Nod1 and Nod2 are intracellular proteins that are involved in host recognition of specific bacterial molecules and are genetically associated with several inflammatory diseases. Nod1 and Nod2 stimulation activates NF-jB through RICK, a caspase-recruitment domain-containing kinase. However, the mechanism by which RICK activates NF-jB in response to Nod1 and Nod2 stimulation is unknown. Here we show that RICK is conjugated with lysine-63-linked polyubiquitin chains at lysine 209 (K209) located in its kinase domain upon Nod1 or Nod2 stimulation and by induced oligomerization of RICK. Polyubiquitination of RICK at K209 was essential for RICK-mediated IKK activation and cytokine/chemokine secretion. However, RICK polyubiquitination did not require the kinase activity of RICK or alter the interaction of RICK with NEMO, a regulatory subunit of IjB kinase (IKK). Instead, polyubiquitination of RICK was found to mediate the recruitment of TAK1, a kinase that was found to be essential for Nod1-induced signaling. Thus, RICK polyubiquitination links TAK1 to IKK complexes, a critical step in Nod1/Nod2-mediated NF-jB activation.

Autophagy, an evolutionally conserved homeostatic process for catabolizing cytoplasmic components, has been implicated in the elimination of intracellular pathogens during mammalian innate immune responses. However, the mechanisms underlying cytoplasmic infection-induced autophagy, and the role of autophagy in host survival against intracellular pathogens are unknown. Here we report that in drosophila, recognition of diaminopimelic acid-type peptidoglycans by the pattern recognition receptor PGRP-LE is crucial for the induction of autophagy, and that autophagy prevents the intracellular growth of Listeria monocytogenes and promotes host survival against this infection. Autophagy induction occurs independently of the Toll and IMD innate signaling pathways. These findings define a clear pathway leading from the intracellular pattern recognition receptors to the induction of autophagy to host defense.

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Nod1 and Nod2 are intracellular proteins that are involved in recognition of bacterial molecules and their genetic variations have been linked to several inflammatory diseases that are strongly affected by environmental factors. However, the distribution of Nod1-and Nod2-stimulatory molecules in different bacterial species and environments is unknown. Here we established a quantitative bioassay to screen and characterize Nod1-and Nod2-stimulatory activities in different environmental sites and bacterial species. Using this system, we found that common environments including foods and soils contain high levels of Nod1-and Nod2-stimulatory activities. Several Bacillus species were identified to possess the highest Nod1-stimulatory activity among soil bacteria. Unlike other immunostimulatory molecules, the higher level of Nod1-stimulatory activity was found in the culture supernatant and not in extracts from whole cell bacteria. Nod1-stimulatory molecules were highly stable at extreme pH and boiling conditions and were synthesized in an amidaseand sltY-independent manner. These results suggest a novel mechanism by which bacteria present in the environment stimulate the host immune system through Nod1.

Carbohydrate recognition is essential for growth, cell adhesion and signalling in all living organisms. A highly conserved carbohydrate binding module, LysM, is found in proteins from viruses, bacteria, fungi, plants and mammals. LysM modules recognize polysaccharides containing N-acetylglucosamine (GlcNAc) residues including peptidoglycan, an essential component of the bacterial cell wall. However, the molecular mechanism underpinning LysM–peptidoglycan interactions remains unclear. Here we describe the molecular basis for peptidoglycan recognition by a multimodular LysM domain from AtlA, an autolysin involved in cell division in the opportunistic bacterial pathogen Enterococcus faecalis. We explore the contribution of individual modules to the binding, identify the peptidoglycan motif recognized, determine the structures of free and bound modules and reveal the residues involved in binding. Our results suggest that peptide stems modulate LysM binding to peptidoglycan. Using these results, we reveal how the LysM module recognizes the GlcNAc-X-GlcNAc motif present in polysaccharides across kingdoms.

Nod1 is a member of family of intracellular proteins that mediate host recognition of bacterial peptidoglycan. To characterize immune responses mediated by Nod1, synthetic ligand compounds possessing enhanced ability to stimulate Nod1 were developed to study the function of Nod1. Stimulation of epithelial cells with Nod1 stimulatory molecules induced chemokines and other proinflammatory molecules that are important for innate immune responses and recruitment of acute inflammatory cells. Administration of Nod1 ligands into mice induced chemokines and recruitment of acute inflammatory cells, an activity that was abolished in Nod1-null mice. Microarray analysis revealed that Nod1 stimulation induces a restricted number of genes in intestinal epithelial cells compared with that induced by tumor necrosis factor (TNF) α. Nod1 stimulation did not induce TNFα, interleukin 12, and interferon γ, suggesting that the primary role of Nod1 is to induce the recruitment of immune cells. These results indicate that Nod1 functions as a pathogen recognition molecule to induce expression of molecules involved in the early stages of the innate immune response.

Mesothelial cells that line the serous cavities and outer surface of internal organs are involved in inflammatory responses induced by microbial stimuli and bacterial infection. Upon exposure to bacterial products, mesothelial cells secrete chemokines, but the signaling pathways by which these cells recognize bacteria to mediate innate immune responses remain largely unknown. We report that stimulation of primary peritoneal mesothelial cells via nucleotide-binding oligomerization domain (Nod)1, a member of the intracytoplasmic Nod-like receptor family, induced potent secretion of the chemokines CXCL1 and CCL2 as well as expression of inducible NO synthase and such responses required the kinase RICK. Mesothelial cells also produced chemokines in response to TLR2, TLR3, TLR4, and TLR5 agonists, but unlike that induced by Nod1 stimulation, the TLR-mediated responses were independent of RICK. Yet, Nod1 stimulation of mesothelial cells via RICK enhanced chemokine secretion induced by LPS or IFN-γ and cooperated with IFN-γ in the production of NO. The i.p. administration of KF1B, a synthetic Nod1 agonist, elicited chemokine production in the serum and peritoneal fluid as well as the recruitment of neutrophils into the peritoneal cavity of wild-type mice, but not RICK-deficient mice. Finally, infection of mesothelial cells with Listeria monocytogenes induced production of CXCL1 and this response was significantly reduced in Nod1- or RICK-deficient cells. These results define mesothelial cells as microbial sensors through TLRs and Nod-like receptors and identify Nod1 and RICK as important mediators of chemokine and antimicrobial responses in mesothelial cells.

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