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 knock-out approach, we show that inactivation of the Cwp84 gene in C. difficile 630DeltaErm results in a bacterial phenotype in which only immature, single chain SlpA comprises the S-layer. The Cwp84 knock-out mutants (CDDeltaCwp84) displayed significantly different colony morphology compared with the wild-type strain and grew more slowly in liquid medium. SlpA extracted from CDDeltaCwp84 was readily cleaved into its mature subunits by trypsin treatment. Addition of trypsin to the growth medium also cleaved SlpA on CDDeltaCwp84 and increased the growth rate of the bacterium in a dose-dependent manner. Using the hamster model for C. difficile infection, CDDeltaCwp84 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.

FIGURE 1.

A, confirmation of intron integration in cwp84 gene by PCR. Lane 1, 4.3-kbp product from CDΔCwp84₃₄₇. Lane 2, 2.4-kbp product from C. difficile 630ΔErm. B, demonstration of erythromycin retrotransposition-activated marker (Erm-RAM) self-splicing, an event strictly coupled to group II intron integration. Lane 1, 900-bp product from CDΔCwp84₃₄₇. Lane 2, 1300-bp product from native pMTL007-EC2 shuttle vector. C, RT-PCR of cwp84 mRNA flanking intron integration site in stationary phase cultures. Lane 1, C. difficile 630ΔErm, RT+; lane 2, CDΔCwp84_347, RT+. D, PCR confirmation that CD1751 (a cysteine protease with homology to Cwp84) is unaffected by the knock-out of cwp84. Lane 1, C. difficile 630ΔErm; lane 2, CDΔCwp84₃₄₇.

FIGURE 2.

Difference in colony morphology of wild-type C. difficile 630ΔErm and CDΔCwp84₃₄₇ grown on solid phase medium.

FIGURE 3.

A, analysis of extracted SLPs from wild-type C. difficile 630ΔErm, CDΔCwp84₃₄₇, CDΔCwp84₆₇₇, and CDΔCwp84₂₀₅₄. Lanes 5 and 6, 24-h culture supernatant fluids from C. difficile 630ΔErm (Cul sup WT), CDΔCwp84₃₄₇ (Cul sup Cwp84), respectively. B, analysis of extracted SLPs by silver-stained SDS-PAGE from wild-type C. difficile 630ΔErm and CDΔCwp84₃₄₇.

FIGURE 4.

A, cleavage of extracted SlpA with trypsin. Cleavage of SLP extracts from CDΔcwp84₃₄₇ with 1 μg/ml trypsin (DPCC-treated, bovine pancreas; Sigma T1005) in 50 mm HEPES pH 7.4 containing 0.15 m NaCl at 22 °C for various times. B, extracted SLPs from 24-h cultures of CDΔcwp84₃₄₇ grown in sBHI supplemented with 0, 1, 10, and 100 μg/ml trypsin. Prior to low pH extraction of SLPs, cultures were washed with 10 mm HEPES containing 100 mm NaCl, pH 7.4, and 0.5 mg/ml trypsin inhibitor and then extracted in 0.2 m glycine, pH 2.2, buffer containing 0.1 mg/ml trypsin inhibitor.

FIGURE 5.

Virulence of C. difficile 630ΔErm and CDΔCwp84₃₄₇ in hamsters. Data show time from challenge to severe disease/death in the hamster model for CDI. For CDΔCwp84₃₄₇, the data represent three test groups of 10 animals and for C. difficile 630ΔErm, two test groups of 10 animals.

FIGURE 6.

Alignment of predicted maturation cleavage sites within SlpA from 14 C. difficile ribotypes (31). Maturation of SlpA is generally predicted to occur C-terminal to a consensus motif TKS or TYX (31). Potential cleavage sites for trypsin or chymotrypsin are shown in bold.