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Clostridium difficile intestinal disease is mediated largely by the actions of toxins A (TcdA) and B (TcdB), whose production occurs after the initial steps of colonization involving different surface or flagellar proteins. In B. subtilis, the sigma factor SigD controls flagellar synthesis, motility, and vegetative autolysins. A homolog of SigD encoding gene is present in the C.difficile 630 genome. We constructed a sigD mutant in C. difficile 630 ∆erm to analyze the regulon of SigD using a global transcriptomic approach. A total of 103 genes were differentially expressed between the wild-type and the sigD mutant, including genes involved in motility, metabolism and regulation. In addition, the sigD mutant displayed decreased expression of genes involved in flagellar biosynthesis, and also of genes encoding TcdA and TcdB as well as TcdR, the positive regulator of the toxins. Genomic analysis and RACE-PCR experiments allowed us to characterize promoter sequences of direct target genes of SigD including tcdR and to identify the SigD consensus. We then established that SigD positively regulates toxin expression via direct control of tcdR transcription. Interestingly, the overexpression of FlgM, a putative anti-SigD factor, inhibited the positive regulation of motility and toxin synthesis by SigD. Thus, SigD appears to be the first positive regulator of the toxin synthesis in C. difficile.

Clostridium difficile is the major etiologic agent of antibiotic-associated intestinal disease. Pathogenesis of C. difficile is mainly attributed to the production and secretion of toxins A and B. Unlike most clostridial toxins, toxins A and B have no signal peptide, and they are therefore secreted by unusual mechanisms involving the holin-like TcdE protein and/or autolysis. In this study, we characterized the cell surface protein Cwp19, a newly identified peptidoglycan-degrading enzyme containing a novel catalytic domain. We purified a recombinant His 6 -tagged Cwp19 protein and showed that it has lytic transglycosylase activity. Moreover, we observed that Cwp19 is involved in cell autolysis and that a C. difficile cwp19 mutant exhibited delayed autolysis in stationary phase compared to the wild type when bacteria were grown in brain heart infusion (BHI) medium. Wild-type cell autolysis is correlated to strong alterations of cell wall thickness and integrity and to release of cytoplasmic material. Furthermore, we demonstrated that toxins were released into the extracellular medium as a result of Cwp19-induced autolysis when cells were grown in BHI medium. In contrast, Cwp19 did not induce autolysis or toxin release when cells were grown in tryptone-yeast extract (TY) medium. These data provide evidence for the first time that TcdE and bacteriolysis are coexisting mechanisms for toxin release, with their relative contributions in vitro depending on growth conditions. Thus, Cwp19 is an important surface protein involved in autolysis of vegetative cells of C. difficile that mediates the release of the toxins from the cell cytosol in response to specific environment conditions. IMPORTANCE Clostridium difficile -associated disease is mainly known as a health care-associated infection. It represents the most problematic hospital-acquired infection in North America and Europe and exerts significant economic pressure on health care systems. Virulent strains of C. difficile generally produce two toxins that have been identified as the major virulence factors. The mechanism for release of these toxins from bacterial cells is not yet fully understood but is thought to be partly mediated by bacteriolysis. Here we identify a novel peptidoglycan hydrolase in C. difficile , Cwp19, exhibiting lytic transglycosylase activity. We show that Cwp19 contributes to C. difficile cell autolysis in the stationary phase and, consequently, to toxin release, most probably as a response to environmental conditions such as nutritional signals. These data highlight that Cwp19 constitutes a promising target for the development of new preventive and curative strategies. Clostridium difficile -associated disease is mainly known as a health care-associated infection. It represents the most problematic hospital-acquired infection in North America and Europe and exerts significant economic pressure on health care systems. Virulent strains of C. difficile generally produce two toxins that have been identified as the major virulence factors. The mechanism for release of these toxins from bacterial cells is not yet fully understood but is thought to be partly mediated by bacteriolysis. Here we identify a novel peptidoglycan hydrolase in C. difficile , Cwp19, exhibiting lytic transglycosylase activity. We show that Cwp19 contributes to C. difficile cell autolysis in the stationary phase and, consequently, to toxin release, most probably as a response to environmental conditions such as nutritional signals. These data highlight that Cwp19 constitutes a promising target for the development of new preventive and curative strategies.

The nucleotide sequence of atlL, a gene encoding a putative Staphylococcus lugdunensis peptidoglycan hydrolase, was determined using degenerate consensus PCR and genome walking. This 3837-bp gene encodes a protein, AtlL, that appears as a putative bifunctional autolysin with a 29-amino acid putative signal peptide and two enzymatic putative centres (N-acetylmuramoyl- l-alanine amidase and N-acetylglucosaminidase) interconnected with three imperfect repeated sequences displaying glycine-tryptophan motifs. In order to determine whether both lytic domains were functional, and verify their exact enzymatic activities, gene fragments harbouring both putative domains, AM (N-acetylmuramoyl- l-alanine amidase enzymatic centre plus two repeated sequences) and GL (N-acetylglucosaminidase enzymatic centre plus one repeated sequence), were isolated, subcloned, and expressed in Escherichia coli. Purified recombinant AM and GL protein truncations exhibited cell wall lytic activity in zymograms performed with cell walls of Micrococcus lysodeikticus, Bacillus subtilis, and S. lugdunensis. AtlL is expressed during the whole growth, with an overexpression in the early-exponential stage. Liquid chromatography-mass spectrometry analysis of muropeptides generated by digestion of B. subtilis cell walls demonstrated the hydrolytic bond specificities and confirmed both of the acetyl domains' activities as predicted by sequence homology data. AtlL is the first autolysin described in S. lugdunensis, with a bifunctional enzymatic activity involved in peptidoglycan hydrolysis.

Housekeeping genes encoding metabolic enzymes may provide alternative markers to 16S ribosomal DNA (rDNA) for genotypic and phylogenetic characterization of bacterial species. We have developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay, targeting the triosephosphate isomerase ( tpi) gene, which allows the differentiation of twelve pathogenic Clostridium species. Degenerate primers constructed from alignments of tpi sequences of various Gram-positive bacteria allowed the amplification of a 501 bp target region in the twelve Clostridium type strains. A phylogenetic tree constructed from the nucleotidic sequences of these tpi amplicons was well correlated with that inferred from analysis of 16S rDNA gene sequences. The analysis of tpi sequences revealed restriction sites of enzyme Alu I that could be species-specific. Indeed, Alu I digestion of amplicons from the twelve type strains provided distinct restriction patterns. A total of 127 strains (three to sixteen strains for each species) was further analyzed by PCR-RFLP of the tpi gene, and confirmed that each species could be characterized by one to three restriction types (RTs). The differences between RTs within species could be explained by point mutations in Alu I restriction sites of the tpi sequences. PCR-restriction analysis of the tpi gene offers an accurate tool for species identification within the genus Clostridium, and provides an alternative marker to 16S rDNA for phylogenetic analyses.

An optimized multilocus enzyme electrophoresis method, which involves polyacrylamide–agarose gel electrophoresis followed by electrophoretic transfers on nitrocellulose sheets, was developed for the analysis of enzyme polymorphism in several aerobic and anaerobic bacterial species including Staphylococcus aureus, Streptococcus pneumoniae, S. agalactiae, Klebsiella pneumoniae and K. oxytoca, Clostridium bifermentans and C. sordellii, and Prevotella bivia. Serial electrophoretic transfers (during 5–15 min each) from a single polyacrylamide gel could be achieved for most enzymes studied, and allowed an increased definition of enzyme bands on nitrocellulose as compared to migration gels. Four enzymes, which could not be blotted in such conditions, could still be stained in gels after blotting. Thus, the method allowed the combined analysis of several enzymes after a single gel electrophoresis separation. The analysis of enzyme polymorphism in the various species studied raised the interest of polymorphic loci such as esterase or glutamic-oxaloacetic transaminase for epidemiologic studies. The method characterized a genetic diversity of enzyme loci of S. pneumoniae higher than previously reported, and is thus convenient for the analysis of genetic relationships between related isolates. Since the present method reduces the tediousness of multilocus enzyme electrophoresis and requires experimental conditions that are not specific for the bacterial population studied, it may be proposed for rapid population genetics analysis of a wide variety of bacteria.

Abstract An optimized multilocus enzyme electrophoresis method, which involves polyacrylamide–agarose gel electrophoresis followed by electrophoretic transfers on nitrocellulose sheets, was developed for the analysis of enzyme polymorphism in several aerobic and anaerobic bacterial species including Staphylococcus aureus, Streptococcus pneumoniae, S. agalactiae, Klebsiella pneumoniae and K. oxytoca, Clostridium bifermentans and C. sordellii, and Prevotella bivia. Serial electrophoretic transfers (during 5–15 min each) from a single polyacrylamide gel could be achieved for most enzymes studied, and allowed an increased definition of enzyme bands on nitrocellulose as compared to migration gels. Four enzymes, which could not be blotted in such conditions, could still be stained in gels after blotting. Thus, the method allowed the combined analysis of several enzymes after a single gel electrophoresis separation. The analysis of enzyme polymorphism in the various species studied raised the interest of polymorphic loci such as esterase or glutamic-oxaloacetic transaminase for epidemiologic studies. The method characterized a genetic diversity of enzyme loci of S. pneumoniae higher than previously reported, and is thus convenient for the analysis of genetic relationships between related isolates. Since the present method reduces the tediousness of multilocus enzyme electrophoresis and requires experimental conditions that are not specific for the bacterial population studied, it may be proposed for rapid population genetics analysis of a wide variety of bacteria.

ABSTRACT Clostridium difficile is the major etiologic agent of antibiotic-associated intestinal disease. Pathogenesis of C. difficile is mainly attributed to the production and secretion of toxins A and B. Unlike most clostridial toxins, toxins A and B have no signal peptide, and they are therefore secreted by unusual mechanisms involving the holin-like TcdE protein and/or autolysis. In this study, we characterized the cell surface protein Cwp19, a newly identified peptidoglycan-degrading enzyme containing a novel catalytic domain. We purified a recombinant His6-tagged Cwp19 protein and showed that it has lytic transglycosylase activity. Moreover, we observed that Cwp19 is involved in cell autolysis and that a C. difficile cwp19 mutant exhibited delayed autolysis in stationary phase compared to the wild type when bacteria were grown in brain heart infusion (BHI) medium. Wild-type cell autolysis is correlated to strong alterations of cell wall thickness and integrity and to release of cytoplasmic material. Furthermore, we demonstrated that toxins were released into the extracellular medium as a result of Cwp19-induced autolysis when cells were grown in BHI medium. In contrast, Cwp19 did not induce autolysis or toxin release when cells were grown in tryptone-yeast extract (TY) medium. These data provide evidence for the first time that TcdE and bacteriolysis are coexisting mechanisms for toxin release, with their relative contributions in vitro depending on growth conditions. Thus, Cwp19 is an important surface protein involved in autolysis of vegetative cells of C. difficile that mediates the release of the toxins from the cell cytosol in response to specific environment conditions. IMPORTANCE Clostridium difficile-associated disease is mainly known as a health care-associated infection. It represents the most problematic hospital-acquired infection in North America and Europe and exerts significant economic pressure on health care systems. Virulent strains of C. difficile generally produce two toxins that have been identified as the major virulence factors. The mechanism for release of these toxins from bacterial cells is not yet fully understood but is thought to be partly mediated by bacteriolysis. Here we identify a novel peptidoglycan hydrolase in C. difficile, Cwp19, exhibiting lytic transglycosylase activity. We show that Cwp19 contributes to C. difficile cell autolysis in the stationary phase and, consequently, to toxin release, most probably as a response to environmental conditions such as nutritional signals. These data highlight that Cwp19 constitutes a promising target for the development of new preventive and curative strategies.

Clostridium difficile intestinal disease is mediated largely by the actions of toxins A (TcdA) and B (TcdB), whose production occurs after the initial steps of colonization involving different surface or flagellar proteins. In B. subtilis, the sigma factor SigD controls flagellar synthesis, motility, and vegetative autolysins. A homolog of SigD encoding gene is present in the C.difficile 630 genome. We constructed a sigD mutant in C. difficile 630 Delta erm to analyze the regulon of SigD using a global transcriptomic approach. A total of 103 genes were differentially expressed between the wild-type and the sigD mutant, including genes involved in motility, metabolism and regulation. In addition, the sigD mutant displayed decreased expression of genes involved in flagellar biosynthesis, and also of genes encoding TcdA and TcdB as well as TcdR, the positive regulator of the toxins. Genomic analysis and RACE-PCR experiments allowed us to characterize promoter sequences of direct target genes of SigD including tcdR and to identify the SigD consensus. We then established that SigD positively regulates toxin expression via direct control of tcdR transcription. Interestingly, the overexpression of FlgM, a putative anti-SigD factor, inhibited the positive regulation of motility and toxin synthesis by SigD. Thus, SigD appears to be the first positive regulator of the toxin synthesis in C. difficile.