Pleiotropic roles of <i>Clostridium difficile sin</i> locus

Clostridium difficile is the primary cause of nosocomial diarrhea and pseudomembranous colitis. It produces dormant spores, which serve as an infectious vehicle responsible for transmission of the disease and persistence of the organism in the environment. In Bacillus subtilis, the sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition. In B. subtilis, SinR mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, so their interaction determines whether SinR can inhibit its target gene expression. The C. difficile genome carries two sinR homologs in the operon that we named sinR and sinR’, coding for SinR (112 aa) and SinR’ (105 aa), respectively. In this study, we constructed and characterized sin locus mutants in two different C. difficile strains R20291 and JIR8094, to decipher the locus’s role in C. difficile physiology. Transcriptome analysis of the sinRR’ mutants revealed their pleiotropic roles in controlling several pathways including sporulation, toxin production, and motility in C. difficile. Through various genetic and biochemical experiments, we have shown that SinR can regulate transcription of key regulators in these pathways, which includes sigD, spo0A, and codY. We have found that SinR’ acts as an antagonist to SinR by blocking its repressor activity. Using a hamster model, we have also demonstrated that the sin locus is needed for successful C. difficile infection. This study reveals the sin locus as a central link that connects the gene regulatory networks of sporulation, toxin production, and motility; three key pathways that are important for C. difficile pathogenesis.

艰难梭菌（Clostridium difficile）是医院获得性腹泻（nosocomial diarrhea）与伪膜性肠炎（pseudomembranous colitis）的首要致病菌。该菌可产生休眠孢子（dormant spores），孢子作为感染载体（infectious vehicle）介导疾病传播，并维持该菌在环境中的存续。在枯草芽孢杆菌（Bacillus subtilis）中，编码SinR与SinI的sin基因座（sin locus）负责抑制孢子形成，其中SinR含113个氨基酸，SinI含57个氨基酸。枯草芽孢杆菌内，SinR主要作为靶基因的转录阻遏蛋白，调控孢子形成、生物膜形成与细胞自溶过程；SinI为SinR的抑制剂，二者的相互作用决定了SinR能否阻遏其靶基因的表达。
艰难梭菌基因组的某操纵子（operon）中携带两个sinR同源基因，我们将其分别命名为sinR与sinR’，对应的编码产物分别为含112个氨基酸的SinR与含105个氨基酸的SinR’。本研究在R20291与JIR8094两株不同的艰难梭菌中构建了sin基因座突变体并完成表征，旨在解析该基因座在艰难梭菌生理过程中的功能。对sinRR’突变体的转录组分析（transcriptome analysis）显示，该基因座在调控艰难梭菌的多条通路中发挥多效性作用，涵盖孢子形成、毒素产生与运动能力调控。
通过一系列遗传与生化实验，我们证实SinR可调控上述通路中关键调控因子的转录，包括sigD、spo0A与codY。研究发现，SinR’可通过阻断SinR的阻遏活性，作为SinR的拮抗蛋白发挥功能。利用仓鼠感染模型（hamster model），我们还证实sin基因座对于艰难梭菌的成功感染不可或缺。本研究揭示，sin基因座是连接孢子形成、毒素产生与运动能力这三大艰难梭菌致病关键通路的基因调控网络的核心枢纽。