Contribution of three tRNA modification enzymes to Proteus mirabilis fitness and catheter-associated urinary tract infection.

RNA modifications play essential roles in cellular physiology by modulating RNA structure, stability, and translation efficiency. Among these, dihydrouridine is a conserved tRNA modification synthesized by the flavin-dependent enzymes DusA, DusB, and DusC, which introduce flexibility at defined uridine positions in the D-loop. While structural and biochemical studies in Escherichia coli have clarified dihydrouridine synthase (Dus) enzyme substrate specificities, their functional relevance in other bacteria remains uncharacterized. Here, we present the first functional characterization of the contribution of Dus enzymes to an important urinary tract pathogen, Proteus mirabilis. Mutants were generated in dusA, dusB, and dusC in P. mirabilis strain HI4320 for evaluation of virulence-relevant phenotypes. All mutants grew similarly to wild type under multiple conditions, although loss of dusB caused a competitive fitness defect. Disruption of dusB also resulted in increased biofilm biomass, impaired swimming motility, enhanced outer membrane permeability, and increased susceptibility to detergents and antibiotics, while disruption of dusA or dusC only impacted biofilm formation and susceptibility to detergents. In a murine model of catheter-associated urinary tract infection, dusB was critical for fitness in all organs during co-challenge against wild type as well as overall colonization during independent challenge. In contrast, dusC did not contribute to fitness, and dusA showed a modest, compartment-specific contribution to fitness, as the dusA::Kan mutant was outcompeted in the bladder. Together, these findings identify DusB as a key regulator of motility, membrane integrity, and host fitness in P. mirabilis, linking Dus enzymes to virulence-associated phenotypes, potentially through effects on RNA modification and/or additional protein functions.