BILE ACID METABOLISM BY INTESTINAL ANAEROBIC BACTERIA

肠道厌氧菌的胆汁酸代谢

• 批准号: 6201846
• 负责人: PHILLIP B HYLEMON
• 金额: $ 14.86万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6201846
• 关键词: Propionibacteriaceae; Pseudomonas; SDS polyacrylamide gel electrophoresis; autoradiography; biotransformation; chenodeoxycholate; cholestane compound; enzyme activity; feces analysis; gene expression; human tissue; hydroxylation; hydroxysteroid dehydrogenases; molecular cloning; northern blottings; nucleic acid probes; nucleic acid sequence; operon; protein sequence; steroid 7alpha hydroxylase; steroid metabolism; thin layer chromatography; transcription termination; transport proteins; ursodeoxycholate

The bacterial alpha-dehydroxylation (7alphaDOH) of cholic acid (CA) and chenodeoxycholic acid (CDCA) generates the secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. Secondary bile acids are believed to normally represent about 20 to 25% of the total bile acid pool in man. However, DCA concentrations have been reported to vary from 5% to 45% of the bile acid pool. There is a high correlation between the DCA concentration and the cholesterol saturation index (CSI) of gallbladder bile in man. A high CSI is a known risk factor for cholesterol gallstone disease. Secondary bile acids are much more toxic to eukaryotic cells than their corresponding primary bile acids and have been implicated in plying a role in cholesterol gallstone formation, cholestatic liver disease and colon carcinogenesis. Secondary bile acids are also strong activators of protein kinase C. Our long range goal is to try and understand the basic enzymology and genetics of intestinal bacterial 7 alphaDOH and to develop specific inhibitors of this biotransformation. We are also interested in testing the hypothesis that colonization of individuals with certain strains of bile acid 7 alpha-dehydroxylating bacteria is a major risk factor for cholesterol gallstone disease. The specific aims of this application are: 1) Complete the cloning, sequencing, and analysis of a large bile acid inducible operon which is involved in 7alphaDOH in Eubacterium sp. VIP 12708. Clone, sequence and analyze a second putative bile acid inducible operon in this bacterium; 2) Determine the function each polypeptide encoded by these operons plays in bile acid 7 alpha/7betaDOH using both biochemical and genetic approaches. These include subcloning and expression of each gene in the operons, reconstitution of the pathway in vitro, and purification of the various bile acid inducible activities. Specifically, we propose to: a) subclone and express the baiE and baiF genes and determine if these genes encode bile acid alpha and 7beta- dehydratases; b) purify, cline and sequence the the genes encoding bile acid delta4 and delta6-reductases; c) subclone and express the baiG gene, which has high amino acid sequence identity with the tetracycline resistance gene, and determine if this gene encodes a bile acid transporter. Finally, we propose to isolate, characterize, and identify bile acid alpha-dehydroxylating bacteria from cholesterol gallstone patients having high (>30%) deoxycholic acid levels and determine if they are unique bacterial species. If these bacteria are unique species, we will attempt to develop specific DNA probes in order to easily detect and quantitate these organs in fecal samples.

胆酸的细菌α-脱羟基化（7alphadoH） （CA）和Chendoxycholic Acid（CDCA）产生次级 胆汁酸，脱氧胆酸（DCA）和岩性酸（LCA），，，， 分别。 据信二级胆汁酸通常代表 大约20％至25％的人类胆汁酸池。 但是，DCA 据报道，浓度从5％到45％ 胆汁酸池。 DCA之间存在很高的相关性 浓度和胆固醇饱和指数（CSI） 男人的胆囊胆汁。 高CSI是已知的风险因素 胆固醇胆结石疾病。 次生胆汁酸更多 对真核细胞的毒性比其相应的原代胆汁酸有毒 并与在胆固醇胆石中发挥作用有关 形成，胆汁淤积性肝病和结肠癌发生。 次生胆汁酸也是蛋白激酶C的强激活剂。 我们的远距离目标是尝试了解基本的酶学 和肠道细菌7α的遗传学并发展 该生物转化的特定抑制剂。 我们也很感兴趣 在检验以下假设时，有一定的个体定植 胆汁酸7α-脱羟基化细菌是主要风险 胆固醇胆结石疾病的因素。 这个特定的目的 应用是：1）完成克隆，测序和分析 大型胆汁酸诱导的操纵子，与7alphadoH有关 Eubacterium sp。 VIP 12708。克隆，序列和分析第二个 该细菌中推定的胆汁酸诱导操纵子； 2）确定 这些操纵子在胆汁中播放的每个多肽的功能 酸7α/7betadoH使用生化和遗传 方法。 这些包括每个基因的亚克隆和表达 在操纵子中，体外途径重建， 纯化各种胆汁酸诱导活性。 具体来说， 我们建议：a）亚克隆并表达baie和baif基因以及 确定这些基因是否编码胆汁酸α和7beta-- 脱水酶； b）纯化，cline和序列编码的基因 胆汁酸delta4和delta6-还原酶； c）亚克隆并表达 Baig Gene，其具有高氨基酸序列身份 四环素抗性基因，并确定该基因是否编码胆汁 酸转运蛋白。 最后，我们建议隔离，表征和 鉴定胆固醇的胆汁酸α-脱氢细菌 胆结石患者的脱氧胆酸水平高（> 30％）和 确定它们是否是独特的细菌物种。 如果这些细菌是 独特的物种，我们将尝试开发特定的DNA探针 为了在粪便样品中轻松检测和定量这些器官。