Requirements for Nitric Oxide Generation from Isoniazid Activation In Vitro and Inhibition of Mycobacterial Respiration In Vivo

Abstract

Isoniazid (INH), a front-line antituberculosis agent, is activated by mycobacterial catalase-peroxidase KatG, converting INH into bactericidal reactive species. Here we investigated the requirements and the pathway of nitric oxide (NO˙) generation during oxidative activation of INH by Mycobacterium tuberculosis KatG in vitro.We also provide in vivo evidence that INH-derived NO˙can inhibit key mycobacterial respiratory enzymes, which may contribute to the overall antimycobacterial action of INH.Mycobacterium tuberculosis infections are of serious concern; they cause 2 million deaths every year and latently persist in over a billion individuals worldwide (41). Isoniazid (isonicotinic acid hydrazide [INH]) remains a front-line antituberculosis agent some 50 years after its development, and millions of doses are prescribed worldwide. M. tuberculosis is exceptionally sensitive to INH (12, 44), a prodrug, which is peroxidatively activated intracellularly by the M. tuberculosis catalase-peroxidase KatG to produce a range of reactive radicals that act as damaging species within the bacteria. Although the mechanism(s) of action and cellular targets of KatG-activated INH continue to be uncovered (22,27), its full range of effects on mycobacterial cells still remains to be resolved (20,33).The critical role of INH activation via KatG is clearly apparent based on the findings that the katG gene represents the main site for mutations causing INH resistance in M. tuberculosis (24, 45). Several INH-derived intermediates generated from isoniazid activation, such as isonicotinic acyl NADH (27), and mycobacterial targets including enzymes from the mycobacterial type II fatty acid synthase system (1, 22) have been identified. Other work on INH activation has centered upon INH-derived free radicals as important antimycobacterial intermediates (18,31,38). Despite these advances, the exact mechanism(s) of INH action that results in its exceptional and specific potency against M. tuberculosis are not yet fully delineated, as multiple targets and pathways have been considered (12,20,23,32).A range of reactive nitrogen species, such as nitric oxide (NO˙) and peroxynitrite (ONOO Ϫ ) are known to have various levels of activity against M. tuberculosis (8, 21, 43). In addition to the action of exogenously added NO˙immune-derived NOḟ rom the action of inducible nitric oxide synthase is considered to contribute to defenses against mycobacterial infection (29). Previous reports of tyrosine nitration during oxidation of INH (34) and NO˙formation from peroxidative activation of hydroxyurea (16, 17) led us to hypothesize that NO˙might be generated as a result of INH activation by KatG. We further hypothesized that the exceptional sensitivity of M. tuberculosis to NO˙could potentially account for at least some of the potency of INH (8,21,43). Here we used spin trapping techniques to document KatG generation of nitric oxide during activation of INH and to probe potential pathways for its production from INH. We also present in vivo analysis indica...

Mycobacterium tuberculosis persistence mutants identified by screening in isoniazid-treated mice

“…Because nitric oxide generated during the oxidative activation of INH by the M. tuberculosis catalase can inhibit bacterial respiratory enzymes (32), impaired cytochrome bd oxidase function might account for the enhanced INH-mediated killing of the cydC:: Tn mutant. However, this interpretation would not explain why this enhancement was observed only when the mutant bacteria were grown in vivo.…”

Stimulation of Phagocytic Activity of Alveolar Macrophages Toward Artificial Microspheres by Infection with Mycobacteria

“…The effect of NO might be consistent due to its steady generation mediated by inducible NO synthase, but that of ROS is regarded to be transit due to its short life-time (25). In addition, it is suggested that the effect of NO generated by infection with MTB is more influential than that of ROS on the phagocytic activity of M7 cells (26). Hence, we examined the NO production on phagocytosis of microbes.…”

Depletion of antibiotic targets has widely varying effects on growth

“…In our study, we found that depletion of drug targets results in varying effects. The cidal effects of reducing the level of InhA protein seen, albeit more slowly, with an inducible promoter substitution (17) suggest that, although isoniazid may target multiple enzymes (27)(28)(29), inhibition of InhA alone could account for antibiotic activity. The rapid cell death seen with InhA depletion and the growth cessation with even only mild depletion of RpoB suggest that these biochemical processes are hypersensitive to inhibition.…”