile 630⌬Erm results in a bacterial phenotype in which only immature, single chain SlpA comprises the S-layer. The Cwp84 knock-out mutants (CD⌬Cwp84) displayed significantly different colony morphology compared with the wild-type strain and grew more slowly in liquid medium. SlpA extracted from CD⌬Cwp84 was readily cleaved into its mature subunits by trypsin treatment. Addition of trypsin to the growth medium also cleaved SlpA on CD⌬Cwp84 and increased the growth rate of the bacterium in a dose-dependent manner. Using the hamster model for C. difficile infection, CD⌬Cwp84 was found to be competent at causing disease with a similar pathology to the wild-type strain. The data show that whereas Cwp84 plays a role in the cleavage of SlpA, it is not an essential virulence factor and that bacteria expressing immature SlpA are able to cause disease.

A real-time quantitative PCR assay for evaluating Clostridium difficile adherence to differentiated intestinal Caco-2 cells

“…The high-molecular-mass S-layer protein is largely responsible for C. difficile binding to both human GI tissues and to components of the extracellular matrix (Calabi et al, 2002). In addition, Cwp84, a putative colonization factor and cysteine protease, has recently been implicated in a process whereby SlpA is cleaved into lowand high-molecular-mass counterparts (Janoir et al, 2007;Kirby et al, 2009). Several other cell-surface colonization factors including Cwp66, Fbp68 and GroEL, may also all play a role in C. difficile adherence to intestinal epithelial cells (Hennequin et al, , 2003Waligora et al, 2001).…”

Clostridium difficile Has Two Parallel and Essential Sec Secretion Systems

“…In addition to the S-layer, C. difficile also possesses a large family of noncovalently anchored cell wall proteins (CWPs) that are related to SlpA and are characterized by the presence of three Pfam04122 cell wall-binding motifs (10). Functions have been assigned to a few of these proteins, including Cwp84, the cysteine protease responsible for SlpA cleavage (11)(12)(13); CwpV, a phase-variable autoaggregating factor (14,15); and Cwp66, a putative adhesin (16). However, despite a number of these proteins being implicated in bacterium-host interactions (16 -18), their exact roles in pathogenesis have yet to be elucidated.…”

ClosTron-Mediated Engineering of Clostridium

“…By way of example, it has been used to establish the role of bacterial factors (toxins, flagella and adhesins) in infection, 23,[26][27][28][29] to garner a greater understanding of the role and activities of enzymes in primary metabolism, [30][31][32][33] to begin to elucidate mechanistic details of the developmental process of spore formation/ germination 21,34,35 and provide fundamental information on regulatory processes important in virulence factor and metabolite production. [36][37][38][39] Its availability has provided the research community with a sequence of the re-targeted region used to generate the insertion should be given.…”