Bacterial membrane vesicles transport their DNA cargo into host cells

© 2017 The Author(s). Bacterial outer membrane vesicles (OMVs) are extracellular sacs containing biologically active products, such as proteins, cell wall components and toxins. OMVs are reported to contain DNA, however, little is known about the nature of this DNA, nor whether it can be transported into host cells. Our work demonstrates that chromosomal DNA is packaged into OMVs shed by bacteria during exponential phase. Most of this DNA was present on the external surfaces of OMVs, with smaller amounts located internally. The DNA within the internal compartments of Pseudomonas aeruginosa OMVs were consistently enriched in specific regions of the bacterial chromosome, encoding proteins involved in virulence, stress response, antibiotic resistance and metabolism. Furthermore, we demonstrated that OMVs carry DNA into eukaryotic cells, and this DNA was detectable by PCR in the nuclear fraction of cells. These findings suggest a role for OMV-associated DNA in bacterial-host cell interactions and have implications for OMV-based vaccines

Molecular interactions at the surface of extracellular vesicles

Extracellular vesicles such as exosomes, microvesicles, apoptotic bodies, and large oncosomes have been shown to participate in a wide variety of biological processes and are currently under intense investigation in many different fields of biomedicine. One of the key features of extracellular vesicles is that they have relatively large surface compared to their volume. Some extracellular vesicle surface molecules are shared with those of the plasma membrane of the releasing cell, while other molecules are characteristic for extracellular vesicular surfaces. Besides proteins, lipids, glycans, and nucleic acids are also players of extracellular vesicle surface interactions. Being secreted and present in high number in biological samples, collectively extracellular vesicles represent a uniquely large interactive surface area which can establish contacts both with cells and with molecules in the extracellular microenvironment. Here, we provide a brief overview of known components of the extracellular vesicle surface interactome and highlight some already established roles of the extracellular vesicle surface interactions in different biological processes in health and disease

Staphylococcus aureus

Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host

Interaction of FkpA, a peptidyl-prolyl cis/trans isomerase with EspP autotransporter protein

The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent a large class of proteases with contributions to virulence. They are synthesized with a C-terminal domain that forms a β-barrel pore in the outer membrane implicated in translocation of the N-terminal ‘passenger’ domain across the outer membrane. The most recent model for autotransporter secretion comprises entry to the periplasm via the Sec apparatus, followed by the insertion of the C-terminus into the outer membrane as a β-barrel protein and accompanied by translocation of the passenger domain to the bacterial cell surface, all of this with the assistance of the Bam complex insertase/foldase and periplasmic chaperone proteins. We have recently observed direct involvement of periplasmic chaperones in the biogenesis of EspP, a prototypical autotransporter protein produced by Escherichia coli O157:H7. Using molecular and biophysical approaches we demonstrated for the first time, direct protein-protein interactions between the periplasmic SurA and DegP chaperones and either the EspP-β or EspP passenger domains. Such chaperone interactions took place on conserved aromatic residues on the SPATE family. In this report, we now demonstrate direct binding of the periplasmic chaperone FkpA to the EspP passanger domain in Surface Plasmon Resonance experiments with relatively high affinity. We also provide evidence of interaction between the SurA and Skp chaperones with the Bam. These findings in conjunction with newly published data support the role of chaperones in preventing misfolding of AT passenger domains before translocation throughout the Bam complex