Role of Bile Acids and Gut Bacteria in GI Diseases

描述（由申请人提供）： 人类结肠拥有已知最密集的细菌生态系统之一（>1011个细菌/g粪便）。胆酸（CA）和鹅去氧胆酸（CDCA）是由肝脏中的胆固醇合成的主要胆汁酸。胆汁酸每天进行数次肝肠循环。进入结肠的初级胆汁酸被解结合，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），分别产生二级胆汁酸脱氧胆酸（DCA）和石胆酸（LCA）。疏水性次级胆汁酸对哺乳动物细胞具有高毒性，并且可以在某些个体的胆汁酸库中积累至高水平（>70%），因为人类肝脏不能7-羟基化次级胆汁酸。血液、胆汁和粪便中高水平的DCA与某些患者结肠癌和胆固醇结石疾病的风险增加相关。次级胆汁酸的形成是由梭菌属的几个物种进行的，并且粪便中这些细菌的水平与胆汁中的DCA水平相关。本课题组已阐明了胆汁酸7 <$-DeOH的生化途径。然而，许多基因编码的酶在这一途径还没有被分离和表征，也没有确定的3D结构的关键酶在这一途径。一些菌株7？β-脱羟基细菌可以将糖皮质激素转化为C-19雄激素化合物，编码这些酶的基因尚未分离。这些基因的分离是至关重要的分子技术（RT-PCR）的发展，以方便地监测肠道胆汁酸和糖皮质激素代谢和药理学或饮食的方式，以减少形成二级胆汁酸和C-19雄激素化合物。Subaim 1A.通过全转录组方法（RNAseq）鉴定来自人胆汁酸7-脱羟基细菌Clostridium glandens ATCC 35704的CA、allo-CA和糖皮质激素诱导基因。Subaim 1B.开发定量PCR检测方法，用于检测allo-DCA和C-19雄激素形成中的关键基因。目标二。表征分别催化异DCA酸和C-19雄激素形成的来自Clostridium glandens ATCC 35704的异DCA和糖皮质激素诱导酶。目的3：确定Clostridium dens中胆汁酸7 <$/7 <$-DeOH的限速酶胆汁酸7 <$$>和7 <$-脱氢酶的三维结构。这些研究的结果将为我们提供进行临床研究的工具，以确定减少胆汁酸池中DCA的最有效方法，目的是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.