Bacterial outer membrane vesicles enter epithelial cells via endocytosis, inducing a NOD1-dependent autophagy response — ASN Events
  1. Schedule
  2. Division 4 – Microbial Genetics, Physiology and Pathogenesis
  3. Symposium 3 - Unique Aspects of the Cell Structures of Pathogenic Bacteria; Biology and Pathogenesis
  4. Bacterial outer membrane vesicles enter epithelial cells via endocytosis, inducing a NOD1-dependent autophagy response

Bacterial outer membrane vesicles enter epithelial cells via endocytosis, inducing a NOD1-dependent autophagy response (#63)

Aaron Irving 1 , Lorinda Turner , Hitomi Mimuro 2 , Chihiro Sasakawa 2 , Thomas Kufer 3 , Dana Philpott 4 , Richard Ferrero 5 , Maria Liaskos 5
  1. Centre for Cancer Research, Monash Institute of Medical Research, Monash University, Clayton, VIC, Australia
  2. Department of Microbiology and Immunology; Institute of Medical Science, University of Tokyo, Tokyo, Japan
  3. Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany
  4. Department of Immunology, University of Toronto, Toronto, Canada
  5. Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Clayton, VIC, Australia
A common feature of Gram negative bacteria is that they naturally shed extracellular outer membrane vesicles (OMVs) as part of their normal growth process. OMVs are spherical, bi-layered membrane nano-structures ranging from 20 to 300 nm in size, and their release occurs naturally both in vitro and in vivo. We recently reported that all Gram negative pathogens deliver peptidoglycan (PG) into non-phagocytic epithelial cells via a novel mechanism involving outer membrane vesicles (OMVs). We showed that upon entry into host cells, PG containing OMVs were detected by the cytosolic host pathogen recognition molecule nucleotide oligomerization domain 1 (NOD1), and that NOD1 was essential for the development of OMV-specific immune responses in vivo. The aim of the current work was to elucidate the mechanism(s) of OMV entry and processing in non-phagyocytic epithelial cells, to further understand their role in bacterial pathogenesis and the development of OMV-specific immune responses.In this study, we treated human epithelial cells with chemical inhibitors targeting specific components of the macropinocytosis and endocytosis pathways. We demonstrated that OMVs enter epithelial cells via macropinocytosis, clathrin- and caveolin-mediated endocytosis, and that their size dictates their route of entry.  We determined once internalized, bacterial OMVs induced aggregates of the autophagosome component LC3 in epithelial cells. LC3-GFP puncta did not form in OMV-stimulated macrophages, indicating that this response was cell-specific. Using siRNA NOD1 knockdown or NOD1 knockout cells, we showed that NOD1 was essential for the induction of OMV-induced autophagy. Furthermore, OMV-stimulation of LC3 or ATG5 knockdown cells resulted in reduced inflammatory responses when compared to control cells. Collectively, our findings suggest that OMVs enter epithelial cells in a size dependent manner, and that once intracellular NOD1 is essential for the induction of autophagy and inflammation. Expanding our understanding of the inflammatory nature of OMVs, their mechanisms of host cell entry and their degradation is fundamental to elucidating their role in disease.