Nod1 and Nod2 are mammalian proteins implicated in the intracellular detection of pathogen-associated molecular patterns. Recently, naturally occurring peptidoglycan (PG) fragments were identified as the microbial motifs sensed by Nod1 and Nod2. Whereas Nod2 detects GlcNAc-MurNAc dipeptide (GM-Di), Nod1 senses a unique diaminopimelate-containing GlcNAc-MurNAc tripeptide muropeptide (GM-Tri DAP ) found mostly in Gram-negative bacterial PGs. Because Nod1 and Nod2 detect similar yet distinct muropeptides, we further analyzed the molecular sensing specificity of Nod1 and Nod2 toward PG fragments. Using a wide array of natural or modified muramyl peptides, we show here that Nod1 and Nod2 have evolved divergent strategies to achieve PG sensing. By defining the PG structural requirements for Nod1 and Nod2 sensing, this study reveals how PG processing and modifications, either by host or bacterial enzymes, may affect innate immune responses. Peptidoglycan (PG)1 is a major constituent of the cell wall of Gram-positive bacteria and consists of glycan chains of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) that are cross-linked to each other by short peptides, allowing the formation of a rigid polymer that surrounds the bacterial cell. During bacterial life cycle, peptidoglycan is constantly degraded by specific hydrolases, and newly synthesized subunits are integrated into the polymeric structure, allowing biological processes such as cell division. In Gram-negative bacteria, a thin layer of peptidoglycan is also found in the periplasmic space. Apart from the thickness and the degree of stem peptides cross-linking, an important difference between Gram-positive and Gram-negative peptidoglycans resides in the nature of the third amino acid of the peptides. In Gram-positive bacteria, this amino acid is commonly a lysine, whereas a diaminopimelic acid is found in most Gramnegative bacteria. Extensive analysis from many bacteria has revealed that, in steady state conditions, peptidoglycans from each bacterial strain have somehow fixed proportions of di-, tri-, tetra-, or pentapeptide substituted to the MurNAc sugar moiety.

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions through selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist infection by Gram-negative bacteria. The bacterial components recognized by these pathways remain to be defined. Here we report that Gram-negative diaminopimelic acid-type peptidoglycan is the most potent inducer of the Imd pathway and that the Toll pathway is predominantly activated by Gram-positive lysine-type peptidoglycan. Thus, the ability of Drosophila to discriminate between Gram-positive and Gram-negative bacteria relies on the recognition of specific forms of peptidoglycan.