Effect of the Extracellular Vesicle RNA Cargo From Uropathogenic Escherichia coli on Bladder Cells

Abstract

Iron restriction in mammals, part of innate antimicrobial defense, may be sensed as a signal by an infecting pathogen. Iron-dependent regulators not only activate the pathogen's specific iron acquisition and storage mechanisms needed for survival but also influence a number of other processes. Bacterial extracellular vesicles (EVs) are a conserved communication mechanism, which can have roles in host colonization, transfer of antimicrobial resistance, modulation of the host's immune response, and biofilm formation. Here we analyze the iron-responsive effect of RNA cargo from Escherichia coli EVs in bladder cells. No differences were found in total RNA quantified from EVs released from representative pathogenic and probiotic strains grown in different iron conditions; nevertheless, lipopolysaccharide (LPS) associated with purified RNA was 10 times greater from EVs derived from the pathogenic strain. The pathogen and probiotic EV-RNA have no substantial toxic effect on the viability of cultured bladder cells, regardless of the iron concentration during bacterial culture. Transcriptomic analysis of bladder cells treated with pathogen EV-RNA delivered in artificial liposomes revealed a gene expression profile with a strong similarity to that of cells treated with liposomes containing LPS alone, with the majority being immune response pathways. EV-RNA from the probiotic strain gave no significant perturbation of gene expression in bladder cells. Cytokine profiling showed that EV-LPS has a role modulating the immune response when internalized by bladder cells, highlighting a key factor that must be considered when evaluating functional studies of bacterial RNA.

The complex, bidirectional role of extracellular vesicles in infection

“…The cargo manifest of bEVs delivered to mammalian cells now goes beyond classical toxins and has identified the possibility of new virulence mechanisms involving small RNAs [22]. Moreover, the delivery of a cargo highlights that pathogenesis will likely result from the combined effects of these virulence factors [12,14,17], and the challenge may be to distinguish the roles of each when other factors are present [23]. EV production is often increased during infection, from both the host and pathogen.…”

“…Uropathogenic E. coli (UPEC) bEVs carry RNA and LPS that inhibit pro-inflammatory cytokine response in human uroepithelial cells [23] Francisella tularensis FtlA lipase carried by bEVs enhances internalisation in mouse macrophages [116] Helicobacter pylori bEVs contain VacA (vacuolating cytotoxin) is internalised by gastric epithelial cells, and may induce pathogenic effects different from that of soluble VacA Protect cells from extracellular ROS-mediated killing [11,117] [118]…”

“…Knowledge of the fate and effect of the delivered bEV cargo is incomplete. In particular, there is a need to better understand the effect of bEV-RNA as, while some studies show the induction of specific cytokines [ 32 , 63 ], others present evidence for the suppression of cytokine secretion by epithelial cells or lymphocytes [ 22 , 23 , 64 ].…”

Bacteroides fragilis outer membrane vesicles preferentially activate innate immune receptors compared to their parent bacteria

“…Interestingly, B. fragilis OMVs contained approximately ten-fold more RNA than DNA ( Figures 1D, E ), which is consistent with previous studies identifying that MVs derived from the Gram-positive commensals Lactobacillus reuteri ( 20 ) and Lactobacillus casei ( 18 ) also packaged significantly more RNA than DNA. While the RNA associated with pathogen-derived BMVs is becoming increasingly recognized as being able to activate innate immune receptors and to modulate cellular functions when delivered into host cells ( 37 , 51 ), knowledge regarding the immunostimulatory and immunomodulatory abilities of RNA delivered by commensal-derived BMVs is limited, highlighting that future research endeavors should focus on broadening our understanding of their functions.…”

Extracellular Vesicles and Host–Pathogen Interactions: A Review of Inter-Kingdom Signaling by Small Noncoding RNA

“…sRNAs secreted in OMVs by Aggregatibacter actinomycetemcomitans, a periodontic pathogen, can cross the blood–brain barrier, and sRNAs in the OMVs stimulate TNF-a production by activating the NF-κB signaling pathways in macrophages, promoting the secretion of pro-inflammatory cytokines in the brain [ 63 , 64 , 65 ]. sRNAs in OMVs secreted by Escherichia coli are transferred into bladder epithelial cells and suppress LPS-induced IL-1a secretion [ 66 ]. In E. coli OMVs, over 90% of sequence reads are mapped to tRNA fragments, which are differentially packaged [ 67 ].…”