Mycofactocin is critical for Mycobacterium tuberculosis adaptation into microaerophilic conditions

Mycofactocin is a new class of peptide-derived redox cofactor whose structural elucidation and functional characterization have recently received enormous focus. Among the six-gene cluster, mftD mediates a probable penultimate step in mycofactocin biosynthesis. MftD is a putative lactate dehydrogenase predicted critical for Mycobacterium tuberculosis survival under oxygen-limited conditions. In this study, mftD transcripts levels were found significantly increased in M. tuberculosis cells adapted to 0.01% oxygen. mftD deletion mutant of M. tuberculosis exhibited survival deficit in in vitro hypoxia and wayne models. However, mftD functions was found dispensable for M. tuberculosis L-Lactate metabolism. Rather surprisingly, the growth fitness of mftD mutant was increased in glucose under aerobic conditions. While the cause of this in vitro phenotype remains unestablished, the levels of NAD(P)H and glucose-6-phosphate dehydrogenase activity was found decreased in ΔmftD when compared to its parental strain. Increased growth fitness did not have any major impact on bacterial cell shape and size except the formation of extracellular fibril-like structures in subpopulations of ΔmftD. Cell-surface proteins analysis showed that genetic deficiency of mycofactocin likely to predispose accumulation of cofactor-free protein aggregates resembling destabilization of the flavoproteome upon riboflavin deprivation. Nevertheless, like in vitro findings, disruption of mftD increased the fitness of M. tuberculosis in C57BL/6J mice at early phase and resulted in growth stasis in a Nos2-/- hypoxia mouse model. Collectively these results establish the relevance of MftD in M. tuberculosis growth, redox and cofactor homeostasis, and pathogenesis.