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ABSTRACTThe pathogenicity of Clostridium difficileis linked to its ability to produce two toxins: TcdA and TcdB. The level of toxin synthesis is influenced by environmental signals, such as phosphotransferase system (PTS) sugars, biotin, and amino acids, especially cysteine. To understand the molecular mechanisms of cysteine-dependent repression of toxin production, we reconstructed the sulfur metabolism pathways of C. difficilestrain 630 in silicoand validated some of them by testing C. difficilegrowth in the presence of various sulfur sources. High levels of sulfide and pyruvate were produced in the presence of 10 mM cysteine, indicating that cysteine is actively catabolized by cysteine desulfhydrases. Using a transcriptomic approach, we analyzed cysteine-dependent control of gene expression and showed that cysteine modulates the expression of genes involved in cysteine metabolism, amino acid biosynthesis, fermentation, energy metabolism, iron acquisition, and the stress response. Additionally, a sigma factor (SigL) and global regulators (CcpA, CodY, and Fur) were tested to elucidate their roles in the cysteine-dependent regulation of toxin production. Among these regulators, only sigLinactivation resulted in the derepression of toxin gene expression in the presence of cysteine. Interestingly, the sigLmutant produced less pyruvate and H2S than the wild-type strain. Unlike cysteine, the addition of 10 mM pyruvate to the medium for a short time during the growth of the wild-type and sigLmutant strains reduced expression of the toxin genes, indicating that cysteine-dependent repression of toxin production is mainly due to the accumulation of cysteine by-products during growth. Finally, we showed that the effect of pyruvate on toxin gene expression is mediated at least in part by the two-component system CD2602-CD2601.

ABSTRACTClostridium difficileis currently the major cause of nosocomial intestinal diseases associated with antibiotic therapy in adults. In order to improve our knowledge of C. difficile-host interactions, we analyzed the genome-wide temporal expression of C. difficile630 genes during the first 38 h of mouse colonization to identify genes whose expression is modulated in vivo, suggesting that they may play a role in facilitating the colonization process. In the ceca of the C. difficile-monoassociated mice, 549 genes of the C. difficilegenome were differentially expressed compared to their expression during in vitrogrowth, and they were distributed in several functional categories. Overall, our results emphasize the roles of genes involved in host adaptation. Colonization results in a metabolic shift, with genes responsible for the fermentation as well as several other metabolic pathways being regulated inversely to those involved in carbon metabolism. In addition, several genes involved in stress responses, such as ferrous iron uptake or the response to oxidative stress, were regulated in vivo. Interestingly, many genes encoding conserved hypothetical proteins (CHP) were highly and specifically upregulated in vivo. Moreover, genes for all stages of sporulation were quickly induced in vivo, highlighting the observation that sporulation is central to the persistence of C. difficilein the gut and to its ability to spread in the environment. Finally, we inactivated two genes that were differentially expressed in vivoand evaluated the relative colonization fitness of the wild-type and mutant strains in coinfection experiments. We identified a CHP as a putative colonization factor, supporting the suggestion that the in vivotranscriptomic approach can unravel new C. difficilevirulence genes.

ABSTRACTGrowth temperature was found to control the expression of toxins A and B in Clostridium difficileVPI 10463,with a maximum at 37°C and low levels at 22 and 42°C in both peptone yeast (PY) and defined media. The up-regulation of toxin A and B mRNA and protein levels upon temperature upshift from 22 to 37°C followed the same kinetics, showing that temperature control occurred at the level of transcription. Experiments with Clostridium perfringensusing gusAas a reporter gene demonstrated that both toxin gene promoters were temperature controlled and that their high activity at 37°C was dependent on the alternative sigma factor TcdD. Furthermore, tcdDwas found to be autoinduced at 37°C. Glucose down-regulated all these responses in the C. perfringensconstructs, similar to its impact on toxin production in C. difficilePY broth cultures. C. difficileproteins induced at 37°C and thus coregulated with the toxins by temperature were demonstrated by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified as enzymes involved in butyric acid production and as electron carriers in oxidation-reduction reactions. The regulation of toxin production in C. difficileby temperature is a novel finding apparently reflecting an adaptation of the expression of its virulence to mammalian hosts.