The pathogenic clostridia cause many human and animal diseases, which typically arise as a consequence of the production of potent exotoxins. Among the enterotoxic clostridia, Clostridium difficile is the main causative agent of nosocomial intestinal infections in adults with a compromised gut microbiota caused by antibiotic treatment. The symptoms of C. difficile infection are essentially caused by the production of two exotoxins: TcdA and TcdB. Moreover, for severe forms of disease, the spectrum of diseases caused by C. difficile has also been correlated to the levels of toxins that are produced during host infection. This observation strengthened the idea that the regulation of toxin synthesis is an important part of C. difficile pathogenesis. This review summarizes our current knowledge about the regulators and sigma factors that have been reported to control toxin gene expression in response to several environmental signals and stresses, including the availability of certain carbon sources and amino acids, or to signaling molecules, such as the autoinducing peptides of quorum sensing systems. The overlapping regulation of key metabolic pathways and toxin synthesis strongly suggests that toxin production is a complex response that is triggered by bacteria in response to particular states of nutrient availability during infection.

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.

The structure of the vegetative cell wall peptidoglycan of Clostridium difficile was determined by analysis of its constituent muropeptides with a combination of reverse-phase high pressure liquid chromatography separation of muropeptides, amino acid analysis, mass spectrometry and tandem mass spectrometry. The structures assigned to 36 muropeptides evidenced several original features in C. difficile vegetative cell peptidoglycan. First, it is characterized by a strikingly high level of N-acetylglucosamine deacetylation. In addition, the majority of dimers (around 75%) contains A(2)pm(3) → A(2)pm(3) (A(2)pm, 2,6-diaminopimelic acid) cross-links and only a minority of the more classical Ala(4) → A(2)pm(3) cross-links. Moreover, a significant amount of muropeptides contains a modified tetrapeptide stem ending in Gly instead of D-Ala(4). Two L,D-transpeptidases homologues encoding genes present in the genome of C. difficile 630 and named ldt(cd1) and ldt(cd2), were inactivated. The inactivation of either ldt(cd1) or ldt(cd2) significantly decreased the abundance of 3-3 cross-links, leading to a marked decrease of peptidoglycan reticulation and demonstrating that both ldt(cd1)-and ldt(cd2)-encoded proteins have a redundant L,D-transpeptidase activity. The contribution of 3-3 cross-links to peptidoglycan synthesis increased in the presence of ampicillin, indicating that this drug does not inhibit the L,D-transpeptidation pathway 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.

In Gram-positive pathogens, surface proteins may be covalently anchored to the bacterial peptidoglycan by sortase, a cysteine transpeptidase enzyme. In contrast to other Gram-positive bacteria, only one single sortase enzyme, SrtB, is conserved between strains of Clostridium difficile. Sortase-mediated peptidase activity has been reported in vitro, and seven potential substrates have been identified. Here, we demonstrate the functionality of sortase in C. difficile. We identify two sortase-anchored proteins, the putative adhesins CD2831 and CD3246, and determine the cell wall anchor structure of CD2831. The C-terminal PPKTG sorting motif of CD2831 is cleaved between the threonine and glycine residues, and the carboxyl group of threonine is amide-linked to the side chain amino group of diaminopimelic acid within the peptidoglycan peptide stem. We show that CD2831 protein levels are elevated in the presence of high intracellular cyclic diGMP (c-diGMP) concentrations, in agreement with the control of CD2831 expression by a c-diGMP-dependent type II riboswitch. Low c-diGMP levels induce the release of CD2831 and presumably CD3246 from the surface of cells. This regulation is mediated by proteolytic cleavage of CD2831 and CD3246 by the zinc metalloprotease ZmpI, whose expression is controlled by a type I c-diGMP riboswitch. These data reveal a novel regulatory mechanism for expression of two sortase substrates by the secondary messenger c-diGMP, on which surface anchoring is dependent.