Abstract / Summary
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.