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 introduces flexibility at defined uridine positions in the D-loop. While structural and biochemical studies in Escherichia coli have clarified Dus enzymes substrate specificities, their functional relevance in other bacterial pathogens remains uncharacterized. Here, we present the first functional characterization of the contribution of Dus enzymes to an important urinary tract pathogen, Proteus mirabilis. Insertional 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 culture conditions, although loss of dusB caused a fitness defect during growth in human urine. Disruption of dusB also resulted in increased biofilm biomass, impaired swimming motility, enhanced outer membrane permeability, and increased susceptibility to detergents and certain 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 also critical for fitness in all organs during co-challenge against wild-type as well as overall colonization and ascending infection 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 tRNA modification to virulence.