Cwp84, a Surface-Associated Protein of Clostridium difficile, Is a Cysteine Protease with Degrading Activity on Extracellular Matrix Proteins

Abstract

Clostridium difficile pathogenicity is mediated mainly by its A and B toxins, but the colonization process is thought to be a necessary preliminary step in the course of infection. The aim of this study was to characterize the Cwp84 protease of C. difficile, which is highly immunogenic in patients with C. difficile-associated disease and is potentially involved in the pathogenic process. Cwp84 was purified as a recombinant His-tagged protein, and specific antibodies were generated in rabbits. Treatment of multiple-band-containing eluted fractions with a reducing agent or with trypsin led to accumulation of a unique protein species with an estimated molecular mass of 61 kDa, corresponding most likely to mature autoprocessed Cwp84 (mCwp84). mCwp84 showed concentration-dependent caseinolytic activity, with maximum activity at pH 7.5. The Cwp84 activity was inhibited by various cysteine protease inhibitors, such as the specific inhibitor E64, and the anti-Cwp84-specific antibodies. Using fractionation experiments followed by immunoblot detection, the protease was found to be associated with the S-layer proteins, mostly as a nonmature species. Proteolytic assays were performed with extracellular matrix proteins to assess the putative role of Cwp84 in the pathogenicity of C. difficile. No degrading activity was detected with type IV collagen. In contrast, Cwp84 exhibited degrading activity with fibronectin, laminin, and vitronectin, which was neutralized by the E64 inhibitor and specific antibodies. In vivo, this proteolytic activity could contribute to the degradation of the host tissue integrity and to the dissemination of the infection.

Clostridium difficile, a gram-positive spore-forming, anaerobic bacterium, is the etiological agent of pseudomembranous colitis and of many cases of nosocomial diarrhea (29). Its pathogenicity is mediated mainly by two exotoxins, toxins A and B, both of which damage human colonic mucosa (28). Recently, highly virulent strains have been isolated in the United States and Canada and have caused epidemic disease associated with high mortality. These isolates produce larger amounts of toxins A and B than typical toxigenic strains and express the binary toxin, a putative virulence factor (9, 21, 37). Whereas toxins A and B have been extensively studied, little is known yet about the role of nontoxin proteins in C. difficile virulence, either during colonization or in the pathogenic process of the bacterium.

Colonization of the host by C. difficile occurs after disturbance of the normal colonic microbiota, following, for example, antibiotic treatment. As in other enteric pathogens, colonization involves different adhesins and presumably hydrolytic and proteolytic enzymes. Some of C. difficile adhesins have been recently characterized. The bacterium expresses an S-layer on the outer cell surface, which is composed of two major proteins of the bacterial surface: the high-molecular-weight protein P47 and the low-molecular-weight protein P36 (6). Both subunits are encoded by the slpA gene and are produced from posttranslational cleavage of a precursor (3, 5). P47 shows strong and specific binding to gastrointestinal tissues and some extracellular matrix proteins (type I collagen, thrombospondin, and vitronectin) (4). Other surface proteins, which mediate in vitro adherence to Vero cells and might be involved in in vivo colonization, have been characterized in our laboratory. These proteins include the Cwp66 protein, expressing the cell wall-anchoring N-terminal domain and the cell surface-exposed C-terminal domain with adhesive properties (36); the flagellar proteins FliC and FliD (34); the heat shock protein GroEL (10); and the fibronectin-binding protein Fbp68 (11). It is also noteworthy that the genes encoding Cwp66 and the S-layer precursor are located near each other in a 37-kb DNA cluster in the C. difficile genome (17).

Apart from the adhesion factors, proteolytic enzymes are frequently involved in the bacterial colonization process, contributing to nutrient acquisition, degradation of host proteins, including immunoglobulins, or processing of bacterial proteins involved in pathogenesis (23). A previous study showed that C. difficile displays a proteolytic activity correlated with strain virulence in the hamster model (26, 31, 32). However, no proteolytic factor of C. difficile has been extensively characterized yet. The cwp84 gene, which displays high levels of homology with genes encoding some cysteine proteases (30), has been located in the same cluster as slpA and cwp66. The 5′ part of this gene cloned into an expression vector conferred a nonspecific proteolytic activity to an Escherichia coli strain (30). An analysis of 28 strains belonging to different serotypes and with different toxigenic status for the presence of cwp84 revealed that this gene was highly conserved among them (30). Moreover, Cwp84 is expressed during the course of human infection, as shown by the presence of specific antibodies in 15/17 sera from patients with C. difficile-associated disease (25). These results suggest that Cwp84 could have an important role in the physiopathology of C. difficile. In particular, Cwp84 could contribute to the cleavage of the extracellular matrix (ECM) host proteins to facilitate the degradation of host tissue integrity and thus dissemination of the infection.

The aim of this study was to further characterize the Cwp84 protease and its putative role in the pathogenesis of C. difficile-associated disease. To address this issue, Cwp84 was purified as a recombinant active protein and specific antibodies were generated in rabbits. The proteolytic activity of Cwp84 was assessed with azocasein and with ECM proteins and was shown to be inhibited by cysteine protease inhibitors and specific antibodies. To the best of our knowledge, this is the first time that a C. difficile protease has been characterized.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

C. difficile toxigenic strain 79-685, isolated from a patient with pseudomembranous colitis in Strasbourg, France, was grown under anaerobic conditions in tryptone glucose yeast infusion broth (Difco Laboratories) and on Columbia agar plates supplemented with 4% horse blood (Biomerieux). The E. coli BL21/pET-28a(+)_cwp84 strain was grown on LB agar or in broth (Difco Laboratories) supplemented with 50 μg/ml kanamycin to maintain the pET plasmid.

Cwp84 purification.

Recombinant Cwp84 (rCwp84) was purified as previously described (25), with some modifications. Briefly, rCwp84 was obtained from the E. coli/pET-28a(+)_cwp84 clone by induction of protein expression with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and subsequent purification by single-step affinity chromatography employing BD TALON cobalt affinity resin (BD Biosciences) as described in protocols supplied by the manufacturer. The eluted fractions containing the recombinant protease (rCwp84) were dialyzed overnight against phosphate-buffered saline and then frozen at −80°C for storage. The recombinant protein concentration was determined by the Bradford method, using the Bio-Rad protein assay reagent with bovine serum albumin as a standard. The different fractions obtained from purification were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting with specific anti-Cwp84 antibodies. The molecular weights of the distinct protein species were determined by comparison with a standard exponential curve.

Maturation of the recombinant protease. (i) Under reducing conditions.

To test for an automaturation process, aliquots of the purified fraction were treated with the reducing agent dithiothreitol (DTT) (known to be a cysteine protease activator) at a concentration of 2 mM in 25 mM Tris buffer (pH 7.5) for 1, 2, 3, 4, 6, 8, and 16 h, and the pattern of each aliquot was then analyzed by SDS-PAGE.

Unless specified otherwise, all experiments described below were performed with the recombinant protease after incubation with 2 mM DTT for 4 h; the resulting protein species was designated mature Cwp84 (mCwp84).

(ii) By action of trypsin. Five micrograms of rCwp84 was incubated with 5 μg of trypsin (Sigma) for various times at 37°C in 25 mM Tris buffer (pH 7.5). The mixture was subsequently resolved by SDS-PAGE.

Production of Cwp84-specific antibodies.

Rabbit polyclonal antibodies were produced and subsequently purified on protein A-Sepharose by Agrobio (France) with rCwp84 purified on nickel-Sepharose resin (25).

Immunoblotting.

Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences) for immunoblotting. The membrane was incubated overnight at room temperature in a blocking buffer consisting of 5% skim milk in TNT (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.05% Tween), and this was followed by 2 h of incubation with anti-Cwp84 diluted previously (1/2,000 dilution). The membrane was then washed three times with TNT. Bound antibodies were detected using goat anti-rabbit immunoglobulin G alkaline phosphatase conjugate (1/20,000 dilution; Sigma) with the substrates nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (Sigma).

Characterization of the enzymatic activity of Cwp84. (i) Azocasein hydrolysis.

The proteolytic activity of mCwp84 was quantified with azocasein (Sigma). Various concentrations of mCwp84 (0.01 to 0.2 mg/ml) were tested by addition of protease (5, 10, 20, 50, or 100 μg) to 500 μl of a 5-mg/ml azocasein solution in 25 mM Tris (pH 7.5); after 16 h of incubation, intact azocasein was removed by 3% trichloroacetic acid precipitation, and the amount of released dye was measured spectrophotometrically at 336 nm. A control was obtained by the same method without addition of protease to the sample. The effect of pH on Cwp84 caseinolytic activity was also determined by using 25 mM Tris buffer at pH 3.5, 7.2, 7.5, 8.0, and 10.0.

(ii) Inhibition assays.

The effects of addition of various protease inhibitors on the Cwp84 caseinolytic activity were investigated. The inhibitors studied included specific cysteine protease inhibitors [trans-epoxysuccinyl-l-leucyl-amido(4-guanidino)butane (E64), N-ethylmaleimide, iodoacetic acid], serine and cysteine protease inhibitors (phenylmethylsulfonyl fluoride, leupeptin, N-p-tosyl-l-lysine chloromethyl ketone [TLCK]), a specific serine protease inhibitor (aprotinin), a specific aspartic protease inhibitor (pepstatin), and the metalloprotease inhibitor EDTA. All inhibitors were purchased from Sigma, and the concentrations tested were the usual concentrations (Table 1). mCwp84 (50 μg) and inhibitors were preincubated for 30 min at 37°C; the experiments with azocasein were then performed by using the conditions described above. All experiments were performed at least in triplicate, and the results are expressed below as the mean values obtained in each separate experiment.

TABLE 1.

Effects of protease inhibitors on caseinolytic activity of mCwp84

Compound	Concn or dilution	Residual proteolytic activity (%)
Specific cysteine protease inhibitors
E64	10 μM	30.5 ± 5.3
	100 μM	0.3 ± 0.6
N-Ethylmaleimide	10 mM	6.8 ± 1.3
Iodoacetic acid	10 mM	15.4 ± 1.5
Serine and cysteine protease inhibitors
TLCK	100 μM	6.8 ± 1.9
Leupeptin	100 μM	9.1 ± 2.3
Phenylmethylsulfonyl fluoride	1 mM	95.5 ± 5.2
	10 mM	6.8 ± 3.5
Specific serine protease inhibitor aprotinin	Equimolar	89.2 ± 1.2
Specific aspartic protease inhibitor pepstatin	10 μM	90.3 ± 2.12
Metalloprotease inhibitor EDTA	5 mM	80.8 ± 4.1
Specific anti-Cwp84 antibodies	1/50 dilution	93.3 ± 6.7
	1/10 dilution	7.9 ± 1.7

(iii) Proteolytic activity of Cwp84 with ECM proteins.

To test for cleavage of human ECM proteins, 5 μg of fibronectin (Roche), vitronectin, laminin, or collagen type IV (Sigma) suspended in 30 μl of 25 mM Tris buffer (pH 7.5) was incubated with various amounts of rCwp84 or mCwp84 (enzyme/substrate ratios, 1:1 to 1:50 [wt/wt]). Experiments were performed at 37°C for 4 or 16 h, with or without 2 mM DTT (Table 2). For negative controls, ECM proteins were incubated in the conditions described above but without Cwp84. In some experiments, to test for inhibition of the Cwp84 activity, E64, the specific cysteine protease inhibitor, was added to the mixture to a final concentration of 10 or 100 μM. The reaction was stopped by adding SDS-PAGE sample buffer, followed by 5 min of boiling. Mixtures were then resolved by 8 to 12% SDS-PAGE. All experiments were performed in triplicate; data from one representative experiment are shown below.

TABLE 2.

Proteolytic activities of rCwp84 and mCwp84 with ECM proteins in different experimental conditions

Protease species	Cwp84/protein ratio (wt/wt)	Reducing conditionsa	Incubation time (h)	Antibodies (dilution)	E64 inhibitor concn (μM)	Cleavage or degradation of ECM proteins by Cwp84b
						Fibronectin (5 μg)	Laminin (5 μg)	Vitronectin (5 μg)	Type IV collagen (5 μg)
rCwp84	1:1	−	16	−		−	−	NDc	−
	1:1	+	16	−		+	+	ND	−
	1:10	+	16	−		+	+	+	−
	1:50	+	16	−		−	ND	−	ND
	1:10	+	16	−	10	±	−	±	ND
				−			−		ND
	1:10	+	16	−	100	−	−	ND	ND
	1:10	+	16	+ (1/50)		±	−	±	ND
	1:10	+	16	+ (1/20)		−	−	ND	ND
	1:10	+	16	+ (1/10)		−	−	−
	1:2	+	4	−		±	+	ND	−
	1:10	+	4	−		−	?	ND	−
mCwp84	1:10	−	4	−		−	−	−	ND
	1:10	+	4	−		+	+	+	ND
	1:10	+	4	−	10	±	−	±	ND
	1:10	+	4	−	100	−	−	−	ND
	1:10	+	4	+ (1/10)		−	−	ND	ND

^aReactions were performed with (+) or without (−) 2 mM DTT.

^bThe presence of total (+), partial (±), or no (−) degrading or cleaving activity of Cwp84 with ECM proteins was determined by SDS-PAGE analysis.

^cND, not done.

Neutralizing activity of specific anti-Cwp84 antibodies.

The neutralizing activity of the specific anti-Cwp84 rabbit immunoglobulin G was tested by monitoring Cwp84-mediated degradation of azocasein. Various amounts of Cwp84 antibodies were added to the azocasein test mixture, resulting in 1/10, 1/20, 1/50, and 1/100 dilutions, and assays were performed as described above. The neutralizing activity of antibodies was also tested by inhibition of the Cwp84 proteolytic activity on ECM components (fibronectin, laminin, and vitronectin). In these experiments, antibodies were added to the reaction after 1/10, 1/20, or 1/50 dilution, and assays were performed as described above. To assess the specificity of the neutralizing activity of the antibodies and to exclude the possibility of a steric hindrance effect, negative control experiments were performed with antibodies directed against the C-terminal domain of the Cwp66 adhesin instead of the anti-Cwp84 using the same dilutions (36).

Localization of Cwp84 in the bacterium.

To assess Cwp84 localization, bacterial proteins from stationary-phase cultures were separated by three different extraction methods. (i) Whole bacterial proteins were separated into four compartments, supernatant, cell wall, membrane, and cytoplasm, as previously described (14). (ii) Surface-associated proteins were obtained by an abrasive method as previously described (38). (iii) A fraction containing the S-layer proteins was obtained by the low-acid glycine extraction method (5). Equal amounts of these various fractions, as measured by the Bradford method, were separated by 12% SDS-PAGE and analyzed by immunoblotting with polyclonal anti-Cwp84 as described above.

To validate the fractionation experiment, anti-Cwp66Cter antibodies for detection of the Cwp66 adhesin in the membrane and the cell wall fraction (36) and anti-GroEL antibodies for detection of the GroEL heat shock protein in the supernatant were used (10).

RESULTS

Purification of rCwp84 and maturation of the protease.

Purification of rCwp84 was performed from an E. coli clone [pET28a(+)Ωcwp84] which displays proteolytic activity on skim milk (25). Since previous purification on a nickel-Sepharose column produced several bands, we purified the recombinant protein on a cobalt-Sepharose column. By SDS-PAGE analysis of the eluted fractions, two major bands were observed, at estimated molecular masses of 68 and 73 kDa; two other bands with low intensity were also observed, at molecular masses of 82 and 58 kDa (Fig. 1A). Immunoblotting revealed that anti-Cwp84 antibodies reacted preferentially with the 68-kDa band and, to a lesser extent, with the 82- and the 73-kDa bands (Fig. 1B). By contrast, anti-Cwp84 antibodies did not react with the 58-kDa band (Fig. 1B).

FIG. 1.

Analysis of the eluted fraction after purification of rCwp84 on a cobalt resin. (A) SDS-PAGE analysis. MW, SDS-PAGE low-range standard (Bio-Rad); rCwp84, eluted fraction, with four distinct protein bands at estimated molecular masses of 82, 73, 68, and 58 kDa. (B) Immunoblot with specific anti-Cwp84 antibodies. Of the four protein bands, the anti-Cwp84 antibodies reacted preferentially with the 68-kDa protein band.

The protein profile of rCwp84 was greatly modified after treatment of the eluted fraction with the reducing agent DTT (2 mM). SDS-PAGE analysis showed that over time there was progressive disappearance of the four distinct protein bands and accumulation of a unique species at an estimated molecular mass of 61 kDa. This molecular species was considered to correspond to the mature active protease, mCwp84 (Fig. 2A). This result suggested that there is sequential autoproteolytic conversion of rCwp84 into the mature enzyme. Cwp84 could be considered fully mature 4 h after the initiation of the autoprocessing reaction. The same result was observed after resolution of the trypsin-rCwp84 reaction; the action of trypsin on rCwp84 led to accumulation of the same unique 61-kDa protein species in less than 1 h (Fig. 2B).

FIG. 2.

Maturation of rCwp84. (A) Autoprocessing of rCwp84 under reducing conditions (2 mM DTT). rCwp84 was incubated with 2 mM DTT in 25 mM Tris buffer (pH 7.5), the reaction was stopped at designated time intervals, and the products were analyzed by SDS-PAGE. MW, SDS-PAGE low-range standard (Bio-Rad); lanes 1 to 8, products from the autoprocessing reaction isolated at different times. (B) Maturation of rCwp84 by trypsin. Lane 1, rCwp84 control (5 μg) incubated in Tris buffer; lane 2, rCwp84 (5 μg) incubated for 30 min with 5 μg of trypsin.

Characterization of the proteolytic activity of Cwp84 on azocasein.

Purified fractions showed weak concentration-dependent proteolytic activity toward azocasein at concentrations between 0.01 and 0.2 mg/ml (Fig. 3A). The protease was active over a wide pH range (pH 3.5 to 8.0); the maximum activity was at pH 7.5 (corresponding to the isoelectric point), and about 80% of the activity was retained at pH 3.5 to 8.0. By contrast, the Cwp84 activity decreased at basic pH values, and only 30% of the activity was retained at pH 10.0 (Fig. 3B).

FIG. 3.

(A) Concentration-dependent activity of mCwp84 on azocasein. The optical density at 336 nm (OD 336 nm) is a direct measure of the amount of azocasein degraded by the protease. (B) pH-dependent activity of Cwp84 on azocasein.

Inhibition assays.

The proteolytic activity of mCwp84 was totally inhibited by E64, the specific cysteine protease inhibitor (100 μM) (Table 1). A similar extent of inhibition was observed with other inhibitors (residual activity, <10%), including the specific cysteine protease inhibitor N-ethylmaleimide and some serine and cysteine protease inhibitors, such as high concentrations of phenylmethylsulfonyl fluoride (10 mM), TLCK (100 μM), and leupeptin (100 μM). The other specific cysteine protease inhibitor tested, iodoacetic acid (10 mM), inhibited the mCwp84 activity to a lesser extent (residual activity, 15%). By contrast, the three other compounds tested, aprotinin, pepstatin, and EDTA, which are specific potent inhibitors of serine, aspartic acid, and metalloproteases, respectively, inhibited the Cwp84 activity only moderately (residual activities, >80%) (Table 1). The polyclonal anti-Cwp84 antibodies inhibited the caseinolytic activity of Cwp84 only at a low dilution (residual proteolytic activity, 10% at a 1/10 dilution and 90% at a 1/100 dilution), probably because of the large amount of mCwp84 used in the azocasein assay (50 μg) (Table 1).

Proteolytic activity on ECM proteins.

In a first set of experiments, we investigated the proteolytic activity against fibronectin to determine the optimal conditions for Cwp84 activity on physiological substrates. Subsequently, in a second set of experiments, we studied the degrading activity of Cwp84 against other ECM proteins, including laminin, vitronectin, and type IV collagen, and the inhibitory effects of the cysteine protease inhibitor E64 and anti-Cwp84 antibodies on Cwp84 activity against these ECM proteins. The results of these experiments are summarized in Table 2.

The cysteine protease Cwp84 (either the nonmature rCwp84 form or mCwp84) was able to cleave fibronectin to produce two bands only in reducing conditions (2 mM DTT), compared with the negative control without protease (Table 2; Fig. 4 A and B, lanes 1 and 2). With the same enzyme/substrate ratio, mCwp84 was more efficient for cleavage of fibronectin than rCwp84 (cleavage in 4 and 16 h, respectively); no degrading activity on fibronectin could be observed with an enzyme/substrate ratio of 1:50, even after a 16-h incubation (Table 2). The optimal enzyme/substrate ratio was determined to be 1:10 and was used for the second set of experiments. Degradation of laminin and vitronectin was observed in reducing conditions after 16 h of incubation with rCwp84 and after only 4 h of incubation with mCwp84 (Table 2; Fig. 4A and B, lanes 5, 6, 9, and 10). The proteolytic activity of Cwp84 against the three ECM proteins was inhibited by the specific cysteine protease inhibitor E64, partially at a concentration of 10 μM and totally at a concentration of 100 μM (Fig. 4A, lanes 3, 4, 7, 8, 11, and 12). In addition, the degrading activity of Cwp84 against ECM proteins was fully inhibited by the anti-Cwp84 antibodies at a 1/10 dilution (Table 2; Fig. 4B, lanes 4, 8, and 12). In contrast, addition of the same amount of anti-Cwp66Cter to the mixture did not result in inhibition of fibronectin cleavage by mCwp84 (data not shown), proving that the inhibition effect was not the result of steric hindrance. With type IV collagen, no degrading activity of Cwp84 could be detected in any of the conditions tested (Table 2).

FIG. 4.

Degrading activity of mCwp84 with the ECM proteins fibronectin, laminin, and vitronectin and inhibition experiments. In all cases, the enzyme/substrate ratio was 1:10, and the reactions were performed under reducing conditions (2 mM DTT). (A) Proteolytic activity of mCwp84 on ECM proteins in a 4-h experiment and inhibition by the E64 inhibitor. Lanes MW, SDS-PAGE standard (Bio-Rad) (high range or low range); lane 1, fibronectin control; lane 2, fibronectin plus mCwp84; lane 3, fibronectin plus mCwp84 and 10 μM E64; lane 4, fibronectin plus mCwp84 and 100 μM E64; lane 5, laminin control; lane 6, laminin plus mCwp84; lane 7, laminin plus mCwp84 and 10 μM E64; lane 8, laminin plus mCwp84 and 100 μM E64; lane 9, vitronectin control; lane 10, vitronectin plus mCwp84; lane 11, vitronectin plus mCwp84 and 10 μM E64; lane 12, vitronectin plus mCwp84 and 100 μM E64. (B) Proteolytic activity of rCwp84 on ECM proteins in a 16-h experiment and inhibition by specific anti-Cwp84 antibodies. Lane 1, fibronectin control; lane 2, fibronectin plus rCwp84; lane 3, fibronectin plus rCwp84 and anti-Cwp84 antibodies (1/50); lane 4, fibronectin plus rCwp84 and anti-Cwp84 antibodies (1/10); lane 5, laminin control; lane 6, laminin plus rCwp84; lane 7, laminin plus rCwp84 and anti-Cwp84 antibodies (1/50); lane 8, laminin plus rCwp84 and anti-Cwp84 antibodies (1/10); lane 9, vitronectin control; lane 10, vitronectin plus rCwp84; lane 11, vitronectin plus rCwp84 and anti-Cwp84 antibodies (1/50); lane 12, vitronectin plus rCwp84 and anti-Cwp84 antibodies (1/10).

Cellular localization of native Cwp84.

As a control for the fractionation scheme, Cwp66 was recovered in the membrane fraction and the cell wall fraction by specific antibodies, as previously described (36), whereas GroEL was detected in the secreted fraction (10) (data not shown). With Cwp84, after fractionation experiments, anti-Cwp84 antibodies did not react with any band either in the cell wall or in the supernatant fraction (data not shown). By contrast, immunoblotting revealed two bands at about 90 and 82 kDa in the cytoplasmic fraction of C. difficile, and the 82-kDa band was also recovered from the membrane fraction. This protein species was also detected in the surface-associated protein extract obtained by the abrasive method (Fig. 5). In the glycine extract containing the S-layer proteins, anti-Cwp84 antibodies reacted strongly with a unique 85-kDa protein and weakly with a 61-kDa band (Fig. 5).

FIG. 5.

Cellular localization of native Cwp84 in C. difficile, as revealed by immunoblotting with polyclonal anti-Cwp84 antibodies. Lane MW, SDS-PAGE low-range standard (Bio-Rad); lane 1, cytoplasmic fraction; lane 2, membrane fraction; lane 3, cell wall-associated protein fraction; lane 4, glycine-extracted fraction containing S-layer proteins.

DISCUSSION

Bacterial proteases are currently recognized as colonization or virulence factors of gram-negative or gram-positive bacteria, especially cysteine proteases produced by Porphyromonas gingivalis, Streptococcus pyogenes, and Staphylococcus aureus (22, 33, 35). However, no work has focused yet on characterization of C. difficile proteases, and to the best of our knowledge, this study is the first attempt to investigate the role of a proteolytic factor of this bacterium.

Previous purification assays of an rCwp84 with a glutathione S-transferase expression system were not successful (30). For this reason, we chose a His tag expression system which allowed us to easily purify the recombinant protein using a cobalt affinity resin. Analysis by SDS-PAGE of purified fractions revealed several bands, including one at the expected molecular mass of 82 kDa (corresponding to the whole gene cloned without the signal sequence). We cannot exclude the possibility that the three bands at low molecular masses corresponded to cleavage products due to E. coli protease activity. However, it is more likely that these bands were generated through a Cwp84 automaturation process, for the following reasons: (i) cysteine proteases are synthesized as inactive proproteins, which are often able to process themselves to the mature enzyme, especially under reducing conditions (18); (ii) incomplete automaturation could be achieved under reducing conditions since treatment of the multiple-band-containing purified fractions with DTT resulted in accumulation of a unique 61-kDa species, which likely corresponds to mCwp84; and (iii) autoprocessing of cysteine proteases could start during the metal affinity purification step, as described previously for the B cathepsin of Schistosoma mansoni (19). A similar automaturation process, involving sequential processing of multiple intermediates, has been observed with the SpeB cysteine protease of S. pyogenes (8). In addition, similar to the SpeB findings, we showed that rCwp84 could be processed to the mature enzyme by the action of trypsin (20); this could have significant implications for the in vivo maturation of the protease.

As expected, the proteolytic activity of Cwp84 was inhibited to various extents by all the cysteine protease inhibitors tested, especially the specific cysteine protease inhibitor E64, but it was quite resistant to the specific inhibitors for serine, aspartic acid, and metalloproteases. This inhibition profile further confirms that Cwp84 is a member of the cysteine protease family. Like most cysteine proteases, Cwp84 is active at pH values between 3.0 and 8.0. The optimal pH for Cwp84 caseinolytic activity was found to be pH 7.5, close to the physiological pH.

Once the protein was characterized, we attempted to localize Cwp84 in the bacterium. The protease in the cytoplasm was found to have a molecular mass (90 kDa) consistent with the structure of the preproprotein (inactive proprotein synthesized with the signal peptide). Removal of the signal peptide sequence led to the 82-kDa species (corresponding to the high-molecular-mass band of rCwp84); this species is believed to be addressed to the membrane and localized to the surface of the bacterium, but it does not seem to be associated with the cell wall fraction. By contrast, Cwp84 was recovered as a 85-kDa molecular species in the glycine-extracted fraction, likely associated with the S-layer proteins. One hypothesis to explain this unexpected high molecular mass is that the protease is glycosylated, like the P47 S-layer protein (5). The localization of Cwp84 at the bacterial surface is in agreement with the prediction given by the PSORT World Wide Web server (http://psort.nibb.ac.jp/). Other bacterial proteases have been shown to be surface associated, including the SpeB cysteine protease of S. pyogenes, which exists as a secreted and tightly surface-bound protein (12), the C5a peptidase of group B streptococci (2), and the PrtA serine protease of Streptococcus pneumoniae (1). In vitro, in the conditions of our experiments, we detected Cwp84 mostly as an inactive proprotein. In vivo, Cwp84 could very well be expressed in the active 61-kDa conformation after a maturation process due to reducing conditions in the colon or to the action of trypsin. Further experiments should be performed to confirm this hypothesis.

One role of bacterial proteases is to degrade host connective tissues and to contribute directly to tissue lesions observed in some infections (15, 16, 24, 27). C. difficile infections induce destruction of enterocytes associated with a significant inflammatory response, mainly due to the proinflammatory action and cytosketon disorganization mediated by toxins A and B (28). However, direct proteolytic degradation of the basement membrane could also contribute to the intestinal epithelium necrosis. A surface-located protease might be able to interact with proteins of the ECM at a high local concentration without a dilution effect. Type IV collagen, fibronectin, vitronectin, and laminin are glycoproteins of the ECM. Type IV collagen forms a two-dimensional reticulum and is a major component of the basal lamina. Fibronectin, vitronectin, and laminin are important components of the basement membrane and are involved in the cellular adhesion process; the latter anchors the cell surface to the basal lamina. Cwp84 does not display any collagenolytic activity but cleaves fibronectin and degrades vitronectin and laminin; therefore, it could affect the integrity of host tissue, potentiating toxins through facilitation of their diffusion. The cwp84 gene seems to be present and expressed in all C. difficile strains (25, 30); however, the level of expression of the protein could be regulated, and this could account for the observation that strains displaying the greatest proteolytic activity are the most virulent strains in the hamster model (31). In addition, fibronectin has been shown to mediate host-microorganism interactions for many gram-positive bacterial pathogens, like S. aureus, S. pyogenes (13), and recently C. difficile (7, 11). In vivo, cleavage of fibronectin could contribute to the release of bacteria from host tissues and dissemination of the infection. Specific neutralization by anti-Cwp84 antibodies of the Cwp84 ECM protein-degrading activity correlates with the previous observation that production of anti-Cwp84 antibodies in patients with C. difficile-associated disease contributes to infection resistance (25). In conclusion, all these results strongly suggest that Cwp84 has a role in the pathogenic process of C. difficile, and further experiments are in progress to better define its putative role in vivo.