The Isoniazid Paradigm of Killing, Resistance, and Persistence in Mycobacterium tuberculosis

Abstract

Isoniazid (INH) was the first synthesized drug that mediated bactericidal killing of the bacterium Mycobacterium tuberculosis, a major clinical breakthrough. To this day, INH remains a cornerstone of modern tuberculosis (TB) chemotherapy. This review describes the serendipitous discovery of INH, its effectiveness on TB patients, and early studies to discover its mechanisms of bacteriocidal activity. Forty years after its introduction as a TB drug, the development of gene transfer in mycobacteria enabled the discovery of the genes encoding INH resistance, namely, the activator (katG) and the target (inhA) of INH. Further biochemical and x-ray crystallography studies on KatG and InhA proteins and mutants provided comprehensive understanding of INH mode of action and resistance mechanisms. Bacterial cultures can harbor subpopulations that are genetically or phenotypically resistant cells, the latter known as persisters. Treatment of exponentially growing cultures of M. tuberculosis with INH reproducibly kills 99% to 99.9% of cells in 3 days. Importantly, the surviving cells are slowly replicating or non-replicating cells expressing a unique stress response signature: these are the persisters. These persisters can be visualized using dual-reporter mycobacteriophages and their formation prevented using reducing compounds, such as N-acetylcysteine or vitamin C, that enhance M. tuberculosis' respiration. Altogether, this review portrays a detailed molecular analysis of INH killing and resistance mechanisms including persistence. The phenomenon of persistence is clearly the single greatest impediment to TB control, and research aimed at understanding persistence will provide new strategies to improve TB chemotherapy.

Keywords: isoniazid; mechanism; mycobacteriophage; persister.

Fig. 1.

Isoniazid (INH) structure and killing of M. tuberculosis. (A) INH structure, analogs, and active form. Ethionamide, nicotinamide, and pyrazinamide are INH analogs, and INH-NAD is the active form of INH. (B) Biphasic killing of M. tuberculosis H37Rv by INH. H37Rv grown in Middlebrook 7H9 supplemented with OADC, glycerol, and tyloxapol was treated once with INH (7.3 μM) at day 0. Bacteria survival was followed by taking samples at different time points and plating for colony-forming units (CFU). Slope “a” represents the rapid killing of INH-susceptible M. tuberculosis bacilli, slope “b” illustrates the slowing down of the bacterial killing by INH as persister cells accumulate, and slope “c” shows the emergence of INH-resistant mutants.

Fig. 2.

Use of dual-reporter mycobacteriophages to monitor persister cells. (A) Representation of the dual-reporter mycobacteriophage Φ²DRM9. mVenus (GFP) expression is driven by the constitutive PL(L5) promoter, and tdTomato (RFP) expression is driven by the promoter of a specific gene highly expressed in the INH persister population (dnaK in Φ²DRM9). (B) Time-lapse microscopy of M. tuberculosis infected with Φ ²DRM9 and treated with INH (from Jain et al. [87], with permission from the publisher). (C) Flow cytometry analysis of M. tuberculosis treated with INH (7.3 μM) for 3 days and infected with Φ²DRM9 for 16 h. The RFP population was back-gated to show the distribution of GFP expression in these cells.

Fig. 3.

Vitamin C and N-acetylcysteine enhance INH killing of M. tuberculosis. (A) Sterilization of M. tuberculosis H37Rv with the combination of INH/Vitamin C or INH/N-acetylcysteine. H37Rv grown in Middlebrook 7H9 supplemented with OADC, glycerol, and tyloxapol was treated once with INH (7.3 μM), vitamin C (VitC, 1 mM) or N-acetylcysteine (NAC, 4 mM) at day 0. Bacterial survival was monitored by taking samples at different time points and plating for colony-forming units (CFU). (B) Flow cytometry analysis of M. tuberculosis treated with INH, INH/VitC, or INH/NAC for 3 days and infected with Φ²DRM9 for 16 h. The RFP population was back-gated to show the distribution of GFP expression in these cells.(C) % RFP⁺GFP⁻ population (gate is shown in panel B) representing INH persisters.