Characterization of the SigD Regulon of C. difficile and Its Positive Control of Toxin Production through the Regulation of tcdR

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.

艰难梭菌（Clostridium difficile）肠道疾病主要由毒素A（TcdA）与毒素B（TcdB）的作用介导，这两种毒素的合成发生在涉及多种表面蛋白或鞭毛蛋白的定植初始步骤之后。在枯草芽孢杆菌（B. subtilis）中，σ因子SigD可调控鞭毛合成、运动能力以及营养型自溶素的表达；艰难梭菌630菌株的基因组中存在编码SigD的同源基因。我们在艰难梭菌630 ∆erm菌株中构建了sigD突变体，借助全局转录组学方法解析SigD的调控子。野生型菌株与sigD突变体间共计有103个基因存在差异表达，涵盖了与运动能力、代谢及调控相关的基因。此外，sigD突变体中参与鞭毛生物合成的基因表达量显著下调，编码TcdA、TcdB以及毒素正调控因子TcdR的基因表达量同样出现下降。通过基因组分析与RACE-PCR（快速扩增cDNA末端聚合酶链式反应）实验，我们明确了SigD直接靶基因（包括tcdR）的启动子序列，并鉴定出SigD的保守结合共识序列。后续研究证实，SigD可通过直接调控tcdR的转录，对毒素的表达发挥正调控作用。值得注意的是，推定的抗SigD因子FlgM的过表达可抑制SigD对运动能力与毒素合成的正调控作用。综上，SigD是目前已发现的首个艰难梭菌毒素合成的正调控因子。