Oxidative Stress Increases Susceptibility of Mycobacterium tuberculosis to Isoniazid

Abstract

Isoniazid is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis.Isoniazid is a prodrug requiring oxidative activation by the catalase-peroxidase hemoprotein, KatG. Resistance to isoniazid can be obtained by point mutations in the katG gene, with one of the most common being a threonine-for-serine substitution at position 315 (S315T). The S315T mutation is found in more than 50% of isoniazid-resistant clinical isolates and results in an Ϸ200-fold increase in the MIC of isoniazid compared to that for M. tuberculosis H37Rv. In the present study we investigated the hypothesis that superoxide plays a role in KatG-mediated isoniazid activation. Plumbagin and clofazimine, compounds capable of generating superoxide anion, resulted in a lower MIC of isoniazid for M. tuberculosis H37Rv and a strain carrying the S315T mutation. These agents did not cause as great of an increase in isoniazid susceptibility in the mutant strain when the susceptibilities were assessed by using the inhibitory concentration that causes a 50% decrease in growth. These results provide evidence that superoxide can play a role in isoniazid activation. Since clofazimine alone has antitubercular activity, the observation of synergism between clofazimine and isoniazid raises the interesting possibility of using both drugs in combination to treat M. tuberculosis infections.Entering the 21st century, Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a major cause of morbidity and mortality worldwide. TB causes 1.9 million deaths annually among a pool of infected individuals close to 2 billion people (47).Isoniazid (INH) is one of the most effective and widely used therapeutic agents for the treatment of TB. M. tuberculosis shows exceptional sensitivity to INH, being several orders of magnitude more sensitive than most other bacterial species. Much is known about the mechanism of INH activation, including that fact that it is a prodrug requiring activation by the catalase-peroxidase hemoprotein, KatG (13,18,19), in a process that requires molecular oxygen (49). It has been further shown that the activated form of INH forms a covalent adduct with NAD ϩ to generate a potent inhibitor of the InhA protein of M. smegmatis, an enoyl-acyl carrier protein reductase important in mycolic acid biosynthesis (26,30,40). Recent investigations have led to the hypothesis that the sensitivity of M. tuberculosis to INH may be governed by the level of expression of the alkyl hydroperoxide reductase, AhpC, since M. tuberculosis does not express the ahpC gene due to inactivation of the oxidative stress regulatory gene oxyR (5, 6, 32, 38, 52).Nevertheless, specific details regarding the mechanism of action of INH, such as the chemical nature of the activated form of INH, have yet to be determined. For example, InhA as the primary target for INH in M. tuberculosis has been questioned following the discovery of a covalent complex of INH, acyl carrier protein (AcpM), and the ␤-ketoacyl acyl carrier protein...

In vitro synergic effect of ²-lapachone and isoniazid on the growth of Mycobacterium fortuitum and Mycobacterium smegmatis

“…Quinones generate reactive oxygen species that can damage lipids, proteins and deoxyribonucleic acids. Published works have postulated that the activity of β-lapachone and other quinones against bacteria and Trypanosoma cruzi is due to superoxide and hydrogen peroxide formation (Cruz et al 1978, Bulatovic et al 2002, Salas et al 2008.…”

“…These reactive by-products are important in the action mechanism of isoniazid. Bulatovic et al (2002) described the synergistic activity of plumbagin (a naturally occurring naphthoquinone) with isoniazid against M. tuberculosis and M. smegmatis. This synergic effect was easily prevented by the overexpression of superoxide-dismutase.…”

“…High levels of resistance may be acquired with the loss of catalase-peroxidase activity and the overproduction of superoxide dismutase (Mdluli et al 1998, Bulatovic et al 2002, Gupta et al 2006.…”

The Susceptibility of katG Ser315Thr Mycobacterium tuberculosis mutant Against Anti Tuberculosis Drugs

“…Hazbon suggested that resistance to INH and evolution of MDR strains is very complex and dynamic, involving various genes interaction and anti TB drug resistace phenotype ( Hazbon et al,2006;Bulatovic et al, 2002;Van Solingen et al, 2000). More extensive studies are needed to be able to explain mechanism of resistance to anti TB drugs.…”

“…Minimal inhibitory concentration of INH resistant isolates increase 20-200 times. With position of cytochrome oxidase super family peroxidase-catalase, it is suggested that Ser315 is close to active site of KatG so that Ser315Thr mutation infl uence enzyme activity of KatG (Heym et al, 1998;Bulatovic et al, 2002;Van Solingen et al, 2000.;Luo et al, 2013).…”