Clostridioides difficile is an anaerobic Gram-positive bacterium that can produce the large clostridial toxins toxin A and toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, which positively regulates toxin gene expression, and TcdC, which is a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor; however, several studies failed to show an association between the tcdC genotype and toxin production. Consequently, the TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the oligonucleotide/oligosaccharide binding fold (OB-fold) domain present at the C terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the OB-fold. In this study, we aimed to obtain topological information on the C terminus of TcdC and demonstrate that the C terminus of TcdC is located extracellularly. In addition, we show that the membrane association of TcdC is dependent on a membrane-proximal cysteine residue and that mutating this residue results in the release of TcdC from the bacterial cell. The extracellular location of TcdC is not compatible with the direct binding of the OB-fold domain to intracellular nucleic acid or protein targets and suggests a mechanism of action that is different from that of the characterized anti-sigma factors.

中文翻译：

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艰难梭菌TcdC的C末端域暴露在细菌细胞表面。

艰难梭菌是一种厌氧革兰氏阳性细菌，可产生致病性基因座（PaLoc）中编码的大型梭菌毒素A和B毒素。PaLoc还编码可正向调节毒素基因表达的sigma因子TcdR和可推定的毒素表达负调节剂TcdC。提出将TcdC作为反西格玛因子。但是，一些研究未能显示tcdC之间的关联基因型和毒素产生。因此，尚未完全了解TcdC功能。先前的研究已将TcdC表征为具有结合G四联体结构能力的膜相关蛋白。通过存在于蛋白质C端的寡核苷酸/寡糖结合折叠（OB-fold）域来介导与DNA二级结构的结合。先前还提出该结构域负责对毒素基因表达的抑制作用，暗示了OB-折叠的胞质定位。在这项研究中，我们旨在获得TcdC的C末端的拓扑信息，并证明TcdC的C末端位于细胞外。此外，我们表明，TcdC的膜缔合取决于膜近半胱氨酸残基，并且突变该残基会导致TcdC从细菌细胞释放。TcdC的细胞外位置与OB折叠结构域与细胞内核酸或蛋白质靶标的直接结合不相容，并表明其作用机制不同于所表征的抗σ因子。