Binding and Cellular Activation Studies Reveal That Toll-like Receptor 2 Can Differentially Recognize Peptidoglycan from Gram-positive and Gram-negative Bacteria

Jinkeng Asong,Margreet A Wolfert,Kaustabh K Maiti,Douglas Miller,Geert-Jan Boons

doi:10.1074/jbc.m806633200

Abstract

Although much progress has been made toward the identification of innate immune receptors, far less is known about how these receptors recognize specific microbial products. Such studies have been hampered by the need to purify compounds from microbial sources and a reliance on biological assays rather than direct binding to monitor recognition. We have employed surface plasmon resonance (SPR) binding studies using a wide range of well defined synthetic muropeptides derived from Gram-positive (lysine-containing) and Gram-negative (diaminopimelic acid (DAP)-containing) bacteria to demonstrate that Toll-like receptor 2 can recognize peptidoglycan (PGN). In the case of lysine-containing muropeptides, a limited number of compounds, which were derived from PGN remodeled by bacterial autolysins, was recognized. However, a wider range of DAP-containing muropeptides was bound with high affinity, and these compounds were derived from nascent and remodeled PGN. The difference in recognition of the two classes of muropeptides is proposed to be a strategy by the host to respond appropriately to Gram-negative and -positive bacteria, which produce vastly different quantities of PGN. It was also found that certain modifications of the carboxylic acids of isoglutamine and DAP can dramatically reduce binding, and thus, bacterial strains may employ such modifications to evade innate immune detection. Cellular activation studies employing highly purified PGN from Bacillus licheniformis, Bacillus subtilis, Escherichia coli, Lactobacillus plantarum, Micrococcus luteus, and Staphylococcus aureus support the structure binding relationship. The data firmly establish Toll-like receptor 2 as an innate immune sensor for PGN and provides an understanding of host-pathogen interactions at the molecular level.

Highlights

Much progress has been made toward the identification of key innate immune receptors, far less is known about how these receptors recognize and initiate responses to specific microbial products
PGN is a highly complex structural component of the cell wall of almost all bacteria that has been implicated as a PAMP in innate immune responses
Additional structural heterogeneity is introduced during bacterial growth and division, which requires remodeling of cell wall PGN by bacterial autolysins such as DD- and DL-carboxypeptidases, endopeptidases, and N-acetylmuramidases

Summary

EXPERIMENTAL PROCEDURES

Reagents—Preparation of compound 2c was as follows. Sieber Amide resin (100 mg, 42 ␮mol was swelled in dimethylformamide (5 ml) for 45 min and treated with piperidine in DMF (20%, 3 ϫ 3 ml). The benzyl ester of the resulting compound was removed by stirring under an atmosphere of H2 (1 atm) in presence of palladium in activated carbon (20 mg) in mixture of. Fmoc-DAP(␦-CONH2)-OH was prepared by a cross-metathesis reaction of suitably protected vinyl glycine and allyl glycine followed by reduction of the double bond of the resulting compound. Insoluble B. licheniformis and L. plantarum PGN were isolated from the bacterial cells by treatment with SDS and glass beads and further treatment with ␣-amylase, DNase, and RNase. A mixture of ethanolamine and biotin (10:1 eq) was passed over the ligand and control flow cells to block free streptavidin binding sites.

PGN results in the loss of a terminal

Findings

DISCUSSION