A novel F₄₂₀‐dependent anti‐oxidant mechanism protects Mycobacterium tuberculosis against oxidative stress and bactericidal agents

Abstract

Mycobacterium tuberculosis (Mtb) is an aerobic bacterium that persists intracellularly in host macrophages and has evolved diverse mechanisms to combat and survive oxidative stress. Here we show a novel F420-dependent anti-oxidant mechanism that protects Mtb against oxidative stress. Inactivation of the fbiC gene in Mtb results in a cofactor F420-deficient mutant that is hypersensitive to oxidative stress and exhibits a reduction in NADH/NAD+ ratios upon treatment with menadione. In agreement with the recent hypothesis on oxidative stress being an important component of the pathway resulting in cell death by bactericidal agents, F420− mutants are hypersensitive to mycobactericidal agents such as isoniazid, moxifloxacin and clofazimine that elevate oxidative stress. The Mtb deazaflavin-dependent nitroreductase (Ddn) and its two homologues Rv1261c and Rv1558 encode for an F420H2-dependent quinone reductase (Fqr) function leading to dihydroquinones. We hypothesize that Fqr proteins catalyse an F420H2-specific obligate two-electron reduction of endogenous quinones, thereby competing with the one-electron reduction pathway and preventing the formation of harmful cytotoxic semiquinones, thus protecting mycobacteria against oxidative stress and bactericidal agents. These findings open up an avenue for the inhibition of the F420 biosynthesis pathway or Fqr-class proteins as a mechanism to potentiate the action of bactericidal agents.

The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria

“…F 420 is known to serve as a redox cofactor in an increasing number of endogenous processes in mycobacteria, including central carbon metabolism (Bashiri et al, 2008), cell wall modification (Purwantini and Mukhopadhyay, 2013), antioxidant production (Ahmed et al, 2015) and possibly quinone reduction (Gurumurthy et al, 2013). Although the cofactor is synthesized in high levels under oxic conditions (Figure 2), phenotypic evidence suggests it is particularly important for survival under hypoxia (Gurumurthy et al, 2013). In this condition, mycobacteria may increasingly depend on low-potential cofactors such as F 420 to maintain redox homeostasis Cook et al, 2014).…”

Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions

“…The observation that F 420 is synthesized even in M. leprae, rendered an unculturable, host-dependent organism through massive genome decay (362), suggests that it has an evolutionarily conserved central role in mycobacterial metabolism. In contrast to methanogens, F 420 is not essential for the viability of mycobacteria under ideal conditions: F 420 biosynthesis (fbiC) and reduction (fgd) genes have been successfully deleted or disrupted in M. smegmatis (28,31,132,363), M. tuberculosis (32,35), and M. bovis (72). However, there is a range of evidence that F 420 contributes to the notorious ability of mycobacteria to persist in deprived and challenging environments (56).…”

“…Partly due to its low redox potential, the F 420 H 2 produced is capable of reducing a wide range of organic compounds otherwise recalcitrant to activation as discussed in section 4 (28,54,55). Recent work also indicates that F 420 may be utilized in aerobic bacteria in hypoxic and anoxic environments, potentially substituting for high-potential nicotinamide cofactors (NAD and NADP) (Ϫ320 mV) (30,32,56).…”

“…While cofactors with properties corresponding to F 420 were isolated in mycobacteria and streptomycetes in 1960 (23,24), it was not until decades later that the cofactor was formally identified in these genera (25)(26)(27). As discussed throughout section 4, F 420 is implicated in the catabolic, biosynthetic, and detoxification pathways of both saprophytic actinobacteria (28)(29)(30) and their pathogenic descendants (e.g., Mycobacterium tuberculosis) (31,32). Interest in F 420 metabolism has surged following the discovery that the recently clinically approved antimycobacterial prodrug delamanid is activated by a specific F 420 H 2 -dependent reductase (33)(34)(35)(36).…”

Antioxidants: Friend or foe for tuberculosis patients

“…In case of atypical Mycobacterium species bacterial antioxidants are thought to modulate host derived oxidative killing mechanisms [19]. In Mycobacterium species new antioxidant mechanisms that protect the bacterium from the phagosomal respiratory burst is an established phenomenon [20,21]. Even recombinant BCG over-expressed with superoxide dismutase A confers less protection for development of tuberculosis [22].…”