Clostridium difficile cell wall protein CwpV undergoes enzyme-independent intramolecular autoproteolysis

Clostridium difficile infection is a leading cause of antibiotic-associated diarrhea, placing considerable economic pressure on healthcare systems and resulting in significant morbidity and mortality. The pathogen produces a proteinaceous array on its cell surface known as the S-layer, consisting primarily of the major S-layer protein SlpA and a family of SlpA homologs. CwpV is the largest member of this family and is expressed in a phase-variable manner. The protein is post-translationally processed into two fragments that form a noncovalent, heterodimeric complex. To date, no specific proteases capable of cleaving CwpV have been identified. Using site-directed mutagenesis we show that CwpV undergoes intramolecular autoproteolysis, most likely facilitated by a N-O acyl shift, with Thr-413 acting as the source of a nucleophile driving this rearrangement. We demonstrate that neighboring residues are also important for correct processing of CwpV. Based on protein structural predictions and analogy to the glycosylasparaginase family of proteins, it appears likely that these residues play key roles in determining the correct protein fold and interact directly with Thr-413 to promote nucleophilic attack. Furthermore, using a cell-free protein synthesis assay we show that CwpV maturation requires neither cofactors nor auxiliary enzymes.     

Novel inhibitors of surface layer processing in Clostridium difficile

Clostridium difficile, a leading cause of hospital-acquired bacterial infection, is coated in a dense surface layer (S-layer) that is thought to provide both physicochemical protection and a scaffold for host-pathogen interactions. The key structural components of the S-layer are two proteins derived from a polypeptide precursor, SlpA, via proteolytic cleavage by the protease Cwp84. Here, we report the design, synthesis and in vivo characterization of a panel of protease inhibitors and activity-based probes (ABPs) designed to target S-layer processing in live C. difficile cells. Inhibitors based on substrate-mimetic peptides bearing a C-terminal Michael acceptor warhead were found to be promising candidates for further development.     

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

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.     

Clostridium difficile has two parallel and essential Sec secretion systems

Protein translocation across the cytoplasmic membrane is an essential process in all bacteria. The Sec system, comprising at its core an ATPase, SecA, and a membrane channel, SecYEG, is responsible for the majority of this protein transport. Recently, a second parallel Sec system has been described in a number of gram-positive species. This accessory Sec system is characterized by the presence of a second copy of the energizing ATPase, SecA2; where it has been studied, SecA2 is responsible for the translocation of a subset of Sec substrates. In common with many pathogenic gram-positive species, Clostridium difficile possesses two copies of SecA. Here, we describe the first characterization of the C. difficile accessory Sec system and the identification of its major substrates. Using inducible antisense RNA expression and dominant-negative alleles of secA1 and secA2, we demonstrate that export of the S-layer proteins (SLPs) and an additional cell wall protein (CwpV) is dependent on SecA2. Accumulation of the cytoplasmic precursor of the SLPs SlpA and other cell wall proteins was observed in cells expressing dominant-negative secA1 or secA2 alleles, concomitant with a decrease in the levels of mature SLPs in the cell wall. Furthermore, expression of either dominant-negative allele or antisense RNA knockdown of SecA1 or SecA2 dramatically impaired growth, indicating that both Sec systems are essential in C. difficile.     

Patterns of sequence conservation in the S-Layer proteins and related sequences in Clostridium difficile

Clostridium difficile is the etiological agent of antibiotic-associated diarrhea. Among the factors that may play a role in infection are S-layer proteins (SLPs). Previous work has shown these to consist mainly of two components, resulting from the cleavage of a precursor encoded by the slpA gene. The high-molecular-weight (MW) subunit is related both to amidases from B. subtilis and to at least another 28 gene products in C. difficile strain 630. To gain insight into the functions of the SLPs and related proteins, we have further investigated the pattern of variability both at the slpA locus and at six nearby paralogs. Sequencing of the slpA gene from an S-layer group II strain and a variant S-layer group strain confirms a high degree of divergence in the low-MW SLP, which may result from diversifying selection. A highly conserved motif, however, is found at the C terminus in all low-MW subunits and may be essential for SlpA precursor cleavage. In strain 167, a variant cleavage product is present, suggesting a secondary processing site. Southern blotting analysis shows slpA-like open reading frames (ORFs) 2 to 7 to be conserved in all nine strains tested, with one exception: ORF2, which encodes a 66-kDa polypeptide coextracted at low pH with the main SLPs in strain 630, may be partially deleted in strain 167. Polymorphism within the slpA-ORF7 cluster may be more pronounced in the region proximal to the slpA gene. Unexpectedly, a high-MW subunit probe cross hybridizes to sequences outside the slpA locus, which appear to vary in number in different strains.     

Identification by flagellum display of an epithelial cell- and fibronectin-binding function in the SlpA surface protein of Lactobacillus brevis

Depletion of the SlpA protein from the bacterial surface greatly reduced the adhesion of Lactobacillus brevis ATCC 8287 to the human intestinal cell lines Caco-2 and Intestine 407, the endothelial cell line EA-hy926, and the urinary bladder cell line T24, as well as immobilized fibronectin. For functional analysis of the SlpA surface protein, different regions of the slpA gene were expressed as internal in-frame fusions in the variable region of the fliC(H7) gene of Escherichia coli. The resulting chimeric flagella carried inserts up to 275 amino acids long from the mature S-layer protein, which is 435 amino acids in size. The expression of the SlpA fragments on the chimeric flagella was assessed by immunoelectron microscopy and Western blotting using anti-SlpA antibodies, and their binding to human cells was assessed by indirect immunofluorescence. Chimeric flagella harboring inserts that represented the N-terminal part of the S-layer protein bound to the epithelial cell lines, whereas the C-terminal part of the S-layer protein did not confer binding on the flagella. The shortest S-layer peptide capable of detectable binding was 81 amino acid residues in size and represented residues 96 through 176 in the unprocessed S-layer protein. The bacteria and the chimeric flagella did not show detectable binding to erythrocytes, whereas the SlpA-expressing ATCC 8287 cells as well as the chimeric SlpA 96-245/FliC flagella bound to immobilized fibronectin. The N-terminal SlpA peptide 96-176 or 96-200 fused to FliC was not recognized in Western blotting or immunoelectron microscopy by a polyclonal serum raised against the S-layer protein; the antiserum, however, reacted in immunofluorescence with the ATCC 8287 cells. In contrast, an antiserum raised against the His-tagged peptide 96-245 of SlpA bound to the hybrid flagella with the N-terminal SlpA inserts but did not react with ATCC 8287 cells. The results identify the S-layer of L. brevis ATCC 8287 as an adhesin with affinity for human epithelial cells and fibronectin and locate the receptor-binding region within a fragment of 81 amino acids in the N-terminal part of the molecule, which in native S-layer seems inaccessible to antibodies.     

On the S-layer of Thermus thermophilus and the assembling of its main protein SlpA

We have isolated and analysed the cell envelope of the thermophilic bacterium Thermus thermophilus HB8. Isolated cell walls, characterized by the dominance of the S-layer protein SlpA, are found to be constituted by several protein complexes of high molecular weights. Further isolation steps, starting from the cell wall samples, led to the selective release of the S-layer protein SlpA in solution as confirmed by mass spectrometry. Blue Native gel electrophoresis on these samples showed that SlpA is organized into a specific hierarchical order of oligomeric states that are consistent with the complexes at high molecular weight identified on the total cell wall fraction. The analysis showed that SlpA bases this peculiar organization on monomers and exceptionally stable dimers, leading to the formation of tetramers, heptamers, and decamers. Furthermore, the two elementary units of SlpA, monomers and dimers, are regulated by the presence of calcium not only for the assembling of monomers into dimers, but also for the splitting of dimers into monomers. Finally, the SlpA protein was found to be subjected to specific proteolysis leading to characteristic degradation products. Findings are discussed in terms of S-layer assembling properties as bases for understanding its structure, turn-over and organization.     

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

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 630ΔErm results in a bacterial phenotype in which only immature, single chain SlpA comprises the S-layer. The Cwp84 knock-out mutants (CDΔCwp84) displayed significantly different colony morphology compared with the wild-type strain and grew more slowly in liquid medium. SlpA extracted from CDΔCwp84 was readily cleaved into its mature subunits by trypsin treatment. Addition of trypsin to the growth medium also cleaved SlpA on CDΔCwp84 and increased the growth rate of the bacterium in a dose-dependent manner. Using the hamster model for C. difficile infection, CDΔCwp84 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.     

The Clostridium difficile cell wall protein CwpV confers phase‐variable phage resistance

Bacteriophages are present in virtually all ecosystems, and bacteria have developed multiple antiphage strategies to counter their attacks. Clostridium difficile is an important pathogen causing severe intestinal infections in humans and animals. Here we show that the conserved cell‐surface protein CwpV provides antiphage protection in C. difficile. This protein, for which the expression is phase‐variable, is classified into five types, each differing in their repeat‐containing C‐terminal domain. When expressed constitutively from a plasmid or the chromosome of locked ‘ON’ cells of C. difficile R20291, CwpV conferred antiphage protection. Differences in the level of phage protection were observed depending on the phage morphological group, siphophages being the most sensitive with efficiency of plaquing (EOP) values of < 5 × 10^?7 for phages ?CD38‐2, ?CD111 and ?CD146. Protection against the myophages ?MMP01 and ?CD52 was weaker, with EOP values between 9.0 × 10^?3 and 1.1 × 10^?1. The C‐terminal domain of CwpV carries the antiphage activity and its deletion, or part of it, significantly reduced the antiphage protection. CwpV does not affect phage adsorption, but phage DNA replication is prevented, suggesting a mechanism reminiscent of superinfection exclusion systems normally encoded on prophages. CwpV thus represents a novel ubiquitous host‐encoded and phase‐variable antiphage system in C. difficile.     

A conserved motif in S-layer proteins is involved in peptidoglycan binding in Thermus thermophilus.

There is experimental evidence to suggest that the 100-kDa S-layer protein from Thermus thermophilus HB8 binds to the peptidoglycan cell wall. This property could be related to the presence of a region (SLH) of homology with other S-layer proteins and extracellular enzymes (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224-1233, 1994). By using specific monoclonal antibodies, we show that similar regions are present in different members of the Deinococcus-Thermus phylogenetic group. To analyze the role that the SLH domain plays in vivo and in vitro in T. thermophilus, we have obtained a mutant form (slpA.X) of the S-layer gene (slpA) in which the SLH domain was deleted. The slpA.X gene was inserted into the chromosome of the thermophile by gene replacement, resulting in a mutant which expressed a major membrane protein with the size expected from the construction (90 kDa). This protein was identified as the product of slpA.X by its differential reaction with monoclonal antibodies. Mutants expressing the SlpA.X protein grow as groups of cells, surrounded by a common external envelope of trigonal symmetry that contains the SlpA.X protein as a main component, thus showing the inability of the SLH-defective protein to attach to the underlying material in vivo. In addition, averaged images of SlpA.X-rich fractions showed a regular arrangement, identical to that built up by the wild-type (SlpA) protein in the absence of peptidoglycan. Finally, we demonstrate by Western blotting (immunoblotting) the direct role of the SLH domain in the binding of the S-layer of T. thermophilus HB8 to the peptidoglycan layer.     

Surface proteins from Lactobacillus kefir antagonize in vitro cytotoxic effect of Clostridium difficile toxins

In this work, the ability of S-layer proteins from kefir-isolated Lactobacillus kefir strains to antagonize the cytophatic effects of toxins from Clostridium difficile (TcdA and TcdB) on eukaryotic cells in vitro was tested by cell detachment assay. S-layer proteins from eight different L. kefir strains were able to inhibit the damage induced by C. difficile spent culture supernatant to Vero cells. Besides, same protective effect was observed by F-actin network staining. S-layer proteins from aggregating L. kefir strains (CIDCA 83115, 8321, 8345 and 8348) showed a higher inhibitory ability than those belonging to non-aggregating ones (CIDCA 83111, 83113, JCM 5818 and ATCC 8007), suggesting that differences in the structure could be related to the ability to antagonize the effect of clostridial toxins. Similar results were obtained using purified TcdA and TcdB. Protective effect was not affected by proteases inhibitors or heat treatment, thus indicating that proteolytic activity is not involved. Only preincubation with specific anti-S-layer antibodies significantly reduced the inhibitory effect of S-layer proteins, suggesting that this could be attributed to a direct interaction between clostridial toxins and L. kefir S-layer protein. Interestingly, the interaction of toxins with S-layer carrying bacteria was observed by dot blot and fluorescence microscopy with specific anti-TcdA or anti-TcdB antibodies, although L. kefir cells did not show protective effects. We hypothesize that the interaction between clostridial toxins and soluble S-layer molecules is different from the interaction with S-layer on the surface of the bacteria thus leading a different ability to antagonize cytotoxic effect. This is the first report showing the ability of S-layer proteins from kefir lactobacilli to antagonize biological effects of bacterial toxins. These results encourage further research on the role of bacterial surface molecules to the probiotic properties of L. kefir and could contribute to strain selection with potential therapeutic or prophylactic benefits towards CDAD.     

Distinct affinity of binding sites for S-layer homologous domains in Clostridium thermocellum and Bacillus anthracis cell envelopes

Binding parameters were determined for the SLH (S-layer homologous) domains from the Clostridium thermocellum outer layer protein OlpB, from the C. thermocellum S-layer protein SlpA, and from the Bacillus anthracis S-layer proteins EA1 and Sap, using cell walls from C. thermocellum and B. anthracis. Each SLH domain bound to C. thermocellum and B. anthracis cell walls with a different KD, ranging between 7.1 x 10(-7) and 1.8 x 10(-8) M. Cell wall binding sites for SLH domains displayed different binding specificities in C. thermocellum and B. anthracis. SLH-binding sites were not detected in cell walls of Bacillus subtilis. Cell walls of C. thermocellum lost their affinity for SLH domains after treatment with 48% hydrofluoric acid but not after treatment with formamide or dilute acid. A soluble component, extracted from C. thermocellum cells by sodium dodecyl sulfate treatment, bound the SLH domains from C. thermocellum but not those from B. anthracis proteins. A corresponding component was not found in B. anthracis.     

Two-dimensional gel electrophoresis analyses of pH-dependent protein expression in facultatively alkaliphilic Bacillus pseudofirmus OF4 lead to characterization of an S-layer protein with a role in alkaliphily

The large majority of proteins of alkaliphilic Bacillus pseudofirmus OF4 grown at pH 7.5 and 10.5, as studied by two-dimensional gel electrophoresis analyses, did not exhibit significant pH-dependent variation. A new surface layer protein (SlpA) was identified in these studies. Although the prominence of some apparent breakdown products of SlpA in gels from pH 10.5-grown cells led to discovery of the alkaliphile S-layer, the largest and major SlpA forms were present in large amounts in gels from pH 7.5-grown cells as well. slpA RNA abundance was, moreover, unchanged by growth pH. SlpA was similar in size to homologues from nonalkaliphiles but contained fewer Arg and Lys residues. An slpA mutant strain (RG21) lacked an exterior S-layer that was identified in the wild type by electron microscopy. Electrophoretic analysis of whole-cell extracts further indicated the absence of a 90-kDa band in the mutant. This band was prominent in wild-type extracts from both pH 7.5- and 10.5-grown cells. The wild type grew with a shorter lag phase than RG21 at either pH 10.5 or 11 and under either Na(+)-replete or suboptimal Na(+) concentrations. The extent of the adaptation deficit increased with pH elevation and suboptimal Na(+). By contrast, the mutant grew with a shorter lag and faster growth rate than the wild type at pH 7. 5 under Na(+)-replete and suboptimal Na(+) conditions, respectively. Logarithmically growing cells of the two strains exhibited no significant differences in growth rate, cytoplasmic pH regulation, starch utilization, motility, Na(+)-dependent transport of alpha-aminoisobutyric acid, or H(+)-dependent synthesis of ATP. However, the capacity for Na(+)-dependent pH homeostasis was diminished in RG21 upon a sudden upward shift of external pH from 8. 5 to 10.5. The energy cost of retaining the SlpA layer at near-neutral pH is apparently adverse, but the constitutive presence of SlpA enhances the capacity of the extremophile to adjust to high pH.     

Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial cells

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.     

Designing a Cell Surface Display System of Protein Domains in Lactobacilli Based on S-Layer Proteins of <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> ATCC 367

S-layer proteins of lactobacilli may be utilized for developing a surface display system in these bacteria. In this study, S-layer proteins of Lactobacillus brevis ATCC 367 were identified for the first time. Using the peptide fingerprint method, it was shown that the main protein of the S-layer of this strain, SlpE, having a mass of 52 kDa is the product of translation of the consecutive open reading frames LVIS_2086 and LVIS_2085. Repeated sequencing of a genome region of L. brevis ATCC 367, containing LVIS_2086 and LVIS_2085 loci, has showed that the LVIS_2086 sequence contains the TGG tryptophan codon instead of the TAG stop codon. Thus, LVIS_2085 and LVIS_2086 form a single slpE gene, the nucleotide sequence we deposited in the Genbank database under No. KY273133. The translation product of the slpE gene consists of 465 amino acids and has a calculated mass of 51.6 kDa, which corresponds to the experimentally obtained value. An S-layer protein with a mass of 56 kDa, identified as a form of the SlpE, is probably formed during the posttranslational modification. The concomitant 48 kDa S-protein was proven to be product of the LVIS- 2083 gene. The N-terminal domains of LVIS_2083 and SlpE have 70.7 and 96.5%, respectively, identity to the anchoring N-terminal domain of SlpA from L. brevis ATCC 8287, which is responsible for attachment to the cell wall. In this work, fusion proteins consisting of N-terminal domains of Lvis_2083 and SlpA proteins and the eGFP marker protein were obtained. The ability of fusion proteins SlpA_eGFP and Lvis_2083_eGFP, as well as the recombinant Lvis_2083 protein, to be specifically sorbed on the cell wall of L. brevis ATCC 8287, ATCC 367, and L. acidophilus ATCC 4356 strains has been demonstrated. It was shown that in the chimeric Lvis_2083_eGFP construction the N-terminal domain Lvis_2083 is responsible for an attachment to the cell wall and provides display of the functionally active eGFP protein on its surface. Thus, the N-terminal domain Lvis_2083 can be used as a basis of the protein display system on the cell surface of L. brevis strains in vitro.     

Structural insights into the molecular organization of the S-layer from Clostridium difficile

Clostridium difficile expresses a surface layer (S-layer) which coats the surface of the bacterium and acts as an adhesin facilitating interaction of the bacterium with host enteric cells. The S-layer contains a high-molecular-weight S-layer protein (HMW SLP) and its low-molecular-weight partner protein (LMW SLP). We show that these proteins form a tightly associated non-covalent complex, the H/L complex, and we identify the regions of both proteins responsible for complex formation. The 2.4 Å X-ray crystal structure of a truncated derivative of the LMW SLP reveals two domains. Domain 1 has a two-layer sandwich architecture while domain 2, predicted to orientate towards the external environment, contains a novel fold. Small-angle X-ray scattering analysis of the H/L complex shows an elongated molecule, with the two SLPs arranged 'end-to-end' interacting with each other through a small contact area. Alignment of LMW SLPs, which exhibit high sequence diversity, reveals a core of conserved residues that could reflect functional conservation, while allowing for immune evasion through sequence variation. These structures are the first described for the S-layer of a bacterial pathogen, and provide insights into the assembly and biogenesis of the S-layer.