Tuberculosis remains the largest cause of death in the world from a single infectious disease and is responsible for one in four avoidable adult deaths in developing countries. Infection with drug-sensitive strains of Mycobacterium tuberculosis can be effectively cured with a combination of isoniazid (INH), rifampicin, and pyrazinamide. However, the emergence of multiple drug resistant strains of M. tuberculosis has resulted in fatal outbreaks in the United States. INH was first reported to be active against M. tuberculosis in 1952, when it was shown to have a highly specific activity against M. tuberculosis and M bovis with less but considerable activity against other mycobacteria. Although INH is one of the most widely used anti-tuberculosis drugs for both therapy and prophylaxis, its precise target of action on Mycobacterium tuberculosis is unknown. We have discovered a novel gene, named inhA, in M. tuberculosis and all mycobacterial species examined that encodes a target for both isoniazid and ethionamide. The inhA gene encodes a protein of 32 kDa that shows significant sequence conservation with an E. coli enzyme, EnvM, known to play a role in fatty acid biosynthesis. Cell-free assays indicate the involvement of InhA protein in mycolic acid biosynthesis, previously suggested site of action of INH. In this proposal, we intend to employ a combination of genetic, biochemical, and x-ray crystallographic methods to characterize the InhA protein and elucidate mechanism of inhibition by ethionamide and isoniazid.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project--Cooperative Agreements (U01)
Project #
1U01AI036849-01
Application #
2073343
Study Section
Special Emphasis Panel (SRC (48))
Project Start
1994-08-01
Project End
1998-04-30
Budget Start
1994-08-01
Budget End
1995-04-30
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Rozwarski, D A; Vilcheze, C; Sugantino, M et al. (1999) Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase, InhA, in complex with NAD+ and a C16 fatty acyl substrate. J Biol Chem 274:15582-9
Basso, L A; Zheng, R; Musser, J M et al. (1998) Mechanisms of isoniazid resistance in Mycobacterium tuberculosis: enzymatic characterization of enoyl reductase mutants identified in isoniazid-resistant clinical isolates. J Infect Dis 178:769-75
Miesel, L; Weisbrod, T R; Marcinkeviciene, J A et al. (1998) NADH dehydrogenase defects confer isoniazid resistance and conditional lethality in Mycobacterium smegmatis. J Bacteriol 180:2459-67
Miesel, L; Rozwarski, D A; Sacchettini, J C et al. (1998) Mechanisms for isoniazid action and resistance. Novartis Found Symp 217:209-20;discussion 220-1
Rozwarski, D A; Grant, G A; Barton, D H et al. (1998) Modification of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis. Science 279:98-102
Sacchettini, J C; Blanchard, J S (1996) The structure and function of the isoniazid target in M. tuberculosis. Res Microbiol 147:36-43
Quemard, A; Sacchettini, J C; Dessen, A et al. (1995) Enzymatic characterization of the target for isoniazid in Mycobacterium tuberculosis. Biochemistry 34:8235-41
Dessen, A; Quemard, A; Blanchard, J S et al. (1995) Crystal structure and function of the isoniazid target of Mycobacterium tuberculosis. Science 267:1638-41