Indeed, it clearly shows the importance of ligand acyl chains to bind and induce heterodimerization of the receptors and provides a rationale to tentatively understand the ligand structure-function relationships, although the presence of binding sites other than that of lipopeptides cannot be excluded. For instance, LTA, that bears two acyl chains, has been unambiguously proved, using chemically synthesized analogs, to stimulate TLR2 and recently demonstrated to bind TLR2. However, its role as a physiological TLR2 ligand is still under debate. Indeed, a set of studies focusing on Staphylococcus aureus and using cell wall-derived compounds as well as a mutant lacking acylated lipoproteins, demonstrates that LTA is much less active than Vorinostat lipoproteins and suggests that not LTA but lipoproteins are the dominant immunobiologically active compounds in this Gram-positive bacterium. As a consequence, in a recent review, Za¨ hringer et al propose that lipoproteins/lipopeptides are the only compounds of microorganisms sensed at physiological concentrations by TLR2. Lipoglycans are surface-exposed molecules of mycobacteria that have been described by other and us to be ligands, as purified molecules, of several PRRs, including the C-type lectins Mannose Receptor and DC-SIGN, as well as TLR2. However, their real nature as MAMPs has never been validated by isogenic mycobacterial mutants in the context of a bacterium infection. Their structure is based on a mannosylphosphatidyl-myo-inositol anchor, which, although very similar to the GPI anchors found in eukaryotic cells, is specific of these microorganisms. The biosynthesis of the mannosyl-phosphatidyl-myo-inositol anchor is essential in mycobacteria. The most active lipoglycan, lipomannan, is sensed by TLR2 at concentrations similar to that of mycobacterial lipoproteins and we have shown recently that it can compete for lipopeptide binding to the receptor, suggesting that it shares at least in part the same binding site. Assuming that it is the case, straightforward structure-function relationships can account for the observed TLR2-stimulatory capacity of the various purified LM acyl-forms. Nevertheless, a contamination of lipoglycan fractions by highly active lipopeptides is formally difficult to rule out. Moreover, a Mycobacterium tuberculosis mutant deficient for lipoprotein processing is dramatically altered in its capacity to stimulate TLR2, suggesting, as for S. aureus, a predominant role of lipoproteins in mycobacteria sensing by TLR2. In order to determine whether lipoglycans are i) bona fide MAMPs and most particularly TLR2 ligands and ii) sensed at physiological concentrations in the context of the whole bacterium, we used here the model organism Mycobacterium smegmatis to generate mutants altered for the production of lipoglycans. Since their biosynthesis cannot be fully abrogated, we attempted to construct some strains with either an increased or a reduced production of lipoglycans and we compared their ability to induce innate immune signaling relatively to control strains in reporter cells, macrophage cell line or dendritic cells. Finally, to compare the relative contribution of lipoglycans and lipoproteins in mycobacteria sensing by TLR2, we constructed a mutant deficient for lipoprotein processing.