Staphylococcus aureus membrane vesicles contain immunostimulatory DNA, RNA and peptidoglycan that activate innate immune receptors and induce autophagy

Abstract

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.

Extracellular vesicles derived from the periodontal pathogen Filifactor alocis induce systemic bone loss through Toll‐like receptor 2

“…NTA tends to measure biological EVs larger than that of TEM (Bachurski et al, 2019). The size of F. alocis EVs was similar to that of EVs derived from other Gram-positive bacteria (Bitto et al, 2021;Kim, Lee et al, 2020b). No colonies were observed after plating F. alocis EVs on Columbia agar (data not shown).…”

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

“…BEVs (~20 nm to 400 nm) secreted by Gram-positive bacteria also contain sRNAs (<100 nt, with most in the range of 10 to 40 nt) [ 10 , 19 , 20 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ]. Although recent studies have shown that sRNAs in BEVs affect host cell biology, to date no studies have identified specific sRNAs that mediate the host response to BEVs.…”

“…Staphylococcus aureus secretes sRNA and DNA in BEVs that have been implicated in inducing IFN-β mRNA by activation of Toll-like receptors in mouse macrophages [ 41 ]. S. aureus secrete BEVs that contain immunostimulatory DNA, RNA and peptidoglycan that activate innate immune receptors and induce autophagy in host A549 epithelial cells [ 40 ]. Although Mycobacterium tuberculosis (M.tb) is not considered a Gram-positive or Gram-negative bacterium, BEVs secreted by M.tb contain RNAs that promote IFN-b secretion upon entry into RAW 264.7 macrophages, which leads to M.tb clearance via activation of the macrophage RIG-I/MAVS signaling pathway [ 61 ].…”

“…The role of O-IMV in interdomain signaling is not clear [ 38 , 39 ]. Gram-positive bacteria also secrete EVs, but since Gram-positive bacteria do not contain an outer membrane, these EVs are often described as bacterial extracellular vesicles (bEV or BEV) [ 10 , 19 , 20 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ]. In this review, the terms EVs, OMVs and BEVs will be used to describe EVs secreted by non-microbial cells, Gram-negative bacteria, and Gram-positive bacteria, respectively.…”

Proteome analysis of the Gram-positive fish pathogen Renibacterium salmoninarum reveals putative role of membrane vesicles in virulence

“…While the mechanism behind the formation of MVs in Gram-positive bacteria is not fully understood, proteins involved in cell wall modification, like transpeptidases and autolysins are hypothesized to play an important role in the formation process, as these proteins have been detected in MVs released from Gram-positive bacteria [20][21][22] . Other contents of MVs include lipopolysaccharides 23 , peptidoglycan 24 , nucleic acids 24 , toxins 25 and proteins 26,27 . Some of the proteins identified in MVs were shown to be relatively enriched within the MVs when compared to their parent bacterium, suggesting a selective packing of proteins into the MVs 26,27 .…”