Abstract
Abstract
The pathogenicity of
Mycobacterium tuberculosis
relies on its ability to withstand hostile host environments. Coenzyme F
420
is critical for redox metabolism in
Mycobacterium tuberculosis
, an organism distinguished by its abundant use of this low-redox-potential cofactor and a repertoire of F
420
-dependent enzymes. While F
420
-dependent mechanisms are known to confer protection against antimicrobials and oxidative stress, the specific enzymes responsible remain largely unidentified. In this study, using the F
420
-dependent detoxification of malachite green as a phenotypic marker, we performed a genome-wide screen in
Mycobacterium tuberculosis
using transposon mutagenesis and whole genome sequencing. The results of transposon mutagenesis and whole genome sequencing revealed that, among the 28 genes encoding F
420
-dependent enzymes, only mutations in
fgd
(essential for F
420
H
2
re-generation) and
Rv2061c
, a gene encoding a putative F
420
-dependent oxidoreductase, were highly susceptible to malachite green. Subsequent experiments confirmed that Rv2061c and its homolog MT2120 are required for detoxifying malachite green and crystal violet. Furthermore, the
Rv2061c
mutant exhibited increased susceptibility to isoniazid and pretomanid; as well as menadione-induced oxidative stress. We also identified mutations in the transcriptional repressor Rv0678 as a key determinant of resistance to malachite green and crystal violet. This study establishes Rv2061c as a pivotal F
420
-dependent oxidoreductase that protects
Mycobacterium tuberculosis
from malachite green and other antimicrobial compounds as well as oxidative stress and further supports Rv0678 as a master regulator of multidrug resistance. These findings expand our knowledge of
Mycobacterium tuberculosis
defense mechanisms and highlight F
420
-dependent pathways as a potential target for new drug development.