Dual strategies for peptidoglycan discrimination by peptidoglycan recognition proteins (PGRPs)

Abstract

The innate immune system constitutes the first line of defense against microorganisms in both vertebrates and invertebrates. Although much progress has been made toward identifying key receptors and understanding their role in host defense, far less is known about how these receptors recognize microbial ligands. Such studies have been severely hampered by the need to purify ligands from microbial sources and a reliance on biological assays, rather than direct binding, to monitor recognition. We used synthetic peptidoglycan (PGN) derivatives, combined with microcalorimetry, to define the binding specificities of human and insect peptidogycan recognition proteins (PGRPs). We demonstrate that these innate immune receptors use dual strategies to distinguish between PGNs from different bacteria: one based on the composition of the PGN peptide stem and another that senses the peptide bridge crosslinking the stems. To pinpoint the site of PGRPs that mediates discrimination, we engineered structure-based variants having altered PGN-binding properties. The plasticity of the PGRPbinding site revealed by these mutants suggests an intrinsic capacity of the innate immune system to rapidly evolve specificities to meet new microbial challenges.affinity ͉ bacteria ͉ innate immunity ͉ calorimetry ͉ synthesis T he innate immune system recognizes invading microbes by means of conserved pattern recognition receptors that bind unique products of microbial metabolism not produced by the host (pathogen-associated molecular patterns) (1, 2). Examples of microbial ligands recognized by pattern recognition receptors such as Toll-like receptors, peptidoglycan recognition proteins (PGRPs), and NOD proteins include lipopolysaccharide of Gram-negative bacteria, lipoteichoic acid of Gram-positive bacteria, nonmethylated CpG sequences, flagellin, and peptidoglycan (PGN) of Gram-negative and -positive bacteria. Cellular activation by pattern recognition receptors results in acute inflammatory responses involving cytokine and chemokine production, direct local attack against the invading pathogen, and induction of the adaptive component of the immune system. In humans, overactivation of inflammatory responses can lead to septic shock, which accounts for 100,000 deaths annually in the United States alone. By sitting at the intersection of the pathways of microbial recognition, inflammation, and cell death, the innate immune system offers emerging opportunities for the development of therapeutics to modulate immune responses (3).PGRPs, a newly discovered class of pattern recognition receptors, are highly conserved from insects to mammals (4-7). By detecting PGN from both Gram-negative and -positive bacteria, PGRPs are important contributors to host defense against microbial infections (2, 4). PGNs are polymers of alternating Nacetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) in ␤(134) linkage, crosslinked by short peptide stems composed of alternating L-and D-amino acids (8, 9) (Fig. 1A). Whereas the carbohydrate backbone is conserved a...

Structural Studies on Molecular Interactions between Camel Peptidoglycan Recognition Protein, CPGRP-S, and Peptidoglycan Moieties N-Acetylglucosamine and N-Acetylmuramic Acid

“…5). The binding and structural studies previously carried out with LPS and LTA (4) (40). It has been shown that PGRP-S binds to both Lys-type and DAP-type PGNs (41).…”

Molecular cloning and functional characterization of a short peptidoglycan recognition protein (HcPGRPS1) from the freshwater mussel, Hyriopsis cumingi

“…PGRP-S preferentially binds DAP-type PGN (Swaminathan et al, 2006). In mollusks, rCfPGRPS1 showed high affinity to Lys-type PGN from S. aureus (Yang et al, 2010).…”

Structural insights into the bactericidal mechanism of human peptidoglycan recognition proteins

“…Thermodynamic parameters for the binding of PGRPs to PGN derivatives were determined using a MicroCal VP-ITC titration calorimeter as described in ref. 20. For the present titrations, c values (the product of the initial PGRP concentration and K b ) ranged from 3.06 to 7.99, allowing for precise determination of K b (19,36).…”

“…5), indicating only a minor contribution from the NAG moiety present in both GMPP and GMPP 2 , but not in MPP. Neither PGRP-I␤C nor -I␣C bound MPP-Dap, even under conditions at which interactions with K b Ͼ100 M Ϫ1 should be detectable (19,20). Thus, both these PGRP domains appear highly specific for Lys-type PGN.…”