Modulation of riboflavin biosynthesis enzymes in non-typhoidal Salmonella isolates differentially impacts human MAIT cell activation

Invasive non-typhoidal Salmonella (iNTS) sequence type 313 (ST313) isolates have emerged in sub-Saharan Africa, with a high incidence of multidrug resistance, enhanced intracellular persistence, increased dissemination and immune evasion. ST313 isolate D23580 was shown to avoid activation of mucosal-associated invariant T (MAIT) cells compared with a genetically similar, non-invasive Salmonella Typhimurium ( S .Tm) 4/74 strain, but the mechanism remains unclear. Here, we studied immune responses to infection with typhoidal strains and ST313 isolates using comparative transcriptomic profiling of human peripheral blood mononuclear cells (PBMCs). MAIT cells responded to typhoidal strains but not to D23580. To uncover underlying mechanisms, we engineered an inducible CRISPRi system targeting riboflavin biosynthesis enzymes ( rib ), which process MAIT ligands, in D23580 and 4/74 strains. Several rib hypomorphs exhibited prolonged lag time in minimal media, which was more pronounced in D23580 than in 4/74. In PBMC infection, knockdown of lumazine synthase (RibH) and riboflavin synthase (RibE) resulted in contrasting MAIT cell activation phenotypes. RibH knockdown restored MAIT cell activation in D23580 but did not affect activation in 4/74. Conversely, RibE knockdown diminished MAIT cell activation by 4/74 but had no impact on MAIT cell activation by D23580. Metabolomic analysis revealed accumulation of the biosynthetic intermediate DMRL in RibE knockdown and a differential impact on riboflavin biosynthesis end products between D23580 and 4/74 RibH knockdown, suggesting altered pathway flux between the strains. Our results indicate RibH and RibE as pivotal nodes for strain-specific MAIT-activation during Salmonella infection of human PBMCs, illustrating teir central role in the complex interplay between bacterial metabolism and host immunity.