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Role of Nod proteins in bacterial infection

The Nod-like receptor (NLR) family comprises proteins with a characteristic tripartite domain organization of an N-terminal protein-protein interaction domain, a central nucleotide-binding site (NBS) and a C-terminal series of leucine-rich repeats (LRR; see Figure 1). Their domain organization is highly reminiscent of a large group of proteins in plants called resistance proteins or R proteins. R proteins, for example RPS2 in Arabidopsis, are involved in sensing of microbes that enter the cytoplasmic compartment and initiating a defense response. Because of this similarity with plant R proteins, NLRs were first hypothesized to play a role in host defense in mammalian cells. Indeed, we now know that some of these proteins play a complementary role to Toll-like receptors for sensing of microbial products. NLRs include such family members as IPAF, the 10-15 NALP proteins and NAIP. The best-characterized members, however, are Nod1 and Nod2. These proteins are located in the cytoplasm and are involved in the detection of bacterial “pathogen-associated molecular patterns” (PAMPs) that enter into the cell either through specific transporters or as a consequence of infection with certain pathogenic bacteria.

Nod1 and Nod2 detect peptidoglycan from the bacterial cell wall.

Our recent studies focused on identifying the bacterial ligands that activate Nod1 and Nod2. Both proteins sense peptidoglycan (PG) fragments released from the cell wall of bacteria. For Nod1, we identified a naturally occurring PG fragment of N-acetyl glucosamine-N-acetyl muramic acid linked to a tripeptide where the terminal amino acid is meso-diaminopilemic acid (meso-DAP). The presence of DAP in PG can be considered as a general signature of Gram-negative bacterial infection since most, but not all, Gram-positive organisms have lysine in this position in their PG. What is also striking for Nod1 is that it is highly specific for the tripeptide structure since the presence of an additional amino acid to the DAP greatly diminishes the sensing of this bacterial product by human Nod1. Strikingly, tetrapeptide structures are recognized efficiently by murine Nod1 indicating that species differences exist in ligand specificity.

Like Nod1, Nod2 also senses a PG fragment, however, this protein is specific for muramyl dipeptide or MDP. MDP is the minimal bioactive PG fragment from both Gram-positive and Gram-negative bacteria making Nod2 a general sensor of bacterial infection. Interestingly, MDP has been known for decades for its immunomodulatory action; it is the key component of Freunds complete adjuvant. With the discovery of Nod2 as the host receptor for MDP, the immunostimulatory properties of this compound can now be investigated in more detail.

Role of Nod proteins in bacterial infection.

Highlighting their key role as intracellular innate immune receptors, studies have implicated Nod1 and Nod2 in the antimicrobial response to a variety of different pathogens including Shigella flexneri, enteroinvasive Escherichia coli, Helicobacter pylori and Listeria monocytogenes (see Figure 2 for scheme of Nod1 activation and signal transduction in response to S. flexneri and H. pylori infection). Although increased susceptibility to infection with bacteria in these models has been suggested to stem from either lack of early chemokine production or defensin secretion the exact mechanism for this remains to be clearly defined.

Role of Nod proteins in human disease.

A striking number of proteins within the NLR family are implicated in susceptibility to infection and inflammatory diseases. These include NALP1, which is associated with Muckle Wells syndrome, NAIP, which is associated with murine susceptibility to Legionella infection, and Nod1 and Nod2, both associated with inflammatory bowel disease.

The gene encoding Nod2 was the first susceptibility gene identified for the chronic inflammatory bowel disease, Crohn's disease. The most common mutation in the Nod2 gene associated with Crohn's disease is an insertion mutation at nucleotide position 3020 that leads to a frame-shift and the deletion of the terminal LRR of the protein. Using in vitro studies, mutated Nod2 protein was shown to be unable to detect MDP. Moreover, peripheral blood mononuclear cells isolated from patients with Crohn's disease cannot respond to MDP to initiate an inflammatory response The implications of these findings therefore suggest that the defect in Crohn's disease patients may stem from the inability to respond normally to bacterial products. How this then initiates the development of disease remains unknown.

More recently, polymorphisms in the human Nod1 gene have been linked to asthma, eczema, atopy and inflammatory bowel disease. In this case, the polymorphisms identified map to an intronic region within the Nod1 gene and it is speculated that these mutations may affect the relative expression of spliced variants of Nod1. For the moment, however, it is not clear whether isoform expression is altered in asthma or IBD patients and how this might contribute to disease pathogenesis.

Rationale

Despite their implication in human disease, the function of Nod1 and Nod2 and the mechanisms of their activation are still largely unknown. The major focus of our research is to examine the molecular basis of Nod/elictor interaction, the cell biology of the signal transduction pathways and how these proteins are implicated in both innate and adaptive immunity in bacterial infection as well in auto-immune disease.

Figure 1: Domain  architecture of Nod1 compared to two plant R proteins, Tobacco N and Arabidopsis RPS2. CARD, caspase activating and recruitment domain; NBS, nucleotide-binding domain; LRR, leucine-rich repeat; TIR, Toll/IL-1 receptor; LZ, leucine zipper.

Figure 2: Activation of the Nod1-dependent signaling pathway to NF-kB by Shigella flexneri or Helicobacter pylori. Intracellular S. flexneri releases peptidoglycan (PG) fragments containing “TriDAP” the ligand for Nod1, while H. pylori contaminates the cytosol with these products through the Type IV injectosome. Nod1 is activated by TriDAP leading to its unfolding and subsequent interaction with the kinase, RICK (also known as Rip2). RICK then interacts with the IKK complex leading to the release of active NF-kB, a transcriptional regulator involved in controlling expression of pro-inflammatory products, including interleukin-8 and COX-2.

Biography

Education

1992 – 1996. University of Toronto, Toronto, Ontario Canada
Doctor of Philosophy
Department of Molecular and Medical Genetics; Supervisor: Dr. Philip Sherman

1985 – 1989. University of Calgary, Calgary, Alberta Canada
Bachelor of Science
Cellular, Molecular and Microbial Biology

Professor Research Experience

Present position. University of Toronto, Toronto, Ontario Canada
Assistant Professor
Department of Immunology

2002 – 2006. Institut Pasteur, Paris, France
Group Leader
Groupe d’Immunité Innée et Signalisation, Département de Biologie Cellulaire et Infection,

1997 – 2001. Institut Pasteur, Paris, France
Post-doctoral Fellow
Unité de Pathogénie Microbienne Moléculaire; Supervisor: Dr. Philippe Sansonetti

1996 – 1997. McMaster University, Hamilton, Ontario, Canada
Post-doctoral Fellow
Cellular Microbiology, Department of Pathology; Supervisor: Dr. Mary H. Perdue

Awards

2006 – 2011. Canadian Institutes for Health Research New Investigator Award
2004 – 2005. Prix Charles-Louis de Saulses de Freycinet - Institut de France, Academie des Sciences
2003 – 2006
. EMBO Young Investigator award
2000 – 2001. Canadian Institutes for Health Research Post-Doctoral Fellowship
1998 – 2000. European Community - Marie Curie Research Training Grant.
1997 – 1998. Howard Frank Post-Doctoral Fellowship, Institut Pasteur.
1997 – 2000. European Molecular Biology Organisation (EMBO) Post-Doctoral Research Fellowship (Declined).
1997 – 2000. Medical Research Council of Canada Post-Doctoral Fellowship (Declined).
1996 – 1997. Canadian Association of Gastroenterology / Glaxo Wellcome Canada Post-Doctoral Fellowship.
1994 – 1996. Medical Research Council of Canada Studentship.