ANTISENSE RNA TO AHPC, AN OXIDATIVE STRESS DEFENSE GENE INVOLVED IN ISONIAZID RESISTANCE, INDICATES THAT AHPC OF MYCOBACTERIUM-BOVIS HAS VIRULENCE PROPERTIES

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 vir ulence of Mycobacterium bovis, a member of the Mycobacterium tuberculo sis complex. Antisense RNA constructs of ahpC were made using differen t strength promoters in front of a reversed coding sequence of ahpC. T hese constructs were electroporated into a virulent wild-type M. bovis strain and a moderately virulent INH-resistant M. bovis strain that w as catalase/peroxidase-negative. Down-regulation of protein synthesis occurred and this was visualized by immunoblotting. All strains contai ning antisense RNA were markedly less virulent than their parent strai ns in guinea pigs. M. bovis with an up-regulated ahpC gene was more re sistant to cumene hydroperoxide than its parent strain, which had a wi ld-type ahpC promoter. These results agree with a model of INH resista nce 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 vi rulence of wild-type M. bovis, which has normal catalase/peroxidase le vels.