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1Ag Research, Wal laceville Animal Research Centre, PO Box 40063, Upper Hutt, New Zealand
2Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA
1 Author for correspondence: Theresa Wilson. Tel: + 64 4 528 6089. Fax: + 64 4 528 1380.e-mail: wilsont@agresearch.cri.nz
ABSTRACT
SUMMARY: Antisense RNA is a versatile tool for reducing gene expression. It was used to determine if ahpC, a gene that is involved in defence against oxidative stress and isoniazid (INH) resistance, is important for virulence of Mycobacterium bovis, a member of the Mycobecterium tuberculosis complex. Antisense RNA constructs of ahpC were made using different strength promoters in front of a reversed coding sequence of ahpC. These constructs were electroporated into a virulent wild-type M. bowis strain and a moderately virulent INH-resistant M. bo wis strain that was cata lase/peroxi dase-negat ive. Down- regulation of protein synthesis occurred and this was visualized by immunoblotting. All strains containing antisense RNA were markedly less virulent than their parent strains in guinea pigs. M. bowis with an up-regulated ahpC gene was more resistant to cumene hydroperoxide than its parent strain, which had a wild- type ahpC promoter. These results agree with a model of INH resistance in which overexpression of AhpC compensates in some INH-resistant strains for loss of catalase/peroxidase by maintaining the ability to defend against oxidative stress mediated through organic peroxides. In addition, normal expression of AhpC is crucial for maintaining the virulence of wild-type M. bovis, which has normal catalase/peroxidase levels.
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