Biology subjects

Leo, J. C.

Publications and source records attributed to Leo, J. C..

3 recordsLinked to original sources

bioRxiv2026

Hippurate hydrolases encoded by Klebsiella pneumoniae and Klebsiella oxytoca are functional

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.

microbiology↗

bioRxiv2025

The Omp85 family protein, TamA, exhibits characteristics 1 of a suitable drug target against Pseudomonas aeruginosa

The outer membrane (OM) of Gram-negative bacteria is crucial for cell stability and virulence and acts as a permeability barrier. The biogenesis, assembly, and regulation of proteins in the OM are therefore attractive areas of study that could lead to identifying novel drug targets. The Translocation and Assembly Module (TAM), composed of TamA and TamB, facilitates the insertion of some {beta}-barrel proteins into the OM of Escherichia coli and Klebsiella pneumoniae, and has also been implicated in lipid homeostasis. However, its role in Pseudomonas aeruginosa remains mostly uncharacterized. To investigate the TAMs function and drug target potential in P. aeruginosa, we generated both single- and double-gene TAM knockouts and assessed their fitness using competition growth assays against wild-type (WT) strains. The WT significantly outcompeted the TAM mutants, indicating a fitness defect. Proteomic analysis revealed surprisingly similar profiles between WT and the double knockout strains, while single knockouts showed changes in OM proteins and reduced expression of flagellar components consistent with attenuated swimming motility observed in {Delta}tamA. Single mutants exhibited differential levels of expression of lipoproteins of the {beta}-barrel assembly machinery suggesting compensatory OM remodelling. In vivo infection assays using Galleria mellonella larvae demonstrated significantly higher survival rates when infected with TAM mutants, with tamA mutants showing the greatest attenuation in virulence. Our findings demonstrate a role the TAM plays in P. aeruginosa virulence and identify TamA as a potential drug target for the development of new antimicrobial therapies. Data summaryThe RNAseq data reported on in this article are available from ArrayExpress: E-MTAB-15348 The mass spectrometry proteomics data have been deposited to the ProteomeXchange with the dataset identifier PXD06704.

microbiology↗

bioRxiv2025

Formation of the β-sheet at the extracellular opening of the intimin β-barrel domain is necessary for stability and efficient passenger secretion

Attaching and effacing pathogens, such as enteropathogenic and enterohaemorrhagic E. coli, rely on an adhesin, intimin, for attachment to enterocytes and downstream effects leading to actin pedestal formation. Intimin belongs to the inverse autotransporter family (type 5e secretion systems), which secrete the extracellular adhesive domain or passenger via a hairpin intermediate. While many of the next steps in secretion are now understood, how the hairpin initially forms is not known. We sought to investigate this by making point mutations at several positions in the {beta}-barrel domain of intimin, as this domain forms at least part of the secretion pore. We made the mutations in a wild-type background and in a stalled intermediate caught in the hairpin conformation, which allowed us to uncouple passenger secretion from hairpin formation. Surprisingly, most of the point mutations did not have an appreciable effect on hairpin formation or passenger secretion, and larger changes such as replacing the entire linker region with a flexible glycine-serine stretch showed only a modest reduction in passenger secretion. By contrast, mutations affecting a small {beta}-sheet at the extracellular face of the intimin {beta}-barrel between two extracellular loops and the C-terminus of the linker had a more pronounced effect on secretion, and abolishing this {beta}-sheet prevented hairpin formation and led to complete loss of the wild-type protein. Our results show that the intimin {beta}-barrel is remarkably tolerant to changes and that the {beta}-sheet on the extracellular side of this domain plays a central role in passenger secretion and protein stability.

microbiology↗