Modulation of riboflavin biosynthesis and utilization in mycobacteria

Abstract

Riboflavin (vitamin B2) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including Mycobacterium tuberculosis (Mtb), can biosynthesize riboflavin de novo. Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize microbial metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism, physiology and MAIT cell recognition, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in Mycobacterium smegmatis (Msm) and Mtb by inducible CRISPR interference and analyzed the impact of gene silencing on viability, and on the levels of expression of riboflavin pathway genes, and also on the levels of the pathway proteins as well as riboflavin in Msm. Despite lacking a canonical transporter, we showed that both organisms can import and assimilate exogenous riboflavin when supplied at high concentration. We demonstrated functional redundancy in lumazine synthase activity in Msm and found that silencing of ribA2 or ribF was profoundly bactericidal in Mtb. In Msm, ribA2 silencing resulted in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and was also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, the collection of hypomorphs provides a useful resource for investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria.