Ultralong C100 Mycolic Acids Support the Assignment of Segniliparus as a New Bacterial Genus

Mycolic acid-producing bacteria isolated from the respiratory tract of human and non-human mammals were recently assigned as a distinct genus, Segniliparus, because they diverge from rhodococci and mycobacteria in genetic and chemical features. Using high accuracy mass spectrometry, we determined the chemical composition of 65 homologous mycolic acids in two Segniliparus species and separately analyzed the three subclasses to measure relative chain length, number and stereochemistry of unsaturations and cyclopropyl groups within each class. Whereas mycobacterial mycolate subclasses are distinguished from one another by R groups on the meromycolate chain, Segniliparus species synthesize solely non-oxygenated α-mycolates with high levels of cis unsaturation. Unexpectedly Segniliparus α-mycolates diverge into three subclasses based on large differences in carbon chain length with one bacterial culture producing mycolates that range from C58 to C100. Both the overall chain length (C100) and the chain length diversity (C42) are larger than previously seen for mycolic acid-producing organisms and provide direct chemical evidence for assignment of Segniliparus as a distinct genus. Yet, electron microscopy shows that the long and diverse mycolates pack into a typical appearing membrane. Therefore, these new and unexpected extremes of mycolic acid chemical structure raise questions about the modes of mycolic acid packing and folding into a membrane

Mycobacterium tuberculosis releases an antacid that remodels phagosomes

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Identification de nouveaux antigènes lipidiques mycobactériens et caractérisation des bases moléculaires de leur présentation par les protéines CD1

Les protéines CD1a à CD1d assurent la présentation d'antigènes lipidiques, tels que les composés de l'enveloppe de M. tuberculosis, aux lymphocytes T (LT). En caractérisant la spécificité d'un clone de LT CD1b-restreint, un nouvel antigène mycobactérien a été mis en évidence: le Glycérol Monomycolate (GroMM). Le clone reconnait également le Glucose Monomycolate (GlcMM). Cette réactivité croisée a pu être comprise grâce à la modélisation moléculaire des complexes CD1b/antigène en montrant que ces deux antigènes peuvent adopter un positionnement superposable. De plus, nous avons observé que, par un mécanisme encore inconnu, l'antigénicité du GroMM est fortement accrue par l'addition de lipides qui, individuellement, sont inactifs. La compréhension de cette potentialisation antigénique devrait avoir des applications en termes de formulation vaccinale. Les mécanismes d'apprêtement des antigènes lipidiques mycobactériens ont été étudiés sur le modèle des phosphatidyl-myo-inositol mannosides (PIM). Leur partie osidique est apprêtée par une alpha-mannosidase qui requiert l'assistance de l'isoforme CD1e dont le mode d'action restait incompris. Nous avons montré que CD1e possède des propriétés de " protéine de transfert de lipides " grâce auxquelles elle pourrait extraire les PIM des membranes pour faciliter l'action de l'enzyme. Afin de mettre en évidence d'autres enzymes impliquées dans l'apprêtement de ces antigènes, la description de leur structure minimale active a été entreprise en analysant l'antigénicité de fragments de PIM obtenus par digestion in vitro. L'activité de produits générés par des lipases suggère qu'il existe aussi un apprêtement de la partie lipidique des PIM.The proteins CD1a to CD1d ensure the presentation of lipidic antigens such as compounds of Mycobacterium tuberculosis'envelope, to T lymphocytes. The characterization of the specificity of a CD1b-restricted T cell clone allowed to describe a new mycobacterial antigen: the Glycerol Monomycolate (GroMM). The clone also recognizes the Glucose Monomycolate (GlcMM). This cross-reactivity could be understood through the molecular modelling of CD1b/antigen complexes showing that these two antigens can adopt a stackable position. Besides, we observed that by a still unknown mechanism, the antigenicity of GroMM is strongly enhanced by the addition of individually inactive lipids. Understanding this antigenic potentiation should have applications for vaccine formulation. The processing mechanisms of mycobacterial lipidic antigens were studied on the model of phosphatidyl-myo-inositol mannosides (PIM). Their osidic part is processed by an alpha-mannosidase which requires the assistance of CD1e isoform whose mode of action remains misunderstood. We showed that CD1e owns "Lipid Transfer Protein" properties through which it could extract PIM from membranes to facilitate the action of the enzyme. To highlight other enzymes involved in the processing of these antigens, description of their active minimum structure was undertaken by analyzing the antigenicity of PIM fragments obtained by in vitro digestion. The activity of products generated by lipases suggests that there is also a processing of the lipid part of PIM

Metabolic anticipation in Mycobacterium tuberculosis

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Rifampin Resistance Mutations Are Associated with Broad Chemical Remodeling of Mycobacterium tuberculosis

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Discovery of deoxyceramides and diacylglycerols as CD1b scaffold lipids among diverse groove-blocking lipids of the human CD1 system

Unlike the dominant role of one class II invariant chain peptide (CLIP) in blocking MHC class II, comparative lipidomics analysis shows that human cluster of differentiation (CD) proteins CD1a, CD1b, CD1c, and CD1d bind lipids corresponding to hundreds of diverse accurate mass retention time values. Although most ions were observed in association with several CD1 proteins, ligands binding selectively to one CD1 isoform allowed the study of how differing antigen-binding grooves influence lipid capture. Although the CD1b groove is distinguished by its unusually large volume (2,200 Å3) and the T′ tunnel, the average mass of compounds eluted from CD1b was similar to that of lipids from CD1 proteins with smaller grooves. Elution of small ligands from the large CD1b groove might be explained if two small lipids bind simultaneously in the groove. Crystal structures indicate that all CD1 proteins can capture one antigen with its hydrophilic head group exposed for T-cell recognition, but CD1b structures show scaffold lipids seated below the antigen. We found that ligands selectively associated with CD1b lacked the hydrophilic head group that is generally needed for antigen recognition but interferes with scaffold function. Furthermore, we identified the scaffolds as deoxyceramides and diacylglycerols and directly demonstrate a function in augmenting presentation of a small glycolipid antigen to T cells. Thus, unlike MHC class II, CD1 proteins capture highly diverse ligands in the secretory pathway. CD1b has a mechanism for presenting either two small or one large lipid, allowing presentation of antigens with an unusually broad range of chain lengths

Pancreatic lipase-related protein 2 and lysosomal phospholipase A2 process mycobacterial multiacylated lipid antigens

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Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis

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