Localization of the Clostridium difficile cysteine protease Cwp84 and insights into its maturation process

Abstract

Clostridium difficile is a nosocomial pathogen involved in antibiotic-associated diarrhea. C. difficile expresses a cysteine protease, Cwp84, which has been shown to degrade some proteins of the extracellular matrix and play a role in the maturation of the precursor of the S-layer proteins. We sought to analyze the localization and the maturation process of this protease. Two identifiable forms of the protease were found to be associated in the bacteria: a form of ∼80 kDa and a cleaved one of 47 kDa, identified as the mature protease. They were found mainly in the bacterial cell surface fractions and weakly in the extracellular fraction. The 80-kDa protein was noncovalently associated with the S-layer proteins, while the 47-kDa form was found to be tightly associated with the underlying cell wall. Our data supported that the anchoring of the Cwp84 47-kDa form is presumably due to a reassociation of the secreted protein. Moreover, we showed that the complete maturation of the recombinant protein Cwp84(30-803) is a sequential process beginning at the C-terminal end, followed by one or more cleavages at the N-terminal end. The processing sites of recombinant Cwp84 are likely to be residues Ser-92 and Lys-518. No proteolytic activity was detected with the mature recombinant protease Cwp84(92-518) (47 kDa). In contrast, a fragment including the propeptide (Cwp84(30-518)) displayed proteolytic activity on azocasein and fibronectin. These results showed that Cwp84 is processed essentially at the bacterial cell surface and that its different forms may display different proteolytic activities.

Fig. 1.

Localization of Cwp84 in C. difficile. (A) Immunofluorescence microscopy of Cwp84 (green) by Alexa Fluor 488 in whole bacteria, comparing the 630 and the 630Δerm cwp84_347a::erm strains; the nucleus was stained by DAPI (blue). (B and C) Immunoblot analysis using Cwp84 antibodies was carried out on protein extracts collected from bacteria grown in TY and TYG media (B) or from bacteria isolated from monoxenic C. difficile-associated mice (C). Lane 1, glycine extract; lane 2, cell surface-associated proteins; lane 3, extracellular proteins; lane 4, cell surface-associated proteins from the cwp84 mutant strain. The various fractions were subjected to SDS–12% PAGE. The positions of molecular mass marker proteins (kDa) are indicated (Biolabs for the in vitro experiments and Bio-Rad for the in vivo experiment).

Fig. 2.

Proteolytic process of Cwp84_30-803. The recombinant protease Cwp84_30-803 was purified in the presence or absence of 50 μM E64 and then incubated under reducing conditions with 2 mM DTT. (A) SDS-PAGE analysis. Purification of Cwp84_30-803 in the presence of the cysteine protease inhibitor (lanes 2 to 5) resulted in the detection of intermediate forms at 0, 2, and 4 h and the mature form at 6 h, whereas the purification in the absence of E64 (lanes 6 to 9) resulted in the detection of the mature form from 0 h. Lane 1 corresponds to the protease before dialysis. (B) Immunoblot analysis with anti-His tag antibody. In the presence of E64, the histidine tag fused to the N-terminal end was detected in the total and some of the processed intermediate forms of Cwp84; however, the mature form was not detected, suggesting that proteolytic process of Cwp84 begins at the C-terminal end, and follows at the N-terminal end. The Bio-Rad molecular mass standard (in kilodaltons) was used.

Fig. 3.

Schematic representation of Cwp84 proteolytic processing. cwp84 encodes a protein of 803 amino acid residues. The peptide signal (), the catalytic domain (□), and the domain corresponding to the anchoring domain () are indicated. The scheme shows the three Pfam 04122 motifs and the catalytic triad (Cys-116, His-262, and Asn-287). The amino acids involved in the proteolytic cleavage are indicated.

Fig. 4.

Purification of Cwp84_C116A and transmaturation by Cwp84_30-803. The mutated protease Cwp84_C116A was incubated at 37°C alone or with Cwp84_30-803 under reducing conditions for 0, 2, 4, 6, and 24 h. Cwp84_C116A alone with 2 mM DTT does not show the maturation process (A), whereas incubation of Cwp84_C116A with Cwp84_30-803 at an enzyme/substrate ratio of 1:100 resulted in the increase of intermediate forms at 4 and 6 h and detection of mature form at 24 h (B). The molecular mass standard used was purchased from Fermentas.

Fig. 5.

Proteolytic activities of Cwp84_30-803 and Cwp84_30-518 on fibronectin. The recombinant proteases were incubated for 16 h with fibronectin at enzyme/substrate ratios of 1:1 and 1:10. All experiments were performed under reducing conditions (2 mM DTT). Lane 1, fibronectin control (5 μg); lane 2, Cwp84_30-803 and fibronectin (1:1); lane 3, Cwp84_30-803 and fibronectin (1:10); lane 4, Cwp84_30-518 and fibronectin (1:1); lane 5, Cwp84_30-518 and fibronectin (1:10). The molecular mass standard used was from Fermentas.

Fig. 6.

Reassociation of Cwp84 protein. Bacteria were loaded onto a nitrocellulose membrane and treated with specific anti-Cwp84 or anti-Fbp68 antibodies. (A) C. difficile strains 630Δerm cwp84_347a::erm and 630Δerm fbpA_640a::erm were incubated with 20 μg of Cwp84_92-518 or 20 μg of FbpA (lane 3). Lane 1, positive control Cwp84_92-518; lane 2, negative control 630Δerm cwp84_347a::erm and 630Δerm fbpA_640a::erm not incubated with the recombinant proteins. (B) Different numbers of C. difficile 630Δerm cwp84_347a::erm and B. subtilis bacteria. Samples—5 × 10⁸ (lane 1), 10⁸ (lane 2), 10⁷ (lane 3), 10⁶ (lane 4), 10⁵ (lane 5), or 10⁴ (lane 6)—were incubated with 2 μg of Cwp84_92-518. The samples were centrifuged, and the reassociation of recombinant Cwp84 was analyzed by dot blotting.

Fig. 7.

Cwp84 localization and maturation process model. The Cwp84 native form is composed of a signal peptide, an N-terminal propeptide (dashed line), an N-terminal part (empty circle), and a C-terminal part (partially filled circle). The native protein is exported to the S layer probably via the SecA secretion system, where the signal peptide is removed. Cwp84_33-803 is then associated with the S layer where the N-terminal peptide (dash line) is potentially removed. At this subcellular position, it is likely that Cwp84_30-803 cleaves the SlpA protein and participates in the S-layer turnover. When Cwp84_30-803 is released from the bacterial surface, it is first cleaved at the C-terminal part, leading to Cwp84_30-518, and then after the N-terminal propeptide, leading to Cwp84_92-518. According to our observations in E. coli, at least the final cleavage is probably due to an automaturation mechanism. The mature Cwp84_92-518 is thereafter reassociated with the underlying cell wall. When the mature form is not surface bound, it potentially degrades the ECM host proteins.