Abstract
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.
Highlights
Clostridium difficile is a Gram positive, anaerobic, sporeforming bacterium recognized as the major etiological agent of intestinal diseases associated with antibiotic therapy, with clinical manifestations ranging from diarrhea to pseudomembranous colitis [1]
These results suggest, that unlike B. subtilis, SigD does not control expression of autolysins involved in cell separation during vegetative growth of C. difficile
The aim of our study was to characterize the regulatory properties of SigD in C. difficile, by comparing phenotypic properties and transcriptomic profiles of C. difficile 630∆erm and its sigD mutant
Summary
Clostridium difficile is a Gram positive, anaerobic, sporeforming bacterium recognized as the major etiological agent of intestinal diseases associated with antibiotic therapy, with clinical manifestations ranging from diarrhea to pseudomembranous colitis [1]. TcdE is a holin-like protein required in the release of the toxins from the cells [10], its role has been discussed [11] Several global regulators, such as CcpA, CodY, Spo0A and SigH regulate expression of toxin genes in response to diverse environmental stimuli. The alternative sigma factor SigH, a key element in the control of the transition phase and of the initiation of sporulation, negatively modulates toxin and motility expression [18]. Most of these regulators control toxin genes expression in association with genes encoding major cell functions, suggesting a strong relationship between the physiology of C. difficile and the expression of the virulence factors of this bacterium
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