title: Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis.
abstract: The inhA and kasA genes of Mycobacterium tuberculosis have each been proposed to encode the primary target of the antibiotic isoniazid (INH). Previous studies investigating whether overexpressed inhA or kasA could confer resistance to INH yielded disparate results. In this work, multicopy plasmids expressing either inhA or kasA genes were transformed into M. smegmatis, M. bovis BCG and three different M. tuberculosis strains. The resulting transformants, as well as previously published M. tuberculosis strains with multicopy inhA or kasAB plasmids, were tested for their resistance to INH, ethionamide (ETH) or thiolactomycin (TLM). Mycobacteria containing inhA plasmids uniformly exhibited 20-fold or greater increased resistance to INH and 10-fold or greater increased resistance to ETH. In contrast, the kasA plasmid conferred no increased resistance to INH or ETH in any of the five strains, but it did confer resistance to thiolactomycin, a known KasA inhibitor. INH is known to increase the expression of kasA in INH-susceptible M. tuberculosis strains. Using molecular beacons, quantified inhA and kasA mRNA levels showed that increased inhA mRNA levels corre--lated with INH resistance, whereas kasA mRNA levels did not. In summary, analysis of strains harbouring inhA or kasA plasmids yielded the same conclusion: overexpressed inhA, but not kasA, confers INH and ETH resistance to M. smegmatis, M. bovis BCG and M. tuberculosis. Therefore, InhA is the primary target of action of INH and ETH in all three species.
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Michelle H
authors.initials: MH
authors.lastname: Larsen
authors.forename: Catherine
authors.initials: C
authors.lastname: Vilchèze
authors.forename: Laurent
authors.initials: L
authors.lastname: Kremer
authors.forename: Gurdyal S
authors.initials: GS
authors.lastname: Besra
authors.forename: Linda
authors.initials: L
authors.lastname: Parsons
authors.forename: Max
authors.initials: M
authors.lastname: Salfinger
authors.forename: Leonid
authors.initials: L
authors.lastname: Heifets
authors.forename: Manzour H
authors.initials: MH
authors.lastname: Hazbon
authors.forename: David
authors.initials: D
authors.lastname: Alland
authors.forename: James C
authors.initials: JC
authors.lastname: Sacchettini
authors.forename: William R
authors.initials: WR
authors.lastname: Jacobs
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000235:genetics / Q000378:metabolism*; D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins; D024881:Drug Resistance, Bacterial; D005000:Ethionamide / Q000494:pharmacology*; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D009161:Mycobacterium / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D009163:Mycobacterium bovis / Q000187:drug effects; D020102:Mycobacterium smegmatis / Q000187:drug effects; D009169:Mycobacterium tuberculosis / Q000187:drug effects; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D014169:Transformation, Bacterial
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D010088:Oxidoreductases; C085257:InhA protein, Mycobacterium; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D005000:Ethionamide; D007538:Isoniazid