Message in a vesicle: Cross-kingdom communication between uropathogenic Escherichia coli and epithelial bladder cells via secreted bacterial membrane vesicles and their RNA cargo

Abstract

The rise in antibiotic resistance has highlighted the need to identify previously-unexploited mediators of the infection process. Urinary tract infections (UTIs) are among the most common infectious diseases with a significant contribution to antibiotic use and resistance; uropathogenic Escherichia coli is the predominant UTI pathogen. The identification of novel aspects of UPEC biology that might suggest novel antimicrobials are therefore especially relevant. The aim of this thesis was to investigate if UPEC uses secreted membrane vesicles (MVs) to deliver RNA to human bladder epithelial cells to modify host cell gene expression and aid the pathogen's survival and colonisation of the human host. In the first part of the study, UPEC 536 cultured in the physiologically relevant culture medium RPMI 1640 were demonstrated to release MVs that contain RNA. MV RNA was isolated from large volume UPEC 536 cultures to enable sequencing of the RNA cargo of MVs. MVs and MV RNA isolated were used in functional studies of MV interactions with cells of the human bladder epithelial cell line 5637. Using microscopy techniques, UPEC 536 MVs, and their RNA cargo, were demonstrated to be transferred into bladder cells and MV RNA localised to the cytosol and the nucleus of 5637 cells. During this work, the need to image MV RNA molecules inside host cells with high resolution led to the development of a novel technique for labelling MV RNA with gold nanoparticles, which enabled visualisation of MV RNA with transmission electron microscopy. The second part of the study was dedicated to investigating phenotypic changes induced in the urothelial cells by whole MVs and MV RNA. It was determined that incubation with whole MVs, and transfection of RNA isolated from MVs, can negatively affect bladder cell viability. Investigation of the transcriptional response of 5637 cells identified that genes involved in urothelial cell survival, proliferation and renewal are differentially expressed in response to the intracellular delivery of MVs and their RNA cargo. A microarray-based transcriptional analysis demonstrated that some of the key genes differentially expressed in bladder cells in response to MV RNA are chemokines responsible for neutrophil recruitment to the bladder. In parallel, it was determined that the transcriptional signature of MV-treated bladder cells is dominated by the strong up-regulation of the aryl hydrocarbon receptor (AhR) and pathways involved in synthesis of AhR ligands from tryptophan and lipids. AhR is a ligand-controlled transcription factor with a known role in inducing impairment in neutrophils recruited to the bladder during UPEC-mediated UTIs. Overall, this work provided evidence that interactions of RNA-carrying UPEC 536 MVs with cells of the urothelium may result in inhibition of antibacterial neutrophil responses in the bladder. The final part of this thesis demonstrates that MV RNA co-isolates with bacterial lipopolysaccharide (LPS) and provides evidence for both LPS-independent effects of MV RNA on 5637 cell phenotype, and for a functional association between RNA and LPS. In summary, the body of work presented in this thesis provides evidence that MV RNA is a UPEC virulence factor and identifies possible mechanism and functional consequences of interactions of RNA-carrying MVs with urothelium in the establishment of UTIs.