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

Natalie J Bitto,Andrew F Hill,Rishi Pathirana,Ivan K H Poon,Maria Kaparakis‐Liaskos,Timothy P Stinear,Lesley Cheng,Ella L Johnston,Thanh Kha Phan,Neil M O'Brien‐Simpson

doi:10.1002/jev2.12080

Natalie J Bitto, Andrew F Hill + Show 8 more

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https://doi.org/10.1002/jev2.12080

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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.

Highlights

Bacterial membrane vesicles (BMVs) are ubiquitously produced by bacteria throughout all stages of growth and are identified as a bona fide secretion system for the long-distance delivery of bacterial molecules that can affect functions in host-pathogen interactions and inter-bacterial communication (reviewed in (Zavan et al, 2020))
We identified that DNA, RNA and peptidoglycan associated with MVs were detected by Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain containing 2 protein (NOD2), resulting in nuclear factor-κB (NF-κB) activation and the production of pro-inflammatory cytokines and chemokines by epithelial cells
We identified that MVs produced by all three S. aureus strains contained protein, DNA, RNA and peptidoglycan, and thereby confirming that Gram-positive bacteria package peptidoglycan into MVs

Summary

Introduction

Bacterial membrane vesicles (BMVs) are ubiquitously produced by bacteria throughout all stages of growth and are identified as a bona fide secretion system for the long-distance delivery of bacterial molecules that can affect functions in host-pathogen interactions and inter-bacterial communication (reviewed in (Zavan et al, 2020)). Owing to the differences in the cell envelope of their parent bacteria, membrane vesicles produced by Gram-negative bacteria which possess an outer membrane are termed outer membrane vesicles (OMVs), while those produced by Gram-positive bacteria which lack an outer membrane are termed membrane vesicles (MVs) (Pathirana & Kaparakis-Liaskos, 2016). It was thought that the thick, rigid layer of peptidoglycan comprising the Gram-positive cell wall prohibited the production of MVs from Gram-positive bacteria (Lee et al, 2009). Evidence within the last decade demonstrates that MV production does occur and is broadly conserved among Gram-positive bacteria (Brown et al, 2015; Lee et al, 2009). In comparison to OMVs, our understanding of the biogenesis, composition and biological functions of MVs produced by Gram-positive bacteria is still in its infancy

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