Abstract

Human urine is non-sterile, harbouring its own microbiota (urobiota). Klebsiella species are commensals of the urobiota but can also cause urinary tract infections (UTIs). Gut-microbiota-host co-metabolites (MHCMs) such as hippurate are major components of urine. We previously showed UTI-associated strains of Klebsiella pneumoniae break down hippurate in human urine, though the enzymes responsible for this bioconversion are unknown. Here we sought to confirm that K. pneumoniae hydrolyses hippurate, and to determine whether the enzyme(s) responsible for this bioconversion are present and/or active in other Klebsiella species. Ninhydrin assays confirmed that strains of K. pneumoniae (n=3) and, albeit to a lesser extent, Klebsiella oxytoca (n=4) isolated from human urine could produce glycine from hippurate. Comparative protein (KEGG, UniProt, phylogenetic) and structural (AlphaFold, ChimeraX) analyses were used to predict putative hippurate hydrolases (HHs) encoded by Klebsiella species. Their gene products (n=2 K. pneumoniae; n=3 K. oxytoca) were cloned, expressed and purified. The proteins belonged to three distinct groups: only group 1 and group 2 HHs hydrolysed hippurate under conditions used in this study. Both HHs co-occurred with high prevalence in 15/20 (75 %) Klebsiella species (2885/3012 genomes, 95.8 %). Most Klebsiella species encode two distinct HHs that release glycine and benzoate from hippurate. The functional group 2 HH is predicted to facilitate delivery of amino acids such as glycine to K. pneumoniae cells in the nutrient-limited environment of urine, and its activity demonstrates that members of the urobiota and opportunistic pathogens can contribute to catabolism of MHCMs in human urine.