Gram-Positive Bacterial Lipoglycans Based on a Glycosylated Diacylglycerol Lipid Anchor Are Microbe-Associated Molecular Patterns Recognized by TLR2

Landry Blanc,Alain Vercellone,Jérôme Nigou,Arun K Mishra,Gurdyal S Besra,Iain Sutcliffe,Romain Castanier,Aurélie Ray

doi:10.1371/journal.pone.0081593

Abstract

Innate immune recognition is the first line of host defense against invading microorganisms. It is a based on the detection, by pattern recognition receptors (PRRs), of invariant molecular signatures that are unique to microorganisms. TLR2 is a PRR that plays a major role in the detection of Gram-positive bacteria by recognizing cell envelope lipid-linked polymers, also called macroamphiphiles, such as lipoproteins, lipoteichoic acids and mycobacterial lipoglycans. These microbe-associated molecular patterns (MAMPs) display a structure based on a lipid anchor, being either an acylated cysteine, a glycosylated diacylglycerol or a mannosyl-phosphatidylinositol respectively, and having in common a diacylglyceryl moiety. A fourth class of macroamphiphile, namely lipoglycans, whose lipid anchor is made, as for lipoteichoic acids, of a glycosylated diacylglycerol unit rather than a mannosyl-phosphatidylinositol, is found in Gram-positive bacteria and produced by certain Actinobacteria, including Micrococcus luteus, Stomatococcus mucilaginosus and Corynebacterium glutamicum. We report here that these alternative lipoglycans are also recognized by TLR2 and that they stimulate TLR2-dependant cytokine production, including IL-8, TNF-α and IL-6, and cell surface co-stimulatory molecule CD40 expression by a human macrophage cell line. However, they differ by their co-receptor requirement and the magnitude of the innate immune response they elicit. M. luteus and S. mucilaginosus lipoglycans require TLR1 for recognition by TLR2 and induce stronger responses than C. glutamicum lipoglycan, sensing of which by TLR2 is dependent on TLR6. These results expand the repertoire of MAMPs recognized by TLR2 to lipoglycans based on a glycosylated diacylglycerol lipid anchor and reinforce the paradigm that macroamphiphiles based on such an anchor, including lipoteichoic acids and alternative lipoglycans, induce TLR2-dependant innate immune responses.

Highlights

The innate immune system is genetically programmed to detect molecular signatures of microbes via a limited number of germlineencoded pattern recognition receptors (PRRs) [1,2,3,4,5,6]
Structure/function relationship studies, corroborated by the reports of the structures of several TLR2-lipopeptide complexes determined by X-ray crystallography, have established that the acylated cysteinyl moiety is the structure recognized by the receptors and that triacylated lipoproteins are preferentially recognized by the TLR2/TLR1 complex, whereas diacylated lipoproteins are recognized by the TLR2/TLR6 complex [16,17]
Signaling via TLR2 We first tested the ability of MlLM, C. glutamicum (CgLM) and Stomatococcus mucilaginosus lipomannan (SmLM) to stimulate HEK293 cells stably transfected with human TLR2 and CD14 genes and a NF-kB-inducible reporter system (HEK-TLR2 cells)

Summary

Introduction

The innate immune system is genetically programmed to detect molecular signatures of microbes via a limited number of germlineencoded pattern recognition receptors (PRRs) [1,2,3,4,5,6]. I.e. cell envelope lipid-linked polymers [9,10], namely Gram-negative bacterial lipopolysaccharide (LPS), Gram-positive bacteria lipoteichoic acid (LTA), lipoproteins and mycobacterial lipoglycans, meet PAMP/MAMP criteria and are well suited to innate immune recognition. They are mostly recognized via their lipid anchor by a family of PRRs, named Toll-like receptors (TLRs). Lipoproteins, LTA and mycobacterial lipoglycans, based on a lipid anchor being either an acylated cysteine (a), a glycosylated diacylglycerol (b) or a mannosyl-phosphatidylinositol (c) respectively, and having in common a diacylglyceryl moiety (Figure 1), are recognized by TLR2. By analogy with lipopeptides, that in the case of tri-acylated lipoglycans, both fatty acids of the DAG unit are inserted in the hydrophobic pocket of TLR2 while the third acyl chain esterfying the mannosyl unit of the MPI anchor is inserted in the hydrophobic channel at the surface of TLR1 [8]

Methods

Results

Conclusion