Disruption of riboflavin biosynthesis in mycobacteria establishes 5-amino-6-D-ribitylaminouracil (5-A-RU) as key precursor of MAIT cell agonists
Authors: Chengalroyen Melissa D. , Oketade Nurudeen , Worley Aneta , Lucas Megan , Ramirez Luisa Nieto , Raphela Mabule L. , Swarbrick Gwendolyn M. , Warner Digby F. , Lewinsohn Deborah A. , Mehaffy Carolina , Adams Erin J. , Hildebrand William , Dobos Karen , Mizrahi Valerie , Lewinsohn David M. 
Published: October 2024

AbstractMucosal-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 inMycobacterium smegmatis(Msm) andMycobacterium 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 offbiC). 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 byMycobacterium tuberculosis. In this study, we investigated the effect of deleting individual genes in the riboflavin biosynthesis pathway on MAIT cell activation byMycobacterium tuberculosisorMycobacterium 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 ofM. 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.