Introduction: Uropathogenic E. coli (UPEC) has been reported in literature to use secreted membrane vesicles (MVs) to deliver protein toxins into target human cells. We investigated if UPEC vesicles carry RNA which can be transferred to bladder cells to exert a phenotypic effect.
Methods: MVs were isolated from concentrated UPEC 536 culture media by ultracentrifugation. MV membranes were labelled with a fluorescent dye PKH67 and incubated with CellTracker™ labelled bladder cells. Confocal microscopy was then used to demonstrate vesicle uptake into the bladder cells. To demonstrate the transfer of RNA into the human cells MV RNA was metabolically labelled with 5-ethynyl uridine and then tagged with Alexa Fluor 488 azide or biotin azide using Click chemistry. Confocal microscopy was used to visualise Alexa Fluor 488 labelled vesicle RNA inside bladder cells whilst biotin-labelled RNA was further labelled with streptavidin-nanogold for visualisation with electron microscopy. Changes in bladder cells in response to RNA extracted from vesicles were investigated with functional phenotypic assays.
Results: Imaging studies showed that vesicles and their RNA cargo are delivered into bladder cells. Digital RT-PCR demonstrated the transfer of specific bacterial RNA species. Phenotypic assays showed that bladder cells treated with whole vesicles and vesicle RNA extracts have reduced viability compared to control cells.
Conclusions: Our data has shown for the first time that a human pathogen UPEC 536 secretes membrane vesicles that carry RNA. Importantly, vesicle associated bacterial RNA was shown to be delivered into cultured human bladder cells. Reduced viability of bladder cells in response to RNA extracts from UPEC MVs suggests that bacterial RNA could function as a virulence factor in human cells.