Characterization of the SigD regulon of C. difficile and its positive control of toxin production through the regulation of tcdR

PloS One
Imane El MeoucheJean-Louis Pons

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, inhibit...Continue Reading

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Datasets Mentioned

BETA
GPL10556
GSE29275

Methods Mentioned

BETA
PCR
PCRs
electrophoresis
glycosylation
phosphotransferase
ELISA
electrophoretic mobility shifts
gene

Software Mentioned

OligoArray
ClustalW
Beacon Designer
Bioconductor
ClosTron
R
GenoList
Limma ( Linear Model for Microarray Data )
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