IL-12 is a key cytokine in directing the development of type 1 Th cells, which are critical to eradicate intracellular pathogens such as Mycobacterium tuberculosis. Here, we report that mannose-capped lipoarabinomannans (ManLAMs) from Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium tuberculosis inhibited, in a dose-dependant manner, the LPS-induced IL-12 production by human dendritic cells. The inhibitory activity was abolished by the loss of the mannose caps or the GPI acyl residues. Mannan, which is a ligand for the mannose receptor (MR) as well as an mAb specific for the MR, also inhibited the LPS-induced IL-12 production by dendritic cells. Our results indicate that ManLAMs may act as virulence factors that contribute to the persistence of M. bovis bacillus Calmette-Guérin and M. tuberculosis within phagocytic cells by suppressing IL-12 responses. Our data also suggest that engagement of the MR by ManLAMs delivers a negative signal that interferes with the LPS-induced positive signals delivered by the Toll-like receptors.

Mycobacterial lipids comprise a heterogeneous group of molecules capable of inducing T cell responses in humans. To identify novel antigenic lipids and increase our understanding of lipid-mediated immune responses, we established a panel of T cell clones with different lipid specificities. Using this approach we characterized a novel lipid antigen belonging to the group of diacylated sulfoglycolipids purified from Mycobacterium tuberculosis. The structure of this sulfoglycolipid was identified as 2-palmitoyl or 2-stearoyl-3-hydroxyphthioceranoyl-2′-sulfate-α-α′-d-trehalose (Ac₂SGL). Its immunogenicity is dependent on the presence of the sulfate group and of the two fatty acids. Ac₂SGL is mainly presented by CD1b molecules after internalization in a cellular compartment with low pH. Ac₂SGL-specific T cells release interferon γ, efficiently recognize M. tuberculosis–infected cells, and kill intracellular bacteria. The presence of Ac₂SGL-responsive T cells in vivo is strictly dependent on previous contact with M. tuberculosis, but independent from the development of clinically overt disease. These properties identify Ac₂SGL as a promising candidate to be tested in novel vaccines against tuberculosis.

Complexes between CD1 molecules and self or microbial glycolipids represent important immunogenic ligands for specific subsets of T cells. However, the function of one of the CD1 family members, CD1e, has yet to be determined. Here, we show that the mycobacterial antigens hexamannosylated phosphatidyl- myo -inositols (PIM ₆ ) stimulate CD1b-restricted T cells only after partial digestion of the oligomannose moiety by lysosomal α-mannosidase and that soluble CD1e is required for this processing. Furthermore, recombinant CD1e was able to bind glycolipids and assist in the digestion of PIM ₆ . We propose that, through this form of glycolipid editing, CD1e helps expand the repertoire of glycolipidic T cell antigens to optimize antimicrobial immune responses.

The dimannoside (PIM₂) and hexamannoside (PIM₆) phosphatidyl-myo-inositol mannosides are the two most abundant classes of PIM found in Mycobacterium bovis bacillus Calmette Guérin, Mycobacterium tuberculosis H37Rv, and Mycobacterium smegmatis 607. Recently, these long known molecules received a renewed interest due to the fact that PIM₂ constitute the anchor motif of an important constituent of the mycobacterial cell wall, the lipoarabinomannans (LAM), and that both LAM (phosphoinositol-capped LAM) and PIM are agonists of Toll-like receptor 2 (TLR2), a pattern recognition receptor involved in innate immunity. Due to the biological importance of these molecules, the chemical structure of PIM was revisited. The structure of PIM₂ was recently published (Gilleron, M., Ronet, C., Mempel, M., Monsarrat, B., Gachelin, G., and Puzo, G. (2001) J. Biol. Chem. 276, 34896–34904). Here we report the purification and molecular characterization of PIM₆ in their native form. For the first time, four acyl forms of this molecule have been purified, using hydrophobic interaction chromatography. Mono- to tetra-acylated molecules were identified in M. bovis bacillus Calmette Guérin, M. tuberculosis H37Rv, and M. smegmatis 607 using a sophisticated combination of analytical tools, including matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and two-dimensional homo- and heteronuclear NMR spectroscopy. These experiments revealed that the major acyl forms are similar to the ones described for PIM₂. Finally, we show that PIM₆, like PIM₂, activate primary macrophages to secrete TNF-α through TLR2, irrespective of their acylation pattern, and that they signal through the adaptor MyD88.

The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials. One of the major virulence mechanisms of the tuberculosis bacillus, Mycobacterium tuberculosis, is its ability to resist killing by phagocytic cells of the host immune system, namely the macrophages. Macrophages degrade invading microbes by engulfment inside a vacuole, or phagosome, that progressively acidifies and accumulates hydrolytic properties. M. tuberculosis has the unique ability to block phagosome maturation and acidification. To identify mycobacterial genes involved in phagosome maturation arrest, we developed a novel high-throughput technology based on automated confocal microscopy. We screened a library containing over 11,000 M. tuberculosis mutants, and we could identify 10 mutants that had lost their ability to resist phagosome acidification. Genetic characterization of these mutants revealed that they carried lesions in genes involved in various cell processes, including biogenesis of the cell envelope. In particular, two independent mutants in the same genetic locus showed altered production of two lipids, namely diacyltrehalose (DAT) and sulfoglycolipid (SGL). In vitro experiments showed that SGL can indeed influence phagosome maturation. Our study unravels the role of novel lipid molecules in mycobacterial intracellular parasitism; our approach may be useful to identify virulence genes in other intracellular pathogens, and to identify novel antimicrobials.

We examined the function of the pimA (Rv2610c) gene, located in the vicinity of the phosphatidylinositol synthase gene in the genomes of Mycobacterium tuberculosis and Mycobacterium smegmatis, which encodes a putative mannosyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis. A cell-free assay was developed in which membranes from M. smegmatis overexpressing the pimA gene incorporate mannose from GDP-[(14)C]Man into di- and tri-acylated phosphatidylinositol mono-mannosides. Moreover, crude extracts from Escherichia coli producing a recombinant PimA protein synthesized diacylated phosphatidylinositol mono-mannoside from GDP-[(14)C]Man and bovine phosphatidylinositol. To determine whether PimA is an essential enzyme of mycobacteria, we constructed a pimA conditional mutant of M. smegmatis. The ability of this mutant to synthesize the PimA mannosyltransferase was dependent on the presence of a functional copy of the pimA gene carried on a temperature-sensitive rescue plasmid. We demonstrate here that the pimA mutant is unable to grow at the higher temperature at which the rescue plasmid is lost. Thus, the synthesis of phosphatidylinositol mono-mannosides and derived higher phosphatidylinositol mannosides in M. smegmatis appears to be dependent on PimA and essential for growth. This work provides the first direct evidence of the essentiality of phosphatidylinositol mannosides for the growth of mycobacteria.