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.
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