Mehaffy, C. | Search bioRxiv

bioRxiv2024

Disruption of riboflavin biosynthesis in mycobacteria establishes 5-amino-6-D-ribitylaminouracil (5-A-RU) as key precursor of MAIT cell agonists

Mucosal-associated invariant T (MAIT) cells exhibit an intrinsic ability to recognize and respond to microbial infections. The semi-invariant antigen recognition receptor of MAIT cells specifically detects the non-polymorphic antigen-presenting molecule, major histocompatibility complex class I-related protein 1 (MR1), which primarily binds riboflavin-derived metabolites of microbial origin. To further interrogate the dependence of these antigens on riboflavin biosynthesis in mycobacteria, we deleted individual genes in the riboflavin biosynthesis pathways in Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb) and evaluated the impact thereof on MAIT cell activation. Blocking the early steps of the pathway by deletion of RibA2 or RibG profoundly reduced, but did not completely ablate, MAIT cell activation by Msm or Mtb, whereas deletion of RibC, which catalyzes the last step in the pathway, had no significant effect. Interestingly, deletion of RibH specifically enhanced MAIT cell recognition of Mtb whereas loss of lumazine synthase (RibH) activity had no impact on MAIT cell activation by Msm. MAIT cell activation by Msm was likewise unaffected by blocking the production of the MAIT cell antagonist, Fo (by inhibiting its conversion from the riboflavin pathway intermediate, 5-amino-6-D-ribitylaminouracil (5-A-RU), through the deletion of fbiC). Together, these results confirm a central role for 5-AR-U in generating mycobacterial MR1 ligands and reveal similarities and differences between Msm and Mtb in terms of the impact of riboflavin pathway disruption on MAIT cell activation. Author summaryMucosal-associated invariant T (MAIT) cells are an abundant population of innate-like T-cells which respond to microbial infections. These specialized cells recognize the MR1 molecule, which presents microbial metabolites derived from riboflavin (vitamin B2) biosynthesis. These cells are enriched in the airways and in some cases reduced in the peripheral blood of tuberculosis (TB) infected individuals suggestive of a role in the early response to infection by Mycobacterium tuberculosis. In this study, we investigated the effect of deleting individual genes in the riboflavin biosynthesis pathway on MAIT cell activation by Mycobacterium tuberculosis or Mycobacterium smegmatis. Our findings revealed that disrupting early stages in the pathway profoundly reduced but did not eliminate MAIT cell activation by both mycobacterial species. However, blocking the penultimate step in the pathway, catalyzed by the enzyme, lumazine synthase, led specifically to increased MAIT cell recognition of M. tuberculosis. Our results confirm the pivotal role of the riboflavin pathway intermediate, 5-A-RU, in generating mycobacterial ligands that serve as MAIT cell agonists. By enhancing our understanding of how MAIT cells recognize mycobacterial infections, the results of this study could inform strategies for the development of vaccines and/or immunotherapies for TB.

immunology↗

bioRxiv2023

Modulation of riboflavin biosynthesis and utilization in mycobacteria

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 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. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of ribA2 or ribF is profoundly bactericidal in Mtb; and (iv) in Msm, ribA2 silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria. IMPORTANCEThe pathway for biosynthesis and utilization of riboflavin, precursor of the essential coenzymes, FMN and FAD, is of particular interest in the flavin-rich pathogen, Mycobacterium tuberculosis (Mtb), for two important reasons: (i) the pathway includes potential tuberculosis (TB) drug targets; and (ii) intermediates from the riboflavin biosynthesis pathway provide ligands for mucosal associated invariant T (MAIT) cells, which have been implicated in TB pathogenesis. However, the riboflavin pathway is poorly understood in mycobacteria, which lack canonical mechanisms to transport this vitamin and to regulate flavin coenzyme homeostasis. By conditionally disrupting each step of the pathway and assessing the impact on mycobacterial viability and on the levels of the pathway proteins as well as riboflavin, our work provides genetic validation of the riboflavin pathway as a target for TB drug discovery and offers a resource for further exploring the association between riboflavin biosynthesis, MAIT cell activation and TB infection and disease.

molecular biology↗