Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2021

Staphylococcus aureus membrane vesicles contain immunostimulatory DNA, RNA and peptidoglycan that activate innate immune receptors and induce autophagy. (#63)

Natalie J Bitto 1 2 , Lesley Cheng 2 3 , Ella L Johnston 1 2 , Rishi Pathirana 1 2 , Thanh Kha Phan 2 3 , Ivan K H Poon 2 3 , Neil M O'Brien-Simpson 4 , Andrew F Hill 2 3 , Tim P Stinear 5 , Maria Kaparakis Liaskos 1 2
  1. Department of Physiology, Anatomy and Microbiology, La Trobe University, Melbourne, Victoria, Australia
  2. Research Centre for Extracellular Vesicles, School of Molecular Sciences, La Trobe University, Melbourne, Victoria, Australia
  3. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia
  4. Centre for Oral Health Research, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Parkville, Victoria, Australia
  5. Department of Microbiology and Immunology, Doherty Institute, The University of Melbourne, Parkville, Victoria, Australia

Bacterial membrane vesicles (BMVs) are naturally secreted by all bacteria. The ability of outer membrane vesicles (OMVs) produced by Gram-negative bacteria to deliver immunogenic cargo to host epithelial cells to modulate immune responses and alter cellular processes is well characterised. In comparison, the functions of membrane vesicles (MVs) produced by Gram-positive bacteria remain poorly defined. Therefore, this study aimed to examine the composition, immunostimulatory functions and intracellular fate of MVs produced by Staphylococcus aureus.

 

To do this, MVs were isolated from three S. aureus strains that differ in virulence; two highly virulent bacteraemia isolates, and one poorly virulent laboratory-adapted strain. Electron microscopy and nanoparticle tracking analysis revealed differences in the amount and morphology of MVs produced by each strain. Examination of their cargo content showed that MVs contained peptidoglycan, protein, DNA and RNA including small RNA (sRNA), and that each strain varied in the amount and composition of these microbe-associated molecular patterns (MAMPs). The ability of S. aureus MVs to deliver ligands to pathogen recognition receptors (PRRs) was investigated using HEK-Blue reporter cell lines, demonstrating that their lipoprotein, nucleic acid and peptidoglycan cargo are delivered to Toll-like receptors (TLRs) 2, 7, 8, 9 and nucleotide binding oligomerization domain containing 2 (NOD2) and activate nuclear factor kappa B (NF-κB) and inflammatory cytokine and chemokine release by epithelial cells. Moreover, confocal microscopy showed that S. aureus MVs colocalise with autophagosomes in A549 lung epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta, revealing the ability of the host to degrade MVs via autophagy.

 

Collectively, these findings demonstrate that S. aureus MVs deliver immunostimulatory DNA, RNA and peptidoglycan into host epithelial cells to activate PRRs, cytokine and chemokine responses and facilitate their intracellular degradation via autophagy. These findings advance our understanding of the intracellular fate of Gram-positive bacterial MVs and their roles in mediating bacterial pathogenesis.