Analyse structurale et fonctionnelle des glycolipides pariétaux de Mycobacterium marinum (une mycobactérie modèle dans l'étude de la tuberculose)

Mycobacterium marinum est une mycobactérie pathogène des ectothermes génétiquement proche de M. tuberculosis. Ce modèle peut être utilisé pour mieux comprendre la formation des granulomes tuberculeux et déterminer le rôle des glycoconjugués pariétaux dans la régulation de la réponse immunitaire. Dans ce contexte, nos travaux ont porté sur la purification des glycolipides polaires et apolaires extraits de la paroi de M. marinum et leur caractérisation en utilisant la résonance magnétique nucléaire et la spectrométrie de masse. Nos résultats concernant l analyse structurale de la famille des lipooligosaccharides polaires (LOSs, comprenant les LOS-I à LOS-IV) ont démontré la présence de monosaccharides rares, dont le caryophyllose et un monosaccharide N-acylé, spécifique du LOS-IV. Nous avons également observé que tous les LOSs inhibent la sécrétion macrophagique d une cytokine pro-inflammatoire, le TNF-a . En revanche, seul le LOS-IV stimule l expression d antigènes (ICAM-1 et CD40) à la surface des macrophages et la production de chimiokine (IL-8). Cet effet inducteur du LOS-IV pourrait être lié à la présence du monosaccharide terminal N-acylé. Par ailleurs, les autres glycolipides polaires analysés, incluant les phosphatidyl-myo-inositol mannosides (PIM), le lipomannane (LM) et le lipoarabinomannane (LAM), possèdent des structures similaires à celles de M. tuberculosis.L étude des glycolipides apolaires a permis de préciser la structure des glycolipides phénoliques (PGL) et des tréhaloses di-mycolates (TDM). De plus, une famille de glycolipides méconnue comprenant le Di-Mycolyl-Di-Arabinoglycérol (DMDA) a été identifiée. Le DMDA présente une structure très proche de la partie terminale du mycolyl-arabinogalactane-peptidoglycanne (mAGp), macro-complexe pariétal majoritaire des mycobactéries. Des études complémentaires effectuées chez M. bovis BCG ont démontré que ce glycolipide était lié au métabolisme du mAGp, ce qui ouvre de nouvelles pistes pour la compréhension du mode d action de certaines drogues anti-tuberculeuses telles que la thiacétazone.Mycobacterium marinum is a natural pathogen of ectotherms genetically close to M. tuberculosis. This pathogen model is useful for deciphering the role of mycobacterial cell wall glycolipids in granulomatous infection. In this context, our work focused on the purification of both polar and apolar glycolipids extracted from M. marinum cell wall and their structural characterization using nuclear magnetic resonance and mass spectrometry. Analysis of the polar lipooligosaccharide family (LOSs, including LOS-I to LOS-IV) demonstrated the presence of several rare or even unique monosaccharides including caryophyllose, derivatives and a N-acylated monosaccharide specific of LOS-IV. Biological activity assays showed that LOSs exert an important pro-inflammatory effect by decreasing the TNF-a secretion from macrophages. Moreover, LOS-IV was found to stimulate the expression of the chemokine IL-8 and cell surface antigens (CD40 and ICAM-1) on macrophages. This specific immunostimulatory property was related to the presence of the terminal N-acylated monosaccharide in LOS-IV. In addition, other polar glycolipids analyzed, including phosphatidyl-myo-inositol mannosides (PIM), lipomannane (LM) and lipoarabinomannan (LAM), possess similar structures than M. tuberculosis. The study of apolar glycolipids permitted to precise the structure of phenolic glycolipids (PGL) and trehalose di-mycolates (TDM). Moreover, a family of unusual glycolipids, including Di-Mycolyl-Di-Arabinoglycérol (DMDA), was identified. DMDA structure is very close from the terminal part of peptidoglycan-arabinogalactan-mycolyl (mAGp), the mycobacterial cell wall macro-complex. Additional studies performed in M. bovis BCG showed that this glycolipid is related to mAGp, providing new insights about the mode of action of anti-tuberculous drugs such as thiacetazone.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Lactoferrin Inhibits the Lipopolysaccharide-Induced Expression and Proteoglycan-Binding Ability of Interleukin-8 in Human Endothelial Cells

Interleukin-8 (IL-8), a C-X-C chemokine bound to endothelium proteoglycans, initiates the activation and selective recruitment of leukocytes at inflammatory foci. We demonstrate that human lactoferrin, an antimicrobial lipopolysaccharide (LPS)-binding protein, decreases both IL-8 mRNA and protein expression induced by the complex Escherichia coli 055:B5 LPS/sCD14 in human umbilical vein endothelial cells. The use of recombinant lactoferrins mutated in the LPS-binding sites indicates that this inhibitory effect is mediated by an interaction of lactoferrin with LPS and CD14s that suppresses the endotoxin biological activity. Furthermore, since dimeric IL-8 and lactoferrin are both proteoglycan-binding molecules, the competition between these proteins for heparin binding was investigated. Lactoferrin strongly inhibited the interaction of radiolabeled IL-8 to immobilized heparin, whereas a lactoferrin variant lacking the amino acid residues essential for heparin binding was not inhibitory. Moreover, this process is specific, since serum transferrin, a glycoprotein whose structure is close to that of lactoferrin, did not prevent the interaction of IL-8 with heparin. These results suggest that the anti-inflammatory properties of lactoferrin during septicemia are related, at least in part, to the regulation of IL-8 production and also to the ability of lactoferrin to compete with chemokines for their binding to proteoglycans

Mycobacterial lipomannan induces MAP kinase phosphatase-1 expression in macrophages.

Mycobacterial lipomannan (LM) and lipoarabinomannan (LAM) regulate macrophage activation by interacting with Toll-like receptors (TLRs). The intracellular signalling pathways elicited by these complex molecules are poorly defined. We have demonstrated that LM purified from various mycobacterial species, but not LAM from Mycobacterium kansasii or Mycobacterium bovis BCG, induced expression of the MAP kinase phosphatase 1 (MKP-1) in macrophages. Anti-TLR2 antibodies, as well as specific ERK and p38 MAPK inhibitors, decreased MKP-1 transcription in LM-stimulated cells. These findings suggest that the binding of LM to TLR2 triggers MAPK activation, followed by an up-regulation of MKP-1 expression, which in turn may act as a negative regulator of MAPK activation

Glycosylphosphatidylinositols of <em>Toxoplasma gondii</em> induce matrix metalloproteinase-9 production and degradation of galectin-3

International audienceToxoplasma gondii glycosylphosphatidylinositols (GPIs) bind to galectin-3 to induce TNF-α production in macrophages via Toll-like receptors 2 and 4. Here we show that T. gondii GPIs stimulate human macrophages to synthesize matrix metalloproteinase-9 in a TNF-α-dependent pathway and degrade extracellular galectin-3

Mycobacterium marinum lipooligosaccharides are unique caryophyllose-containing cell wall glycolipids that inhibit tumor necrosis factor-alpha secretion in macrophages.

International audienceEarlier studies have reported a role for lipooligosaccharides (LOSs) in sliding motility, biofilm formation, and infection of host macrophages in Mycobacterium marinum. Although a LOS biosynthetic gene cluster has recently been identified in this species, many structural features of the different LOSs (LOS-I-IV) are still unknown. This clearly hampers assessing the contribution of each LOS in mycobacterial virulence as well as structure-function-based studies of these important cell wall-associated glycolipids. In this study, we have identified an M. marinum isolate, M. marinum 7 (Mma7), which failed to produce LOS-IV but instead accumulated large amounts of LOS-III. Local genomic comparison of the LOS biosynthetic cluster established the presence of a highly disorganized region in Mma7 compared with the standard M strain, characterized by multiple genetic lesions that are likely to be responsible for the defect in LOS-IV production in Mma7. Our results indicate that the glycosyltransferase LosA alone is not sufficient to ensure LOS-IV biosynthesis. The availability of different M. marinum strains allowed us to determine the precise structure of individual LOSs through the combination of mass spectrometric and NMR techniques. In particular, we established the presence of two related 4-C-branched monosaccharides within LOS-II to IV sequences, of which one was never identified before. In addition, we provided evidence that LOSs are capable of inhibiting the secretion of tumor necrosis factor-alpha in lipopolysaccharide-stimulated human macrophages. This unexpected finding suggests that these cell wall-associated glycolipids represent key effectors capable of interfering with the establishment of a pro-inflammatory response

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

International audienceThe 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

Mycobacterial Lipomannan Induces Matrix Metalloproteinase-9 Expression in Human Macrophagic Cells through a Toll-Like Receptor 1 (TLR1)/TLR2- and CD14-Dependent Mechanism

Lipomannans (LM) from various mycobacterial species were found to induce expression and secretion of the matrix metalloproteinase 9 (MMP-9) both in human macrophage-like differentiated THP-1 cells and in primary human macrophages. Inhibition studies using antireceptor-neutralizing antibodies are indicative of a Toll-like receptor 1 (TLR1)/TLR2- and CD14-dependent signaling mechanism. Moreover, LM was shown to down-regulate transcription of the metalloproteinase inhibitor TIMP-1, a major endogenous MMP-9 regulator