Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2014

Identification of the intracellular location and mechanisms of peptidoglycan recognition by the immune receptor NOD1. (#105)

Aaron T Irving 1 , Hitomi Mimuro 2 , Thomas A Kufer 3 , Camden Lo 4 , Richard Wheeler 5 , Lorinda Turner 1 , Belinda Thomas 1 , John Bertin 6 , Ivo G Boneca 5 , Chihiro Sasakawa 2 , Dana J Philpott 7 , Richard L Ferrero 1 , Maria Kaparakis-Liaskos 1
  1. MIMR-PHI Institute of Medical Research, Clayton, VIC, Australia
  2. Division of Bacteriology, The University of Tokyo, Tokyo, Japan
  3. Institute for Medical Microbiology, Immunology and Hygiene , University of Cologne, Cologne, Germany
  4. Monash Micro Imaging, Victoria
  5. Institut Pasteur, Paris, France
  6. GlaxoSmithKline, Collegeville, USA
  7. Department of Immunology, University of Toronto, Toronto, Canada

Nucleotide oligomerization domain 1 (NOD1) is an intracellular host receptor that senses microbial pathogens by detecting a conserved structure of Gram negative bacterial peptidoglycan (PG). Detection of peptidoglycan by NOD1 ultimately results in the establishment of an inflammatory response in the host. However, to date, the intracellular location and the mechanisms whereby NOD1 detects peptidoglycan resulting in the development of inflammatory responses, including autophagy are unknown. In this study, we used peptidoglycan-containing bacterial outer membrane vesicles (OMVs) as a tool to expand our limited knowledge regarding the intracellular location of NOD1 and the mechanisms of NOD1-dependent responses.

Our findings showed that upon entry into host epithelial cells, peptidoglycan-containing OMVs (PG-OMVs) from multiple pathogens induced NOD1-dependent autophagy and inflammatory IL-8 responses. Fluorescent labelling of peptidoglycan contained within bacterial OMVs revealed that upon entry into host cells, peptidoglycan migrated to early endosomes where it 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 PG-OMVs to early endosomes occurred in a NOD1 dependent manner, identifying a previously unknown role for NOD1 in the intracellular migration of peptidoglycan. 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 bacterial peptidoglycan and NOD1 within host cells.

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