Nitric oxide generated from isoniazid activation by KatG: source of nitric oxide and activity against Mycobacterium tuberculosis

Abstract

Isonicotinic acid hydrazide (INH) is a frontline antituberculosis agent. Once taken up by Mycobacterium tuberculosis, INH requires activation by the catalase-peroxidase KatG, converting INH from its prodrug form into a range of bactericidal reactive species. Here we used 15N-labeled INH together with electron paramagnetic resonance spin trapping techniques to demonstrate that nitric oxide (NO*) is generated from oxidation at the hydrazide nitrogens during the activation of INH by M. tuberculosis KatG. We also observed that a specific scavenger of NO* provided protection against the antimycobacterial activity of INH in bacterial culture. No significant increases in mycobacterial protein nitration were detected, suggesting that NOdot; and not peroxynitrite, a nitrating metabolite of NO*, is involved in antimycobacterial action. In conclusion, INH-derived NO* has biological activity, which directly contributes to the antimycobacterial action of INH.

FIG. 1.

Nitric oxide is generated from oxidation hydrazide nitrogen atoms during INH activation by KatG. (a) EPR spectra of NȮ derived from KatG activation of INH. NȮ was spin trapped with 10 mM Fe^II (N-methyl-d-glucamine dithiocarbamate)₂ complex after incubation of 0.471 mg of KatG ml⁻¹ with 35 mM INH and 10 mM H₂O₂ in 10 mM phosphate buffer, pH 7, at 37°C for 5 min. Spectrum i is all [¹⁴N]INH; spectrum ii is hydrazide-labeled [¹⁵N₂, ¹⁵N₃]INH. The EPR spectrometer settings were as follows: microwave power, 10 mW; modulation, 0.2 mT at 100 kHz; x-axis resolution, 1,024 points; conversion time, 82 ms; time constant, 164 ms; sweep, 8 mT; number of scans, 45. (b) Dependence of NȮ production from INH on KatG and H₂O₂. Conditions are the same as in panel a except that the concentration of INH is 10 mM. Data are the results of four experiments.

FIG. 2.

Antimycobacterial action of INH-derived nitric oxide. (a) Survival data for M. tuberculosis var. bovis BCG upon exposure to 1 mM DETA-NONOate. Exponentially growing cultures in aerobic roller bottle culture of M. tuberculosis var. bovis BCG were exposed to 1 mM DETA-NONOate for 7 days at 37°C. Data are normalized to the respective day 0 values and represent triplicate experiments (P = 0.026). (b) The NȮ scavenger CPTIO protects against the antimycobacterial action of INH. Exponentially growing cultures in aerobic roller bottle cultures of M. tuberculosis var. bovis BCG were exposed to 2 mM CPTIO and/or 3.7 μM INH for 7 days at 37°C. CPTIO alone had no effect on viability (data not shown). Cultures were serially diluted and plated on 7H11 plates for CFU determination. Values are normalized to CFU values of untreated controls at day 0 and represent triplicate experiments (P = 0.0009). The actual survival rate in culture with CPTIO was 17.5% compared to day 0 controls. (c) Nitrotyrosine levels upon exposure to INH. Exponentially growing cultures of M. tuberculosis var. bovis BCG (as in panel a) were treated overnight with 73 μM INH with or without 1 mM plumbagin at 37°C. Cell extracts (obtained by bead beating with 0.1-mm ZrSi beads) were assayed for nitrotyrosine by using the Hycult Biotechnology Hbt nitrotyrosine ELISA. Data represent the means of three replicate cultures. No levels of nitrotyrosine were significantly different from others (P < 0.05).

FIG. 3.

Postulated pathway of NȮ production. Oxidation of INH (1) at N₂ (18, 25) to form the hydrazyl radical (2) is followed by oxygen addition to the hydrazyl radical (causing N-O bond formation) (3), followed by as yet undelineated fragmentation or elimination.