Roles of cysteine proteases Cwp84 and Cwp13 in biogenesis of the cell wall of Clostridium difficile

Abstract

Clostridium difficile expresses a number of cell wall proteins, including the abundant high-molecular-weight and low-molecular-weight S-layer proteins (SLPs). These proteins are generated by posttranslational cleavage of the precursor SlpA by the cysteine protease Cwp84. We compared the phenotypes of C. difficile strains containing insertional mutations in either cwp84 or its paralog cwp13 and complemented with plasmids expressing wild-type or mutant forms of their genes. We show that the presence of uncleaved SlpA in the cell wall of the cwp84 mutant results in aberrant retention of other cell wall proteins at the cell surface, as demonstrated by secretion of the proteins Cwp66 and Cwp2 into the growth medium. These phenotypes are restored by complementation with a plasmid expressing wild-type Cwp84 enzyme but not with one encoding a Cys116Ala substitution in the active site. The cwp13 mutant cleaved the SlpA precursor normally and had a wild-type-like colony phenotype. Both Cwp84 and Cwp13 are produced as proenzymes which are processed by cleavage to produce mature enzymes. In the case of Cwp84, this cleavage does not appear to be autocatalytic, whereas in Cwp13 autocatalysis was demonstrated as a Cys109Ala mutant did not undergo processing. Cwp13 appears to have a role in processing of Cwp84 but is not essential for Cwp84 activity. Cwp13 cleaves SlpA in the HMW SLP domain, which we suggest may reflect a role in cleavage and degradation of misfolded proteins at the cell surface.

Fig. 1.

Phenotype and complementation of a cwp84 mutant of C. difficile 630Δerm. (A) SDS-PAGE analysis of cell wall proteins and culture supernatants. Bacteria containing plasmids (see below) were grown in BHI broth. Cell wall proteins were extracted, and culture supernatants were concentrated from bacterial cultures at late exponential phase (OD₆₀₀ = 0.5). Plasmids: –, pMTL960; 84, pCwp84_WT; 84*, pCwp84_C116A. (B) Colony morphologies. Serial dilutions (100 μl) of overnight cultures were plated on BHI agar supplemented with 15 μg of thiamphenicol/ml and grown for 2 days, and representative colonies were photographed.

Fig. 2.

Cell wall protein localization is defective in a cwp84 mutant. C. difficile 630Δerm (WT) and the cwp84 mutant harboring plasmids (see below) were grown in BHI broth to late exponential phase (OD₆₀₀ = 0.5). Cell wall extracts and culture supernatants were prepared and analyzed by SDS-PAGE, followed by Western blotting with antibodies against Cwp66 and Cwp2. Plasmids: –, pMTL960; 84, pCwp84_WT; 84*, pCwp84_C116A.

Fig. 3.

Processing of Cwp84. (A) Detection of Cwp84 in the cwp84 mutant overexpressing either WT or the Cys116Ala protein. C. difficile 630Δerm (WT) and the cwp84 mutant harboring plasmids as indicated were grown in BHI broth to late exponential phase (OD₆₀₀ = 0.5). Cell wall extracts and culture supernatants were prepared and analyzed by SDS-PAGE, followed by Western blotting with antibodies against Cwp84 and the C-terminal His₆ tag. Plasmids: –, pMTL960; 84, pCwp84_WT; 84*, pCwp84_C116A. The upper band (84 kDa) recognized by these antibodies in the cwp84 mutant carrying pCwp84_WT is indicated (◀). The N-terminal sequence of the 77-kDa protein corresponding to the lower band was determined as SSVAY. (B) Line diagram of domain structure of Cwp84 and location of the signal peptide and propeptide domains. At the top is the domain structure of Cwp84 showing the location of the signal peptide (black rectangle), as predicted by SignalP, and the cysteine protease domain (white box), as predicted by Pfam (PF00112). The three cell wall binding motifs (PF04122) are shaded in gray. Below is the N-terminal amino acid sequence of Cwp84 showing the sites of cleavage to release the signal peptide (▾) and the mature protein (▿).

Fig. 4.

Characterization of a cwp13 mutant and processing of Cwp13. (A) Analysis of the effects of an insertional mutation in cwp13 by SDS-PAGE and Western blotting. C. difficile 630Δerm (WT) and the cwp13 mutant harboring plasmids as indicated were grown in BHI broth supplemented with 15 μg of thiamphenicol/ml to late exponential phase (OD₆₀₀ = 0.5). Cell wall extracts and culture supernatants were prepared and analyzed by Coomassie blue staining (top) and Western blotting with antibodies against the C-terminal His₆ tag (6% acrylamide), Cwp84 (6% acrylamide) and LMW SLP. Plasmids: –, pMTL960; 13, pCwp13_WT; 13*, pCwp13_C109A. The 77-kDa Cwp13_WT and 84-kDa Cwp13_C109A proteins detected by Coomassie blue staining and the anti-His₆ tag are indicated (◁ and ◀), and their N-terminal sequences were determined as DNSNT and APTSY, respectively. (B) Domain structure of Cwp13 and location of the signal peptide and propeptide domains. At the top is domain structure of Cwp13 showing the location of the signal peptide (black rectangle) and the cysteine protease domain (white box), as predicted by Pfam (PF00112). The cell wall anchoring domains (PF04122) are shaded in gray. Below is the N-terminal amino acid sequence of Cwp13 showing the sites of cleavage to release the signal peptide (▾) and the mature protein (▿). (C) Domain structure of SlpA. The vertical bar indicates the cleavage site of Cwp84 to produce the mature HMW SLP and LMW SLP. The black triangle (▴) shows the approximate site of cleavage, within a cell wall binding domain, by Cwp13 to generate a 47-kDa N-terminal product.

Fig. 5.

Cwp13 cleaves SlpA but does not complement the cwp84 mutant. C. difficile cwp84 mutant harboring different plasmids was grown in BHI broth supplemented with thiamphenicol (15 μg/ml) till late exponential phase (OD₆₀₀ = 0.5). Cell wall extracts and culture supernatants were prepared and analyzed by Coomassie blue staining (top) and Western blotting with antibody against the LMW SLP (middle and bottom). The bottom panel shows the 34-kDa LMW SLP species on an overexposed Western blot. Plasmids: –, pMTL960; 84, pCwp84_WT; 84*, pCwp84_C116A; 13, pCwp13_WT; 13*, pCwp13_C109A. Open arrow, 77-kDa Cwp84; solid arrow, 78-kDa Cwp13_C109A processed by endogenous Cwp13; ◀, 47-kDa fragment of SlpA.

Fig. 6.

Investigation of cleavage of CwpV by Cwp84 and Cwp13. C. difficile strains containing either pMTL960 vector or pCBR044 carrying cwpV+ were grown overnight in BHI broth supplemented with thiamphenicol (15 μg/ml). Cell wall proteins were extracted and analyzed by SDS-PAGE. Plasmids: –, pMTL960; V, pCBR044 CwpV+.

Fig. 7.

Model for processing and activities of Cwp84 and Cwp13. SlpA, Cwp84, and Cwp13 are produced as preproteins containing signal peptides that are removed during processing by the sec system (step a). The propeptides of Cwp84 and Cwp13 are removed (step b), either by autocatalysis in the case of Cwp13 or by an unknown activity together with Cwp13 activity in the case of Cwp84, to form the active enzyme species that are incorporated into the S-layer (step c). Mature Cwp84 cleaves the SlpA precursor (step d), which results in the formation of the H/L complex (step e). Misfolded proteins are recognized by Cwp13 and are cleaved in their cell wall binding domains to prevent incorporation into the S-layer, resulting in detachment from the cell and deposition into the growth medium (step f). S-L, S-layer; PG, peptidoglycan; Mem, membrane; Cyt, cytoplasm.