Role of Bile Acids and Gut Bacteria in GI Diseases

描述(由申请人提供)： 人类结肠是已知的最密集的细菌生态系统之一(1011个细菌/克粪便)。胆酸(CA)和鹅去氧胆酸(CDCA)是肝脏中由胆固醇合成的主要胆汁酸。胆汁酸每天要经历几次肝-肠循环。进入结肠的初级胆汁酸被去结合，而游离胆汁酸被肠道细菌生物转化为20多种不同的代谢物。主要的生物转化是CA和CDCA的7？-脱羟基(7？-DeOH)，分别产生次级胆汁酸脱氧胆酸(DCA)和石胆酸(LCA)。疏水性次级胆汁酸对哺乳动物细胞有很强的毒性，由于人体肝脏不能使次级胆汁酸羟化，因此可在某些个体的胆汁酸池中积累到高水平(70%)。在一些患者中，血液、胆汁和粪便中高水平的DCA与结肠癌和胆固醇结石疾病的风险增加相关。次生胆汁酸的形成是由梭状芽孢杆菌属的少数几种细菌完成的，这些细菌在粪便中的水平与胆汁中的DCA水平有关。本课题组已经阐明了胆汁酸7？-脱氢酶的生化途径。然而，许多编码这一途径中的酶的基因还没有被分离和鉴定，也没有确定这一途径中关键酶的三维结构。一些7？脱羟基细菌菌株可以将糖皮质激素转化为C-19雄激素化合物，而编码这些酶的基因还没有分离出来。这些基因的分离对于分子技术(RT-PCR)的发展至关重要，该技术可以方便地监测肠道胆汁酸和糖皮质激素代谢，并通过药物或饮食方法减少次级胆汁酸和C-19雄激素化合物的形成。苏巴伊姆1号楼。通过全转录组学方法(RNAseq)从人胆汁酸脱羟化细菌Clostridium scindens atcc 35704中鉴定CA、allo-CA和糖皮质激素诱导基因。苏巴伊姆1B。建立涉及allo-DCA和C-19雄激素形成的关键基因的定量聚合酶链式反应分析。目的2.鉴定Clostridium scindens ATCC 35704产生的allo-CA和糖皮质激素诱导酶，分别催化形成allo-DCA酸和C-19雄激素。目的3：确定斯氏梭菌胆汁酸7？/7？脱氢酶中限速酶7？和7？-脱水酶的三维结构。这些研究的结果将为我们提供进行临床研究的工具，以确定减少胆酸池中DCA的最有效方法，以降低某些患者患结肠癌和胆固醇结石的风险。

DESCRIPTION (provided by applicant): The human colon harbors one of the most densely populated bacterial ecosystems known (>1011 bacteria/g feces). Cholic acid (CA) and chenodeoxycholic acid (CDCA) are primary bile acids synthesized from cholesterol in the liver. Bile acids undergo enterohepatic circulation several times each day. Primary bile acids entering the colon are deconjugated and free bile acids are biotransformed to more than 20 different metabolites by gut bacteria. The major biotransformation is 7¿-dehydroxylation (7¿- DeOH) of CA and CDCA yielding the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. Hydrophobic secondary bile acids are highly toxic to mammalian cells and can accumulate to high levels (>70%) in the bile acid pool of some individuals as the human liver cannot 7¿-hydroxylate secondary bile acids. High levels of DCA in blood, bile, and feces have been correlated with an increase risk of colon cancer and cholesterol gallstone disease, in some patients. The formation of secondary bile acids is carried out by a few species of the genus Clostridium and the level of these bacteria in feces is correlated with DCA levels in bile. The biochemical pathway of bile acid 7¿-DeOH has been elucidated by our group. However, many of the genes encoding enzymes in this pathway have not been isolated and characterized nor have the 3D structure of key enzymes in this pathway determined. Some strains of 7¿- dehydroxylating bacteria can convert glucocorticoids into C-19 androgenic compounds and the genes encoding these enzymes have not been isolated. Isolation of these genes is crucial for the development of molecular techniques (RT-PCR) to easily monitor intestinal bile acid and glucocorticoid metabolism and pharmacological or dietary ways to decrease formation of secondary bile acids and C-19 androgenic compounds. Subaim 1A. Identify CA, allo-CA, and glucocorticoid inducible genes from the human bile acid 7¿-dehydroxylating bacterium, Clostridium scindens ATCC 35704, via a wholetranscriptome approach (RNAseq). Subaim 1B. Develop quantitative PCR assays for key genes involved in allo-DCA and C-19 androgen formation. Aim 2. Characterize the allo-CA and glucocorticoid inducible enzymes from Clostridium scindens ATCC 35704 catalyzing the formation of allo-DCA acid and C-19 androgens, respectively. Aim 3: Determine the 3D structure of bile acid 7¿ and 7¿-dehydratases, the rate limiting enzymes in bile acid 7¿/7¿-DeOH, from Clostridium scindens. The results of these studies will give us the tools to carry out clinical studies to determine the most effective ways to decrease DCA in the bile acid pool with the aim of decreasing the risk of colon cancer and cholesterol gallstones, in some patients.