DataSheet1_Investigation of metabolic crosstalk between host and pathogenic Clostridioides difficile via multiomics approaches.xlsx

Clostridioides difficile is a gram-positive anaerobic bacterium that causes antibiotic-associated infections in the gut. C. difficile infection develops in the intestine of a host with an imbalance of the intestinal microbiota and, in severe cases, can lead to toxic megacolon, intestinal perforation, and even death. Despite its severity and importance, however, the lack of a model to understand host-pathogen interactions and the lack of research results on host cell effects and response mechanisms under C. difficile infection remain limited. Here, we developed an in vitro anaerobic-aerobic C. difficile infection model that enables direct interaction between human gut epithelial cells and C. difficile through the Mimetic Intestinal Host–Microbe Interaction Coculture System. Additionally, an integrative multiomics approach was applied to investigate the biological changes and response mechanisms of host cells caused by C. difficile in the early stage of infection. The C. difficile infection model was validated through the induction of disaggregation of the actin filaments and disruption of the intestinal epithelial barrier as the toxin-mediated phenotypes following infection progression. In addition, an upregulation of stress-induced chaperones and an increase in the ubiquitin proteasomal pathway were identified in response to protein stress that occurred in the early stage of infection, and downregulation of proteins contained in the electron transfer chain and ATP synthase was observed. It has been demonstrated that host cell energy metabolism is inhibited through the glycolysis of Caco-2 cells and the reduction of metabolites belonging to the TCA cycle. Taken together, our C. difficile infection model suggests a new biological response pathway in the host cell induced by C. difficile during the early stage of infection at the molecular level under anaerobic-aerobic conditions. Therefore, this study has the potential to be applied to the development of future therapeutics through basic metabolic studies of C. difficile infection.

艰难梭菌（Clostridioides difficile）是一种革兰氏阳性厌氧菌（gram-positive anaerobic bacterium），可引发肠道抗生素相关性感染。当宿主肠道菌群失衡时，易发生艰难梭菌感染，重症病例可进展为中毒性巨结肠（toxic megacolon）、肠穿孔（intestinal perforation）甚至死亡。尽管该感染危害严重且临床意义重大，但目前仍缺乏用于解析宿主-病原体相互作用的研究模型，且关于艰难梭菌感染后宿主细胞的效应及应答机制的研究成果亦相对匮乏。为此，本研究借助模拟肠道宿主-微生物相互作用共培养系统（Mimetic Intestinal Host–Microbe Interaction Coculture System），构建了一种可实现人肠道上皮细胞与艰难梭菌直接相互作用的体外厌氧-需氧联合感染模型。此外，本研究采用整合多组学方法（integrative multiomics approach），探究了感染早期阶段宿主细胞受艰难梭菌诱导产生的生物学变化及应答机制。通过观察感染进程中毒素介导的表型（toxin-mediated phenotypes）——肌动蛋白丝解聚（disaggregation of the actin filaments）与肠上皮屏障破坏（disruption of the intestinal epithelial barrier），验证了该感染模型的有效性。进一步分析发现，感染早期宿主细胞响应蛋白应激时，应激诱导分子伴侣（stress-induced chaperones）表达上调、泛素蛋白酶体通路（ubiquitin proteasomal pathway）活性增强；同时，电子传递链（electron transfer chain）及ATP合酶（ATP synthase）相关蛋白表达下调。研究证实，Caco-2细胞的糖酵解（glycolysis）过程受抑、三羧酸循环（TCA cycle）代谢物水平降低，宿主细胞能量代谢因此受到抑制。综上，本研究构建的艰难梭菌感染模型，在厌氧-需氧条件下于分子层面揭示了感染早期宿主细胞受艰难梭菌诱导的全新生物学应答通路。本研究通过对艰难梭菌感染的基础代谢研究，有望为未来治疗药物的开发提供理论依据。