Disruption of riboflavin biosynthesis in mycobacteria establishes 5-amino-6-D-ribitylaminouracil (5-A-RU) as key precursor of MAIT cell agonists
Melissa Dalcina ChengalroyeniD, Nurudeen OketadeiD, Aneta H. WorleyiD, Megan Lucas, Luisa María Nieto RamíreziD, Mabule L. Raphela, Gwendolyn M. SwarbrickiD, Paul S. SomaiD, Mandisa Zuma, Digby Francis WarneriD, Deborah A. LewinsohniD, Carolina MehaffyiD, Erin J. AdamsiD, William H. HildebrandiD, Karen M. DobosiD, Valerie MizrahiiD, David Michael LewinsohniD
Abstract
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.
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Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (5 Oct 2024) · CC BY 4.0
Deleting early riboflavin genes (ribA2 in both species, ribG in M. smegmatis) sharply reduced MAIT cell activation, while ribH deletion raised recognition of M. tuberculosis only.
Riboflavin-pathway ligands drive MAIT cell activation, but residual gene expression in CRISPRi hypomorphs blurs which intermediate matters in mycobacteria. Clean deletions in M. smegmatis and M. tuberculosis let the roles of 5-A-RU, DMRL and riboflavin be separated.
Key results
ShownIn M. smegmatis, deleting ribA2 or ribG, which blocks 5-A-RU production, strongly reduced IFN-γ responses of all four MR1-restricted T cell clones, including the TRAV1-2-negative clone. Complementation reversed this.
ELISPOT with dendritic cells at MOI 10: 82-99% reduction in recognition for all clones tested. Representative of n=3 independent experiments; one-way ANOVA with Sidak's test. · Deletion of ribA2 or ribG selectively ablates…; Figure legends
ShownIn M. tuberculosis, ribA2 deletion similarly cut recognition by all three MAIT clones, but some activation remained. This points to minor agonists that do not come from the riboflavin pathway.
87-97% reduction in recognition by the three clones, restored by complementation. Responses normalized to the HLA-restricted Mtb8.4-specific CD8 clone D454 H1-2; MOI 10; n=3 independent experiments. · Opposite effects of ribA2 vs. ribH deletion on…; Discussion; Figure legends
ShownDeleting ribH (lumazine synthase) increased MAIT clone recognition of M. tuberculosis. The M. smegmatis ΔribH2ΔribH1 mutant did not show this; TRAV1-2+ clones recognized it like wildtype.
M. tuberculosis ΔribH: 3-4-fold higher recognition by D426-G11, D481-C7 and D481-F12. M. smegmatis ΔribH2ΔribH1: no effect on the three TRAV1-2+ clones; the D520-E10 response fell but was not rescued by complementation. · Deletion of ribA2 or ribG selectively ablates…; Opposite effects of ribA2 vs. ribH deletion on…; Discussion
ShownDeleting ribC (riboflavin synthase, last pathway step) had no discernible effect on MR1T activation by either species. So enzymatic riboflavin production is not needed for agonist generation.
ELISPOT: no significant impact of ΔribC on any clone for M. smegmatis (Fig 5) or M. tuberculosis (Fig 6), tested against wildtype by one-way ANOVA with Sidak's test. · Deletion of ribA2 or ribG selectively ablates…; Opposite effects of ribA2 vs. ribH deletion on…; Discussion
ShownKnockouts and complements were validated by growth, transcript and protein measurement, showing riboflavin auxotrophy for the key mutants and some cross-regulation among pathway genes, such as higher ribH2 in the M. smegmatis ΔribH1 mutant.
M. smegmatis ΔribH1: 3-fold higher ribH2 transcript and protein. M. tuberculosis ΔribA2 and its complement: ribH transcript and protein 12-to-16-fold above wildtype. ΔribA2, ΔribH and ΔribC of M. tuberculosis were auxotrophic. · Construction of deletion mutants in the…
Methods, data and reproducibility
Gene deletions (allelic exchange or ORBIT) and complemented strains of riboflavin pathway genes in M. smegmatis and M. tuberculosis were checked by growth, qRT-PCR, DIA/PRM proteomics and sequencing. MR1-restricted T cell clone IFN-γ ELISPOT responses were then compared with wildtype.
- M. smegmatis mc^2155 deletion and complemented strains (ΔribA2, ΔribG, ΔribH1, ΔribH2, ΔribH2ΔribH1, ΔribC, ΔfbiC) · Mycobacterium smegmatis · n = two biological replicates (qRT-PCR, DIA proteomics)
- M. tuberculosis H37RvMA deletion and complemented strains (ΔribA2, ΔribH, ΔribC) · Mycobacterium tuberculosis · n = three biological replicates (qRT-PCR, proteomics)
- MR1-restricted T cell clones: D426-G11, D481-C7, D481-F12 (TRAV1-2+ MAIT) and D520-E10 (TRAV19-1) · human · n = four MR1T clones; ELISPOT representative of n=3 independent experiments
- HLA-B*15:01-restricted CD8 clone D454 H1-2 (Mtb8.4-specific), used to normalize M. tuberculosis ELISPOT responses · human · n = one clone
- Whole genome sequencing of knockout and complemented strains, aligned to the M. tuberculosis H37Rv and M. smegmatis mc^2155 reference genomes · Mycobacterium tuberculosis; Mycobacterium smegmatis · NC_000962.3 (Mtb H37Rv reference); CP000480.1 / NZ_CP054795.1 (Msm mc^2155 reference)
- Methods & tools
- ORBIT recombineering, Allelic exchange (p2NIL/pGOAL vectors), qRT-PCR (QuantStudio 3, sigA-normalized ΔΔCt), DIA-PASEF on timsTOF Pro, FragPipe v. 19.1 (MSFragger 3.7, IonQuant 1.8.10, Philosopher 4.8.1), Mycobrowser Release 4 protein database, Skyline PRM-PASEF with heavy-labeled peptides, IFN-γ ELISPOT, NovaSeq 6000 WGS; Bowtie2, samtools, lofreq, GraphPad Prism 10 (one-way ANOVA; Sidak, Dunnett)
- Reagents & environment
- FragPipe v. 19.1 (MSFragger 3.7, IonQuant 1.8.10, Philosopher 4.8.1); Mycobrowser Release 4 FASTA; GraphPad Prism 10 (Prism 9 also cited).
- Reproducibility gaps
- No code repository or public data deposit (raw proteomics, WGS reads) is given. Processed proteomics and SNP results are in supplementary tables (S6 Table, S9 Tables).
Limitations (5)
- MAIT agonist/antagonist balance could only be inferred indirectly from T cell readouts of wildtype vs. mutant strains grown in vitro with riboflavin; ligand levels were not measured directly. · stated by the authors
- ribH1 and ribH2 complementation was incomplete in M. smegmatis (growth not restored), so the reduced D520-E10 response in those mutants could not be cleanly assigned to loss of lumazine synthase. · stated by the authors
- The M. tuberculosis ΔribA2 mutant and its complement carry a 12-to-16-fold RibH increase, likely from a vector-borne promoter. This confounds ΔribA2 phenotypes given that ribH loss itself raises recognition. · noted in this summary
- M. smegmatis qRT-PCR and DIA proteomics rest on two biological replicates; with ELISPOT shown as representative of n=3 experiments, statistical power for small differences is limited. · noted in this summary
- Residual MAIT activation by ΔribA2 and ΔribG mutants implies unidentified antigens independent of 5-A-RU, which this study did not identify. · stated by the authors
Who should read:Read this if you study MAIT or MR1-restricted T cell antigens in mycobacteria and want genetic evidence on which riboflavin pathway steps drive T cell activation.
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