Abstract
BackgroundBacterium-to-host signalling during infection is a complex process involving proteins, lipids and other diffusible signals that manipulate host cell biology for pathogen survival. Bacteria also release membrane vesicles (MV) that can carry a cargo of effector molecules directly into host cells. Supported by recent publications, we hypothesised that these MVs also associate with RNA, which may be directly involved in the modulation of the host response to infection.Methods and ResultsUsing the uropathogenic Escherichia coli (UPEC) strain 536, we have isolated MVs and found they carry a range of RNA species. Density gradient centrifugation further fractionated and characterised the MV preparation and confirmed that the isolated RNA was associated with the highest particle and protein containing fractions. Using a new approach, RNA-sequencing of libraries derived from three different ‘size’ RNA populations (<50nt, 50-200nt and 200nt+) isolated from MVs has enabled us to now report the first example of a complete bacterial MV-RNA profile. These data show that MVs carry rRNA, tRNAs, other small RNAs as well as full-length protein coding mRNAs. Confocal microscopy visualised the delivery of lipid labelled MVs into cultured bladder epithelial cells and showed their RNA cargo labelled with 5-EU (5-ethynyl uridine), was transported into the host cell cytoplasm and nucleus. MV RNA uptake by the cells was confirmed by droplet digital RT-PCR of csrC. It was estimated that 1% of MV RNA cargo is delivered into cultured cells.ConclusionsThese data add to the growing evidence of pathogenic bacterial MV being associated a wide range of RNAs. It further raises the plausibility for MV-RNA-mediated cross-kingdom communication whereby they influence host cell function during the infection process.
Highlights
Bacteria communicate to one another using diffusible signals [1] and to the host during the infection process using well-studied proteins, amino acids and lipids and small molecule signals [2]
Using the uropathogenic Escherichia coli (UPEC) strain 536, we have isolated membrane vesicles (MV) and found they carry a range of RNA species
Density gradient centrifugation further fractionated and characterised the MV preparation and confirmed that the isolated RNA was associated with the highest particle and protein containing fractions
Summary
Bacteria communicate to one another using diffusible signals [1] and to the host during the infection process using well-studied proteins, amino acids and lipids and small molecule signals [2]. This cross-kingdom signaling allows the bacterium to manipulate the host defense mechanisms to promote its survival. Other UPEC strains release another toxin, cytotoxic necrotizing factor type 1 (CNF1) in MVs, which targets the Rho GTPase signaling pathway to modulate the acute inflammatory response in host cells [6]. Bacterium-to-host signalling during infection is a complex process involving proteins, lipids and other diffusible signals that manipulate host cell biology for pathogen survival. We hypothesised that these MVs associate with RNA, which may be directly involved in the modulation of the host response to infection
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