Les glycolipides mycobactériens multi-acylés : de la structure à la présentation antigénique

Les lipides peuvent être antigéniques et sont alors présentés aux lymphocytes T par les protéines CD1 (CD1a à CD1d), exprimées à la surface des cellules présentatrices d'antigènes. Dans le cadre de la tuberculose, dont l'agent étiologique est Mycobacterium tuberculosis, un certain nombre d'antigènes lipidiques ont été décrits et il faut remarquer que la plupart sont présentés par l'isoforme CD1b. Ainsi, les phosphatidyl-myo-inositol mannosides (PIM) et les sulfoglycolipides di-acylés (Ac2SGL) sont deux familles de glycolipides de l'enveloppe mycobactérienne capables, in vitro, de stimuler des clones de lymphocytes T. Un premier axe de mes travaux de thèse a consisté d'une part à déterminer la structure des espèces antigéniques appartenant à ces familles de glycolipides et d'autre part, à mieux caractériser les mécanismes d'apprêtement intracellulaire qui gouvernent la présentation des PIM dans lesquels CD1e, cinquième isoforme non présentatrice des protéines CD1, joue un rôle central non défini. Le second axe concerne la recherche de nouveaux antigènes afin d'étendre le répertoire des lipides immunogènes impliqués dans l'immunité anti-mycobactérienne. Les PIM sont présents sous plusieurs formes glycosylées, PIM2 à PIM6 (PIM possédant 2 à 6 unités mannosyles), mais aussi sous plusieurs formes acylées, de mono- à tétra-acylées. Pour être présentés et reconnus par les lymphocytes T, les PIM6 doivent être apprêtés en PIM2 par une mannosidase lysosomale exprimée par les cellules présentatrices, et cette dégradation nécessite la protéine CD1e. Au cours du travail présenté, nous avons montré que CD1e possède in vitro des propriétés de " protéine de transfert de lipides " spécifiques de certaines acyl-formes de PIM: les PIM di-acylés. Cependant, seules les formes tétra-acylées se sont avérées antigéniques, donc capables de stimuler les lymphocytes T. En fait, nous avons pu démontrer qu'une dégradation de la partie lipidique existe également au sein des cellules présentatrices d'antigènes. L'ensemble des données obtenues indiquent qu'in vivo, les PIM6 tétra-acylés seraient d'abord apprêtés au niveau de leur partie lipidique pour générer des formes di-acylées non conventionnelles que CD1e serait ensuite capable d'extraire des membranes. Par cette extraction, CD1e permettrait la digestion par une alpha-mannosidase des PIM6 en PIM2 di-acylés antigéniques que CD1e pourrait également transférer vers l'isoforme présentatrice CD1b. Les sulfoglycolipides (SGL), contrairement aux PIM, sont spécifiques de l'enveloppe de M. tuberculosis. Parmi les SGL, seuls les Ac2SGL ont été décrits comme des antigènes immunogènes pouvant in vitro activer des lymphocytes T CD8+ aux propriétés bactéricides. Les Ac2SGL sont constitués d'un corps tréhalose sulfaté estérifié par un acide gras banal (acide palmitique ou stéarique) et un acide hydroxyphthiocéranoïque de structure variable, pouvant atteindre jusqu'à 50 atomes de carbone. Afin de caractériser quelle(s) espèce(s) active(nt) les lymphocytes T spécifiques des Ac2SGL, nous avons amélioré leur méthode de purification et analysé finement la structure de chaque espèce moléculaire constituant cette famille de glycolipides par spectrométrie de masse et par spectroscopie RMN. Ainsi, il a notamment pu être mis en évidence de nouvelles populations minoritaires d'Ac2SGL. La caractérisation de la contribution des différentes populations dans la réponse T anti-mycobactérienne est un objectif de ce travail. Nous avons également initié la purification et la caractérisation d'un nouvel antigène produit par M. tuberculosis. Il s'agit d'un lipopeptide dont l'épitope est reconnu par un clone de lymphocytes T restreint par les molécules du CMH-II. Selon la structure de l'antigène, les processus d'apprêtement ainsi que son mode d'interaction avec la molécule du CMH-II devront être étudiés et, à plus long terme, son immunogénicité devra être évaluée.Antigenic lipids are presented to T-cells by CD1 proteins (CD1a to CD1d) expressed at the antigen presenting cell surface. In tuberculosis context, which the etiological agent is Mycobacterium tuberculosis, several lipid antigens have been described and it is important to note that most of them are presented by the isoforme CD1b. Thus, phosphatidyl-myo-inositol mannosides (PIM), and di-acylated sulfoglycolipids (Ac2SGL) are two glycolipid families of the mycobacterial cell-wall able to stimulate T-cell clones in vitro. The first line of my thesis work has consisted in the structural characterization of the antigenic species belonging to these two glycolipid families and, in an other way, to decipher intracellular processing mechanisms that manage PIM presentation in which CD1e, the fifth isoform of CD1 proteins, plays a key and non-presenting role not defined. Secondly, my research has consisted in looking for new antigens to expand the repertoire of immunogenic lipids involved in the anti-mycobacterial immunity. PIM are present in several glycosylated forms, PIM2 to PIM6 (possessing 2 to 6 mannosyl units), but also in different acylated forms, mono- to tetra-acylated. To be presented and recognized by T-cells, PIM6 have to be processed in PIM2 by a lysosomal mannosidase expressed in presenting cells. This degradation requires the CD1e protein. In this study, we have shown that CD1e has properties of lipid transfer protein, in vitro, specifically towards some PIM acyl-forms: di-acylated PIM. However, only tetra-acylated forms are antigenic and are able to stimulate T cells. Actually, we have demonstrated that a degradation of the lipidic part exist also in antigen presenting cells. All the data show that, in vivo, tetra-acylated PIM6 would be firstly processed in their lipidic part in order to generate non-conventional di-acylated forms that CD1e could be able to extract them from membranes. With this extraction, CD1e could allow PIM6 digestion by an a-mannosidase in an antigenic di-acylated PIM2 that CD1e could also transfer to the presenting isoform, CD1b. Sulfoglycolipids (SGL), contrary to PIM, are specific to M. tuberculosis. Among SGL, only Ac2SGL have been described as immunogenic antigens able to, in vitro, activate CD8+ T-cells that have bactericide properties. Ac2SGL are constituted by a sulfated trehalose esterified by a classical fatty acid (palmitic acid or stearic acid) and a hydroxyphthioceranoic acid with a variable structure with up to 50 carbon atoms. In order to characterize which species can activate Ac2SGL specific T-cells, we improved the purification method and we analyzed with accuracy the structure of each molecular species constituting this glycolipid family by mass spectrometry and NMR spectroscopy. Thus, it has been highlighted new minority populations of Ac2SGL. The characterization of each population contribution in the anti-mycobacterial T response is an aim of this work. We also initiated the purification and the characterization of a new antigen produced by M. tuberculosis. This antigen is a lipopeptide which the epitope is recognized by T-cell clone restricted by MHC-II molecule. Depending of the antig

Les glycolipides mycobactériens multi-acylés (de la structure à la présentation antigénique)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

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