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Human Vγ9δ2 T lymphocytes are suggested to play an important role in the immune response to various microbial pathogens. In contrast to αβ T cells, γδ T lymphocytes recognize small, non-protein, phosphate-bearing antigens (phosphoantigens) in a major histocompatibility complex-independent manner. Four different phosphoantigens termed TUBag1 to TUBag4 with a common 3-formyl-1-butyl-pyrophosphate moiety and isopentenyl-pyrophosphate have been isolated and identified from mycobacteria. However, natural occurring γδ T cell ligands from other bacterial species were not characterized so far. Here, we describe the structural identification of the two compounds responsible for the γδ T cell-stimulating capacity of Escherichia colias similar to the mycobacterial phosphoantigens 3-formyl-1-butyl-pyrophosphate and itsMr275 homologue TUBag2. In addition,E. coliphosphoantigens exert bioactivities on γδ T cells with similar potencies to the mycobacterial phosphoantigens at 5–15 nmconcentration. Furthermore, our results clearly prove that the deoxyxylulose 5-phophate pathway (also referred to as Rohmer metabolic route of isoprenoid biosynthesis) is essential for the biosynthesis of the phosphoantigens in E. coli. Because this pathway is absent from human cells, it proves an ideal target for focusing efficiently the antimicrobial selectivity of human γδ T lymphocytes.

Most human blood gammadelta T cells react without major histocompatibility complex restriction to small phosphorylated nonpeptide antigens (phosphoantigens) that are abundantly produced by mycobacteria and several other microbial pathogens. Although isopentenyl pyrophosphate has been identified as a mycobacterial antigen for gammadelta T cells, the structure of several other stimulating compounds with bioactivities around 1000-fold higher than isopentenyl pyrophosphate remains to be elucidated. This paper describes the structural identification of 3-formyl-1-butyl-pyrophosphate as the core of several non-prenyl mycobacterial phosphoantigens bioactive at the nM range. Recognition of this molecule by gammadelta T cells is very selective and relies on its aldehyde and pyrophosphate groups. This novel pyrophosphorylated aldehyde most probably corresponds to a metabolic intermediate of the non-mevalonate pathway of prenyl phosphate biosynthesis in eubacteria and algae. The reactivity to 3-formyl-1-butyl-pyrophosphate supports the view that human gammadelta T cells are physiologically devoted to antimicrobial surveillance.

A novel O-methyl-2,6-dideoxyhexose was isolated from the major phenolic glycolipid (previously called mycoside A) of Mycobacterium kansasii. Its molecular weight (162) was determined by gas chromatography-mass spectrometry analysis (chemical ionization with ammonia as reactant gas) of its underivatized reducing form. The methoxyl group was located by electron impact-mass spectrometry of its alditol acetate. The configuration was established by 1H NMR of its peracetylated derivative. The structure 2,6-dideoxy-4-O-methyl-arabino-hexopyranose is proposed for this new sugar. Evidence is also presented that the phenolic glycolipid previously called mycoside A is an antigen of M. kansasii since it reacts with rabbit antisera raised against whole M. kansasii.

Data from the literature indicate the presence, in the Mycobacterium kansasii wall, of a phenolic glycolipid called mycoside A. A tentative trisaccharide structure was proposed for the oligosaccharide part, whereas the aglycone was found to correspond to a phenol phthiocerol residue esterified by two mycocerosic acids. In the present work, structural information mainly arising from fast atom bombardment-mass spectrometry, tandem mass spectrometry, and 1H NMR of native and chemically degraded phenolic glycolipid indicates a tetrasaccharide structure for the inherent oligosaccharide. This structure is now determined as: 2,6-dideoxy-4-O-methyl-arabino-hexopyranosyl(1 alpha—3)2-O-methyl-4-O- acetylfucopyranosyl(1 alpha—3)2-O-methyl-rhamnopyranosyl(1 alpha—3)2-4-di- O-methylrhamnopyranosyl-1 alpha—phenolglycol. Evidence for the structure of the dideoxyhexose, unique in mycobacteria, is presented in the following paper (Fournié, J.-J., Rivière, M., Papa, F., and Puzo, G. (1987) J. Biol. Chem. 262, 3180-3184).

Using high-performance liquid chromatography, a new kind of phenolic glycolipid quantitatively minor, called phenolic glycolipid-II, was isolated from a lipidic fraction of Mycobacterium kansasii. The structure was determined by fast atom bombardment-mass spectrometry and proton nuclear magnetic resonance spectroscopy, as: 2,4-di-O-Me-alpha-D-Manp(1—3) 4-O-Ac-2-O-Me-alpha-L-Fucp(1—3)2-O-Me- alpha-L-Rhap(1—3) 2,4-di-O-Me-alpha-L-Rhap 1—phenolphthiocerol dimycocerosate. Phenolic glycolipids I and II differ only by their distal monosaccharide hapten which is 2,6-dideoxy-4-O-Me-alpha-D-arabinohexopyranosyl and the 2,4-di-O-Me-alpha-D-mannopyranosyl, respectively. This sugar appears to be characteristic and apparently unique in the Mycobacterium genus. Moreover, phenolic glycolipids I and II constitute with the lipooligosaccharides two classes of antigens of M. kansasii.