Table_4_Differential View on the Bile Acid Stress Response of Clostridioides difficile.XLSX

Clostridioides difficile is an intestinal human pathogen that uses the opportunity of a depleted microbiota to cause an infection. It is known, that the composition of the intestinal bile acid cocktail has a great impact on the susceptibility toward a C. difficile infection. However, the specific response of growing C. difficile cells to diverse bile acids on the molecular level has not been described yet. In this study, we recorded proteome signatures of shock and long-term (LT) stress with the four main bile acids cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA). A general overlapping response to all tested bile acids could be determined particularly in shock experiments which appears plausible in the light of their common steroid structure. However, during LT stress several proteins showed an altered abundance in the presence of only a single or a few of the bile acids indicating the existence of specific adaptation mechanisms. Our results point at a differential induction of the groEL and dnaKJgrpE chaperone systems, both belonging to the class I heat shock genes. Additionally, central metabolic pathways involving butyrate fermentation and the reductive Stickland fermentation of leucine were effected, although CA caused a proteome signature different from the other three bile acids. Furthermore, quantitative proteomics revealed a loss of flagellar proteins in LT stress with LCA. The absence of flagella could be substantiated by electron microscopy which also indicated less flagellated cells in the presence of DCA and CDCA and no influence on flagella formation by CA. Our data break down the bile acid stress response of C. difficile into a general and a specific adaptation. The latter cannot simply be divided into a response to primary and secondary bile acids, but rather reflects a complex and variable adaptation process enabling C. difficile to survive and to cause an infection in the intestinal tract.

艰难梭菌（Clostridioides difficile）是一种人类肠道致病菌，可借助肠道微生物群耗竭的契机引发感染。已知肠道胆汁酸混合物的组成对艰难梭菌感染的易感性具有显著影响，但目前尚未在分子层面阐明增殖态艰难梭菌对各类胆汁酸的特异性应答机制。本研究针对四种主要胆汁酸——胆酸（cholic acid, CA）、鹅脱氧胆酸（chenodeoxycholic acid, CDCA）、脱氧胆酸（deoxycholic acid, DCA）及石胆酸（lithocholic acid, LCA），分别采集了其诱导的休克应激与长期（LT）应激下的蛋白质组特征。尤其在休克实验中，我们可观测到所有受试胆汁酸共有的通用应答模式，这一结果与其共同的类固醇结构特征相符，具备生物学合理性。而在长期应激条件下，仅在一种或少数几种胆汁酸存在时，才有部分蛋白质的丰度发生改变，提示存在特异性的适应机制。研究结果显示，groEL与dnaKJgrpE分子伴侣系统（二者均属于I类热休克基因）的诱导模式存在差异。此外，涉及丁酸发酵及亮氨酸还原型Stickland发酵的核心代谢通路也受到影响，不过胆酸诱导的蛋白质组特征与其余三种胆汁酸存在显著区别。进一步的定量蛋白质组学分析显示，在石胆酸诱导的长期应激中，鞭毛蛋白的表达出现缺失。电子显微镜实验验证了这一结果，同时还发现脱氧胆酸与鹅脱氧胆酸处理组的鞭毛细胞数量减少，而胆酸对鞭毛形成无明显影响。本研究数据将艰难梭菌的胆汁酸应激应答划分为通用适应与特异性适应两类。后者无法简单地按照初级与次级胆汁酸的分类进行划分，而是反映出一套复杂且动态的适应过程，助力艰难梭菌在肠道环境中存活并引发感染。