ARTICLES 702 VOLUME 4 NUMBER 7 JULY 2003 NATURE IMMUNOLOGY The innate immune system represents the first line of defense against invading pathogens. Animals and plants possess specialized receptors recognizing conserved molecules that are expressed exclusively by microorganisms or parasites 1–3 . These molecules, known as microbial pathogen–associated molecular patterns, help to establish the distinc- tion between pathogens and host cells. Among vertebrates and inver- tebrates, detection of microbes is mediated by specific host pattern-recognition receptors (PRRs) in intracellular compartments or on the cell surface 2,3 . For example, Toll-like receptors (TLRs) expressed on the surface of innate immune cells have an important role in the recognition of microbial pathogen–associated molecular patterns and activation of innate and adaptive immunity 1,3 . However, little is known about how the host cell can sense and respond to inter- nalized bacteria. Nucleotide-binding oligomerization domain (NOD) proteins, including NOD1 and NOD2, are members of an emerging family that have been implicated in intracellular recognition of bacterial compo- nents 2 . NODs show structural homology to a class of proteins (R pro- teins) that are encoded by plant disease-resistance genes 2 . Plant R proteins recognize distinct effector molecules from invading pathogens and mediate a defense response resulting in plant disease resistance 4 . NOD1, also called CARD4, is composed of an N-terminal caspase- recruitment domain (CARD), a centrally located NOD and multiple C- terminal leucine-rich repeats (LRRs). NOD1 is expressed in multiple tissues 5,6 . Initial studies showed that transient expression of NOD1 in mammalian cells induces NF-κB activation, an activity that is mediated through homophilic CARD-CARD interactions with RIP-like interact- ing CLARP kinase (RICK, also known as CARDIAK or RIP-2), a CARD-containing protein kinase interacting with the IκB kinase com- plex 5,6 . An important role for RICK in NOD1 signaling is supported by analysis of cells derived from mutant mice deficient in RICK 7 . Recent studies have revealed the muramyldipeptide MurNAc-L-Ala- D-isoGln (MDP), a moiety conserved in the cell wall peptidoglycan (PGN) of practically all bacteria, as the essential bacterial structure rec- ognized by NOD2 (refs. 8,9). NOD1 mediates responsiveness to vari- ous Gram-negative bacteria through its C-terminal LRRs 10,11 . However, the precise bacterial molecules recognized by NOD1 remain unknown. Using biochemical and genetic approaches, we demonstrate in this report that PGN containing γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) is uniquely present in certain bacteria including Gram- negative bacilli and particular Gram-positive bacteria, and is recog- nized by the NOD1-mediated pathway. These results indicate that NOD1 acts as an intracellular PRR for a subset of bacteria through the detection of iE-DAP. RESULTS Bacterial LPS in NOD1 stimulation Previous studies showed that NOD1 can mediate MyD88-independent cellular responsiveness to lipopolysaccharide (LPS) preparations from various Gram-negative bacteria including Escherichia coli, but not to 1 Department of Pathology and 2 Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA. 3 Department of Infectious Diseases and Tropical Medicine, Research Institute, International Medical Center of Japan, Tokyo 162-8655, Japan. 4 Advanced Medical Discovery Institute, Ontario Cancer Institute and Department of Medical Biophysics and Immunology, University of Toronto, 620 University Avenue, Toronto, Ontario M5G 2C1, Canada. 5 Department of Chemistry, Graduate School of Science, Osaka University, Machikaneyama 1-1, Toyonaka, Osaka 560-0043, Japan. 6 Departments of Microbiology and Immunology, and Medicine, University of Western Ontario, London, Ontario N6A 5C1, Canada. 7 Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK. 8 These authors contributed equally to this work. Correspondence should be addressed to N.I. (ino@umich.edu). An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid Mathias Chamaillard 1,2,8 , Masahito Hashimoto 3,8 , Yasuo Horie 4,8 , Junya Masumoto 1 , Su Qiu 1 , Lisa Saab 1,2 , Yasunori Ogura 1,2 , Akiko Kawasaki 5 , Koichi Fukase 5 , Shoichi Kusumoto 5 , Miguel A Valvano 6 , Simon J Foster 7 , Tak W Mak 4 , Gabriel Nuñez 1,2 & Naohiro Inohara 1 Nucleotide-binding oligomerization domain protein 1 (NOD1) belongs to a family that includes multiple members with NOD and leucine-rich repeats in vertebrates and plants. NOD1 has been suggested to have a role in innate immune responses, but the mechanism involved remains unknown. Here we report that NOD1 mediates the recognition of peptidoglycan derived primarily from Gram-negative bacteria. Biochemical and functional analyses using highly purified and synthetic compounds indicate that the core structure recognized by NOD1 is a dipeptide, γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP). Murine macrophages deficient in NOD1 did not secrete cytokines in response to synthetic iE-DAP and did not prime the lipopolysaccharide response. Thus, NOD1 mediates selective recognition of bacteria through detection of iE-DAP-containing peptidoglycan. © 2003 Nature Publishing Group http://www.nature.com/natureimmunology