EFFECTS OF OVEREXPRESSION OF THE ALKYL HYDROPEROXIDE REDUCTASE AHPC ON THE VIRULENCE AND ISONIAZID RESISTANCE OF MYCOBACTERIUM-TUBERCULOSIS

Mutations to the regulatory region of the ahpC gene, resulting in over production of alkyl hydroperoxide reductase, were encountered frequent ly in a large collection of isoniazid (INH)-resistant clinical isolate s of Mycobacterium tuberculosis but not in INH-susceptible strains. Ov erexpression of ahpC did not seem to be important for INH resistance, however, as most of these strains were already defective for catalase- peroxidase, KatG, the enzyme required for activation of INH. Transform ation of the INH-susceptible reference strain, M. tuberculosis H37Rv, with plasmids bearing the ahpC genes of M. tuberculosis or M. leprae d id not result in a significant increase in the MIC. Two highly INH-res istant mutants of H37Rv, BH3 and BH8, were isolated in vitro and shown to produce no or little KatG activity and, in the case of BH3, to ove rproduce alkyl hydroperoxide reductase as the result of an ahpC regula tory mutation that was also found in some clinical isolates. The virul ence of H37Rv, BH3, and BH8 was studied intensively in three mouse mod els: fully immunocompetent BALB/c and Black 6 mice, BALB/c major histo compatibility complex class II-knockout mice with abnormally low level s of CD4 T cells and athymic mice producing no cellular immune respons e. The results indicated that M. tuberculosis strains producing catala se-peroxidase were considerably more virulent in immunocompetent mice than the isogenic KatG-deficient mutants but that loss of catalase-per oxidase was less important when immunodeficient mice, unable to produc e activated macrophages, were infected. Restoration of virulence was n ot seen in an INH-resistant M. tuberculosis strain that overexpressed ahpC, and this finding was confirmed by experiments performed with app ropriate M. bovis strains in guinea pigs. Thus, in contrast to catalas e-peroxidase, alkyl hydroperoxide reductase does not appear to act as a virulence factor in rodent infections or to play a direct role in IN H resistance, although it may be important in maintaining peroxide hom eostasis of the organism when KatG activity is low or absent.