Identification by surface plasmon resonance of the mycobacterial lipomannan and lipoarabinomannan domains involved in binding to CD14 and LPS‐binding protein

Abstract

The mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), regulate host defence mechanisms through their interaction with pattern recognition receptors such as Toll-like receptors (TLRs). We have developed a surface plasmon resonance assay to analyse the molecular basis for the recognition of Mycobacterium kansasii LM or LAM, by immobilized CD14 and LPS-binding protein (LBP) both being capable to promote presentation of bacterial glycolipids to TLRs. The affinity of either LM/LAM was higher to CD14 than to LBP. Kinetic and Scatchard analyses were consistent with a model involving a single class of binding sites. These interactions required the lipidic anchor, but not the carbohydrate domains, of LM or LAM. We also provide evidence that addition of recombinant LBP enhanced the stimulatory effect of LM or LAM on matrix metalloproteinase-9 expression and secretion in macrophages, through a TLR1/TLR2-dependent mechanism.

Structural Analysis of an Unusual BioactiveN-Acylated Lipo-Oligosaccharide LOS-IV inMycobacterium marinum

“…This is reminiscent with other studies demonstrating that the fatty acid chains of the mannosyl-phosphatidyl inositol anchor of lipomannan and lipoarabinomannan are required for ligation to either CD14 or LPS binding protein. 68 These results are also consistent with the fact that the immunomodulatory functions of mycobacterial glycolipids/lipoglycans required the integrity of their acyl chains to mediate the formation of micelles and facilitate the multivalency/presentation to their ligands. 69 Therefore, by analogy with other glycolipids, we propose that biological activity of LOS-IV is dependent on its specific carbohydrate domain whereas its lipid moiety plays a crucial role for an efficient presentation to cellular receptors.…”

Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

“…CD14 was a likely candidate. Indeed, natural PIM 2 from M. Kansasii was shown to interact with CD14 [50] and S-LPS binding was shown to depend on the presence of CD14 [51]. We first confirmed that S-LPS binding depended on the presence of CD14 as S-LPS binding was essentially absent in CD14-deficient BMDM macrophages, while it was only slightly reduced in TLR4-deficient macrophages and similar in MD2-deficient and wild-type macrophages (Figure S3).…”

“…Indeed, CD14 is necessary for S-LPS binding to cells and subsequent signalling [51] and CD14 was also implicated as a first step in Pam 3 CSK 4 recognition, inducing physical proximity with TLR2/TLR1 and formation of the TLR2 signalling complex [61]. Natural PIM 2 from M. kansasii was shown to interact with CD14 [50], and CD14 was implicated in mycobacterial LM and H37Ra LAM pro-inflammatory activities [54], [62]. Here, we documented the inhibition of S-LPS binding to soluble CD14 by the anti-inflammatory PIM 1 , isoPIM 1 and PIM 2 mimetic, but not by PI or a deacylated PIM 2 analogue.…”

In vitroimmunomodulatory effects of fractions obtained from aqueous extracts ofLarrea divaricataCav (Jarilla) on mouse peritoneal macrophages

“…Untreated and treated cells were washed with PBS and fixed with cold methanol [34]. Then, cells were incubated for 30 min at 37°C with 20 µg mL −1 LPS-FITC or monoclonal anti-mouse CD14-FITC antibodies (Sigma, San Diego, CA, USA).…”