Uropathogenic Escherichia coli (UPEC) is the most commonly known cause of urinary tract infection (UTI). The possession of many virulence factors and global dissemination of multi-drug resistant UPEC strains is posing a critical risk to treatment worldwide. The most virulent UPEC strains have descended from major lineages such as sequence type (ST) 131, ST38 and ST127. Strains of ST127 exhibit the highest virulence potential of most UPEC lineages, but little is known about their pathogenicity. Previous studies have reported the important role of metabolic adaptations in UPEC virulence. This study investigated the metabolic adaptations of UPEC isolates, with a focus on ST127, using a quantitative proteomics approach. We employed a classical shotgun proteomics approach to analyse proteins extracted from multiple strains of UPEC following growth in various environments (Lysogeny broth, artificial urine medium and co-culture with uroepithelial cells). The digested proteins and peptides from all fractions were separated on a Dionex Ultimate 3000 RSLC nano flow system and analysed in an Orbitrap Velos Pro FTMS. Data were processed using Perseus software. Expression and Gene Ontology Enrichment analysis revealed different proteomic profiles of UPEC ST127 strains cultured in LB and AUM. This study showed that environmental changes have an effect on the metabolic pathways expressed by a specific strain. These conditions can be engineered to simulate the variety of environments UPEC isolates may need to survive in. This methodology was then employed to enhance our understanding of the pathogenesis of UPEC. Building on previous work with UPEC paired isolates (urine and blood), proteomics analysis revealed that members of some STs have a higher metabolic potential that could provide virulence advantages. The study also revealed minimal proteomic variations among the paired isolates for some STs. The methods were then applied during co-culture experiments with UPEC and HT1197 bladder epithelial cells. UPEC strain SA189 caused exfoliation effects in HT1197 not seen with other UPEC strains. Analysis of the UPEC SA189 secretome revealed highly abundant bacterial proteins, including hemolysin A. Proteomic analysis is helping to understand the pathogenic potential of UPEC and may facilitate identification of novel diagnostic or therapeutic targets to reduce UTI and potential subsequent bacteraemia.

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