The bacterial 7 alpha-dehydroxylation of cholic acid and chenodeoxycholic acid generates the secondary bile acids, deoxycholic acid and lithocholic acid, respectively. Secondary bile acids normally represent about 20% to 25% of the total biliary bile acid pool in man. However, deoxycholic acid increases to about 40% of the biliary bile acid pool following removal of the gallbladder (cholecystectomy). Secondary bile acids are much more toxic to eucaryotic cells than their corresponding primary bile acids and have been implicated in playing an important role in certain cholestatic liver diseases and colon carcinogenesis. Treatment of certain cholestatic liver diseases or cholesterol gallstone dissolution by hydrophilic bile acids is complicated by bacterial 7-alpha-dehydroxylation. Our long range goal is to try and understand the basic enzymology and genetics of intestinal bile acid 7-alpha-dehydroxylation and develop a specific inhibitor of this bacterial biotransformation.
The specific aims of this application are: l) Complete the cloning, sequencing and analysis of a large bile acid inducible operon which is involved in 7-alpha- dehydroxylation. 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- dehydroxylation using both biochemical and genetic approaches. These include subcloning and expression of each gene, reconstitution of the bile acid 7-alpha-dehydroxylation pathway in vitro, and purification of various bile acid inducible activities. Specifically, we propose to: l) Subclone, express, purify and characterize the baiA1 and baiA3 gene products which we hypothesize are 3-alpha-hydroxysteroid dehydrogenase isoenzymes; 2) Subclone and express the baiE, and baiF genes, and determine if these genes encode a bile acid 7-alpha and 7-beta dehydratases; 3) Purification, cloning and sequencing of the bile acid delta 4 and delta 6-reductases; Subclone and express the baiG gene, which has high amino acid sequence identity to the tetracycline resistance gene, and determine if this gene encodes a bile acid transporter/exporter.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK040986-06A2
Application #
2141557
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1989-01-01
Project End
1999-06-30
Budget Start
1995-07-25
Budget End
1996-06-30
Support Year
6
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Virginia Commonwealth University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Richmond
State
VA
Country
United States
Zip Code
23298
Mallonee, D H; Hylemon, P B (1999) Use of a short A/T-rich cassette for enhanced expression of cloned genes in Escherichia coli. Mol Biotechnol 11:27-35
Doerner, K C; Takamine, F; LaVoie, C P et al. (1997) Assessment of fecal bacteria with bile acid 7 alpha-dehydroxylating activity for the presence of bai-like genes. Appl Environ Microbiol 63:1185-8
Mallonee, D H; Hylemon, P B (1996) Sequencing and expression of a gene encoding a bile acid transporter from Eubacterium sp. strain VPI 12708. J Bacteriol 178:7053-8
Dawson, J A; Mallonee, D H; Bjorkhem, I et al. (1996) Expression and characterization of a C24 bile acid 7 alpha-dehydratase from Eubacterium sp. strain VPI 12708 in Escherichia coli. J Lipid Res 37:1258-67
Baron, S F; Hylemon, P B (1995) Expression of the bile acid-inducible NADH:flavin oxidoreductase gene of Eubacterium sp. VPI 12708 in Escherichia coli. Biochim Biophys Acta 1249:145-54
Mallonee, D H; Lijewski, M A; Hylemon, P B (1995) Expression in Escherichia coli and characterization of a bile acid-inducible 3 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708. Curr Microbiol 30:259-63
de Prada, P; Setchell, K D; Hylemon, P B (1994) Purification and characterization of a novel 17 alpha-hydroxysteroid dehydrogenase from an intestinal Eubacterium sp. VPI 12708. J Lipid Res 35:922-9
Franklund, C V; Baron, S F; Hylemon, P B (1993) Characterization of the baiH gene encoding a bile acid-inducible NADH:flavin oxidoreductase from Eubacterium sp. strain VPI 12708. J Bacteriol 175:3002-12
Mallonee, D H; Adams, J L; Hylemon, P B (1992) The bile acid-inducible baiB gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A ligase. J Bacteriol 174:2065-71
Baron, S F; Franklund, C V; Hylemon, P B (1991) Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708. J Bacteriol 173:4558-69

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