胆盐水解酶调控嗜酸乳杆菌ATCC43121超胆盐耐受特性机制解析

胆盐水解酶活性在肠道乳杆菌中普遍存在，然而其调控乳杆菌胆盐耐受的机制尚未阐明。嗜酸乳杆菌ATCC43121在人类生理pH条件下已经显示了超胆盐耐受特性。通过基因敲除试验已证明胆盐水解酶是其具有该特性的关键因子。基于结合胆盐在该菌株胞内解离后释放出的氨基酸基团未排到胞外的现象，推测其在胞内进一步代谢形成的产物应在菌株的胆盐耐受过程中发挥核心作用。本项目在此基础上，拟采用14^C标记结合胆盐的氨基酸基团，使用放射自显影技术定位14^C原子在菌株胞内的分布，利用同位素示踪技术追踪其代谢轨迹，基于转录组学、蛋白组学和脂质组学技术挖掘与氨基酸基团代谢物相关的能显著提升菌株胆盐耐受能力的关键蛋白(酶)，并通过分析其对菌株在胆盐胁迫下的细胞膜完整性、跨膜电势、胞内pH和DNA损伤的影响，解析其调节菌株胆盐耐受的机制。本项目将从新的视角揭示胆盐水解酶的生物学意义及机制，为操控乳杆菌在肠道内的存活提供理论依据。

Bile salt hydrolase activity is commonly detected in intestinal lactobacilli. However, the mechanisms by which bile salt hydrolases regulate bile tolerance ability of the lactobacilli remain unclear. Lactobacillus acidophilus ATCC 43121 has shown the ultra-bile-tolerant property at human physiological pH in our previous study. Bile salt hydrolases were demonstrated to be a key factor affecting its ultra-bile-tolerant property based a gene knockout experiment. Based on the phenomenon that the amino acid groups released from the conjugated bile salts via their deconjugation by the bile salt hydrolases in the cells were not transported to the outside of the cells, we speculated that the products forming by further metabolization of the amino acid groups in the cells should play a central role in the ultra bile tolerance of the strain. On this basis, we will label the amino acid groups of the conjugated bile salts with 14^C, locate the 14^C atoms in the cells by autoradiography, track the metabolic pathway of the amino acid groups by isotope tracer technique, explore the key proteins (enzymes) that are associated with the metabolites of the amino acid groups and able to significantly enhance bile tolerance ability of the strain by transcriptomics, proteomics and lipidomics analyses, explain the mechanisms by which the proteins (enzymes) regulate the bile tolerance of the strain by analyzing their effects on cell membrane integrity, transmembrane potential, intracellular pHs, and DNA damage of the strain following exposure to bile. This project will reveal the biological significance and action mechanisms of bile salt hydrolases from a new perspective, and provide a theoretical basis for manipulating survival of lactobacilli in the intestinal tract.