Characterization of the Clostridioides difficile 630Δerm putative Pro-Pro endopeptidase CD1597

Clostridioides difficile is the leading cause of antibiotic-associated infections worldwide. Within the host, C. difficile can transition from a sessile to a motile state by secretion of PPEP-1, which releases the cells from the intestinal epithelium by cleaving adhesion proteins. PPEP-1 belongs to the group of Pro-Pro endopeptidases, which are characterized by their unique ability to cleave proline-proline bonds. Interestingly, another putative member of this group, CD1597, is present in C. difficile. Although it possesses a domain similar to other PPEPs, CD1597 displays several distinct features that suggest a markedly different role for this protein. We investigated the proteolytic activity of CD1597 by testing various potential substrates. In addition, we investigated the effect of the absence of CD1597 by generating an insertional mutant of the cd1597 gene. Using the cd1597 mutant, we sought to identify phenotypic changes through a series of in vitro experiments and quantitative proteomic analyses. Furthermore, we aimed to study the localization of this protein using a fluorogenic fusion protein. Despite its similarities to PPEP-1, CD1597 did not show proteolytic activity. In addition, the absence of CD1597 caused an increase in various sporulation proteins during the stationary phase, yet we did not observe any alterations in the sporulation frequency of the cd1597 mutant. Furthermore, a promoter activity assay indicated a very low expression level of cd1597 in vegetative cells that was independent of the culture medium and growth stage. The low expression was corroborated by our comprehensive proteomics analysis of whole cell cultures, which failed to identify CD1597. However, an analysis of purified C. difficile spores identified CD1597 as part of the spore proteome. Hence, we predict that the protein is involved in sporulation, although we were unable to define a precise role for CD1597 in C. difficile

艰难梭菌（Clostridioides difficile）是全球范围内抗生素相关性感染的首要致病菌。在宿主体内，艰难梭菌可通过分泌PPEP-1从固着态转变为运动态，该蛋白可通过切割黏附蛋白使菌体脱离肠上皮细胞。PPEP-1属于脯氨酸-脯氨酸内切肽酶（Pro-Pro endopeptidases）家族，这类酶的核心特征为可特异性切割脯氨酸-脯氨酸肽键。值得注意的是，艰难梭菌中还存在该家族的另一个推定成员CD1597。尽管CD1597拥有与其他PPEP家族蛋白相似的结构域，但它展现出多项独特的分子特征，提示该蛋白的生物学功能与PPEP-1存在显著差异。本研究通过检测多种潜在底物，探究了CD1597的蛋白水解活性；此外，我们通过构建cd1597基因的插入突变体，分析了CD1597缺失所产生的生物学影响。利用该cd1597突变体，我们通过一系列体外实验与定量蛋白质组学分析，试图鉴定CD1597缺失引发的表型变化；进一步地，我们还借助荧光融合蛋白研究了该蛋白的亚细胞定位情况。尽管CD1597与PPEP-1存在序列相似性，但并未检测到其蛋白水解活性。此外，CD1597的缺失会导致稳定生长期多种芽孢形成相关蛋白的表达水平上调，但我们未观察到cd1597突变体的芽孢形成率出现任何显著性变化。进一步的启动子活性测定结果显示，cd1597在营养体细胞中的表达水平极低，且该表达模式不受培养基类型与生长阶段的影响。我们对全细胞培养物的全面蛋白质组学分析也佐证了这一低表达现象，未能检测到CD1597蛋白。然而，对纯化的艰难梭菌芽孢进行的蛋白质组分析则将CD1597鉴定为芽孢蛋白质组的组分之一。因此，我们推测该蛋白参与芽孢形成过程，尽管目前尚未明确CD1597在艰难梭菌中的具体生物学功能。