Oral Presentation BacPath 13: Molecular Analysis of Bacterial Pathogens Conference 2015

Bacterial outer membrane vesicles migrate to early endosomes, resulting in NOD1-dependent autophagy and inflammation (#21)

Aaron Irving 1 , Hitomi Mimuro 2 , Thomas Kufer 3 , Camden Lo 4 , Chihiro Sasakawa 2 , Richard Ferrero 1 , Maria Liaskos 1
  1. Hudson Institute of Medical Research, Clayton, Victoria, Australia
  2. Institute of Medical Science, University of Tokyo, Tokyo, Japan
  3. Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany
  4. Monash Micro Imaging, Victoria

A common feature of Gram negative bacteria is that they naturally shed extracellular outer membrane vesicles (OMVs) as part of their normal growth process. We previously reported that upon entry into host epithelial cells, peptidoglycan-containing OMVs are detected by the cytosolic host pathogen recognition molecule nucleotide oligomerization domain 1 (NOD1), resulting in the development of OMV-specific innate and humoral immune responses. In this study, we aimed to elucidate the mechanisms of OMV recognition by NOD1, and their subsequent intracellular fate, in order to further understand their role in bacterial pathogenesis.

Using confocal microscopy and live cell imaging, we determined that OMVs induced autophagosome formation in human epithelial cells. Using siRNA NOD1 knockdown or NOD1 knockout cells, we showed that NOD1 was essential for the induction of OMV-induced autophagy and inflammatory IL-8 responses in epithelial cells. Fluorescent labelling of bacterial OMVs revealed that once intracellular, OMVs migrated to early endosomes where they interacted with NOD1 and the NOD1-adaptor protein RIP-2, facilitating the development of an inflammatory response from this location. Using NOD1 knockout cells, we showed that migration of OMVs to early endosomes occurred in a NOD1 dependent manner. Most importantly, using fluorescent lifetime imaging microscopy (FLIM)-fluorescence energy transfer (FRET), we were able to show for the first time the direct interaction between peptidoglycan contained within OMVs and NOD1 within host cells.

Collectively, these data reveal the intracellular location and the early recognition events required for the detection of Gram negative bacterial OMVs by the intracellular host pathogen receptor NOD1. Moreover, this study is the first to visualize a direct interaction between NOD1 and bacterial peptidoglycan. These findings will significantly expand our limited knowledge of the contribution of OMVs and NOD1 in Gram negative bacterial pathogenesis, innate immunity and inflammatory disorders.