Intact glycans from cestode antigens are involved in innate activation of myeloid suppressor cells.

During helminthic infections, strong Th2 type-biased responses concomitant with impaired cell-proliferative responses to parasitic and unrelated antigens are major immunological hallmarks. Parasite glycan structures have been proposed to play a role in modulating these responses. To understand early events related to immune modulation during cestode infection, we have examined the role of intact glycans of antigens from Taenia crassiceps in the recruitment of innate cells. Soluble antigens from this cestode contained higher levels of carbohydrates than proteins. Intraperitoneal injection of the antigens rapidly recruited a cell population expressing F4/80(+)/Gr-1(+)surface markers, which adoptively suppressed naïve T-cell proliferation in vitro in response to anti-CD3/CD28 MAb stimulation in a cell-contact dependent manner. Soluble antigens with altered glycans by treatment with sodium periodate significantly reduced the recruitment of F4/80(+)/Gr1(+)cells, concomitantly their suppressive activity was abrogated, indicating that glycans have a role in the early activation of these suppressor cells. Using C3H/HeJ and STAT6-KO mice, we found that expansion and suppressive activity of F4/80(+)Gr1(+)cells induced by T. crassiceps intact antigens was TLR4 and Th2-type cytokine independent. Together with previous studies on nematode and trematode parasites, our data support the hypothesis that glycans can be involved on a similar pathway in the immunoregulation by helminths.

High-polarity Mycobacterium avium-derived lipids interact with murine macrophage lipid rafts.

Cholesterol- and sphingolipid-rich membrane microdomains (lipid rafts) are widely recognized as portals for pathogenic micro-organisms. A growing body of evidence demonstrates mobilization of host plasma cell membrane lipid rafts towards the site of contact with several pathogens as well as a strict dependence on cholesterol for appropriate internalization. The fate of lipid rafts once the pathogen has been internalized and the nature of the pathogen components that interact with them is however less understood. To address both these issues, infection of the J774 murine cell line with Mycobacterium avium was used as a model. After demonstrating that M. avium induces lipid raft mobilization and that M. avium infects J774 by a cholesterol-dependent mechanism, it is shown here that mycobacterial phagosomes harbour lipid rafts, which are, at least in part, of plasma cell membrane origin. On the other hand, by using latex microbeads coated with any of the three fractions of M. avium-derived lipids of different polarity, we provide evidence that high-polarity, in contrast to low-polarity and intermediate-polarity, mycobacterial lipids or uncoated latex beads have a strong capacity to induce lipid raft mobilization. These results suggest that high-polarity mycobacterial lipid(s) interact with host cell cholesterol-enriched microdomains which may in turn influence the course of infection.

Mycobacterial di-O-acyl-trehalose inhibits mitogen- and antigen-induced proliferation of murine T cells in vitro.

2,3-Di-O-acyl-trehalose (DAT) is a glycolipid located on the outer layer of the Mycobacterium tuberculosis cell envelope. Due to its noncovalent linkage to the mycobacterial peptidoglycan, DAT could easily interact with host cells located in the focus of infection. The aim of the present work was to study the effects of DAT on the proliferation of murine spleen cells. DAT was purified from reference strains of M. tuberculosis, or M. fortuitum as a surrogate source of the compound, by various chromatography and solvent extraction procedures and then chemically identified. Incubation of mouse spleen cells with DAT inhibited in a dose-dependent manner concanavalin A-stimulated proliferation of the cells. Experiments, including the propidium iodide exclusion test, showed that these effects were not due to death of the cells. Tracking of cell division by labeling with 5,6-carboxyfluorescein diacetate succinimidyl ester revealed that DAT reduces the rounds of cell division. Immunofluorescence with an anti-CD3 monoclonal antibody indicated that T lymphocytes were the population affected in our model. Our experiments also suggest that the extent of the suppressive activity is strongly dependent on the structural composition of the acyl moieties in DATs. Finally, the inhibitory effect was also observed on antigen-induced proliferation of mouse spleen cells specific for Toxoplasma gondii. All of these data suggest that DAT could have a role in the T-cell hyporesponsiveness observed in chronic tuberculosis.

Mycobacterium fortuitum glycolipids for the serodiagnosis of pulmonary tuberculosis.

Glycolipids belonging to the family of acylated trehaloses were isolated from Mycobacterium fortuitum, a rapidly growing mycobacterial species, and tested in the serologic diagnosis of human pulmonary tuberculosis by enzyme-linked immunosorbent assay. Di- and tri-O-acylated trehaloses from M. fortuitum reacted with serum antibodies of patients with pulmonary tuberculosis at higher titers than did with sera from healthy donors. With both glycolipids, the sensitivity of the test was above 0.80 at a chosen specificity of 0.98. Individuals with treated tuberculosis showed lower antibody titers compared with their initial reactivities. These data show that M. fortuitum could be used as a surrogate source of antigens for tuberculosis serodiagnosis.

Structures of the glycolipid antigens of members of the third biovariant complex of Mycobacterium fortuitum.

Among the fast-growing mycobacteria, members of the Mycobacterium fortuitum complex are the most-commonly cited opportunistic human pathogens, notably in post-surgical infections. Previous studies showed that this complex was composed of four well-identified species and a group of isolates that did not correspond to recognized species, which has been referred to as the third biovariant complex. The occurrence and chemical structure of the glycolipid antigens of six strains that belong to this latter group were examined in the present study. Based on the TLC profiles, resistance to alkali and seroreactivities of their glycolipids, the examined strains were classified into three groups: one group was devoid of species-specific glycolipid and the two other groups contained alkali-stable or alkali-labile glycoconjugates. The structures of the major glycolipid antigens of the latter two groups were elucidated by fast-atom-bombardment MS, one-dimensional and two-dimensional NMR spectroscopy and conventional chemical analyses. The alkali-stable glycolipids were structurally identical to the C-mycoside-type glycopeptidolipids characterized in the taxonomically related species Mycobacterium peregrinum. The major alkali-labile glycolipid was identified as beta-Glcp-1 --> 6)-alpha-Glcp2Acyl-(1 --> 1)-alpha-GLcp3,4,6Acyl3. The acyl substituents consisted on one acetyl group and three fatty acyl residues composed mainly of tetradecanoyl residues, but significant amounts of 2-methylhexadecanoyl and 2-methyloctadecanoyl substituents were also present. The heterogeneity of the glycolipid content of members of the third biovariant complex of M. fortuitum demonstrated in the present study confirms the heterogeneity of the complex. In addition, the occurrence of a species-specific glycolipid in some strains supports the hypothesis that some strains of this complex of M. fortuitum may belong to a new mycobacterial species.

Interactions of mycobacterial glycopeptidolipids with membranes: influence of carbohydrate on induced alterations.

Glycopeptidolipids (GPLs) are specific constituents of mycobacteria known as opportunistic pathogens. The influence of the carbohydrate moiety on GPL-induced membrane alterations was examined with GPLs bearing 1-5 sugar residues (GPL-1 to GPL-5) and a sulfated GPL (S-GPL-2). GPLs decreased the ADP/O ratio and increased controlled respiration of isolated mitochondria. The more polar GPLs were the less active, with the following order of efficiency: GPL-1 > GPL-2 > S-GPL-2 = GPL-3 = GPL-5. GPL-1 and GPL-2 increased passive permeability of liposomes to carboxyfluorescein (GPL-1 > GPL-2), while GPL-3 and GPL-5 were inactive. GPL-2 and GPL-3 decreased the transmembrane electrical potential (delta psi) in isolated mitochondria (GPL-2 > GPL-3). These results suggest that GPLs uncouple oxidative phosphorylation by increasing the passive permeability of the mitochondrial membrane to protons. Compression isotherms of GPL-2 monolayers showed that, at low surface pressure, the area per GPL-2 molecule was about 5 times that of an acyl chain: it is likely that the peptide moiety was at the air/water interface. With an increase in the surface pressure, its area decreased, down to that of a tightly packed acyl chain. It is postulated that the glycopeptidic moiety can be either at in the interface or dipping into the water.(ABSTRACT TRUNCATED AT 250 WORDS)

Structures of the glycopeptidolipid antigens of Mycobacterium abscessus and Mycobacterium chelonae and possible chemical basis of the serological cross-reactions in the Mycobacterium fortuitum complex.

Mycobacterium abscessus and Mycobacterium chelonae, two members of the Mycobacterium fortuitum complex, contain five major glycolipids. A combination of NMR spectroscopy, fast atom bombardment mass spectrometry and chemical degradation was used to elucidate their structures. All the compounds belong to the family of glycopeptidolipids. A 6-deoxy-alpha-L-talosyl unit, which may bear one or two acetyl groups, invariably occupies the site of glycosylation on the threonine residue in the various compounds. A 3,4-di-O-methyl- or 2,3,4-tri-O-methyl-alpha-L-rhamnosyl unit modifies the alaninol end of the diglycosylated molecules. Both species also contain a multiglycosylated compound consisting of alpha-L-rhamnosyl-(1-->2)-3,4-di-O-methyl-alpha-L-rhamnosyl linked to alaninol, which belongs to the class of new variants of glycopeptidolipids recently described. Using an ELISA, the latter glycolipid as well as the diglycosylated ones (not previously reported to be antigenic), were shown to react with the serum raised against the whole lipid antigens of M. chelonae. A comparative serologic study of the native and chemically modified glycopeptidolipid antigens allowed the identification of their epitope as the 3,4-di-O-methyl-alpha-L-rhamnosyl residue. Similar experiments conducted on the glycopeptidolipids isolated from the serologically cross-reacting species M. peregrinum led to the conclusion that the epitope identified in M. chelonae and M. abscessus was involved in the cross-reactions and demonstrated the existence of a second haptenic moiety in the glycolipids of M. peregrinum, the 3-O-methyl-alpha-L-rhamnosyl unit. In addition to this latter non-shared epitope, the recently described sulfated glycopeptidolipid antigen of M. peregrinum did not react with the M. chelonae serum, thus further explaining the difference in the seroreactivity within the complex.

Structures of the glycopeptidolipid antigens of two animal pathogens: Mycobacterium senegalense and Mycobacterium porcinum.

The structures of the major glycolipid antigens of two animal pathogens Mycobacterium senegalense and Mycobacterium porcinum were elucidated by a combination of fast-atom bombardment mass spectrometry, nuclear magnetic resonance spectroscopy, chemical analyses and radiolabeling experiments. Five glycoconjugates belonging to the class of C-mycoside glycopeptidolipids were characterized in each species. They shared with those recently described in M. peregrinum the same unusual distribution of the disaccharides on the alaninol end of the molecules. Both species showed the presence of the novel sulfated glycopeptidolipid. In addition, some acetylated forms of the glycolipids were also present in the species examined. Identical seroreactivities were observed between the glycolipid antigens extracted from M. senegalense, M. porcinum and M. peregrinum and an antiserum raised against the whole lipid antigens of M. peregrinum. These data reinforce the close taxonomic relationships between the three mycobacterial species and demonstrate the antigenicity of the new variants of mycobacterial glycopeptidolipids.

Structure of a novel sulfate-containing mycobacterial glycolipid.

We described previously the unusual structures of the two major C-mycoside glycopeptidolipids from Mycobacterium fortuitum biovar. peregrinum. More polar glycolipids, potentially more interesting in terms of antigenicity, were also present in the strains. A combination of FAB mass spectrometry, NMR, chemical analyses, and radiolabeling was successfully applied to these glycolipids to arrive at the unexpected and novel structure for the more polar compound. This consisted of the "orthodox" basic structure of the apolar C-mycosides, modified at the alaninol end by the presence of a sulfate group on position 2 of a 3,4-di-O-methylrhamnosyl residue. This novel and second class of sulfate-containing mycobacterial glycolipid may provide a chemical basis for the differentiation and classification of members of the M. fortuitum complex, the main group causing human diseases among the many fast-growing mycobacteria widely distributed in nature.

Structure of mycoside F, a family of trehalose-containing glycolipids of Mycobacterium fortuitum.

Nuclear magnetic resonance spectroscopy, fast-atom bombardment mass spectrometry, gas chromatography-mass spectrometry, as well as chemical degradations were used to elucidate the structure of the major glycolipids of Mycobacterium fortuitum. Three main glycoconjugates were detected and their structures established as 2,3-diacyl, 2,3,4- and 2,3,6-triacyl trehalose. The characteristic infrared spectrum which led to their original designation as mycoside F, a family of glycolipids limited in distribution to M. fortuitum, was due to the nature of the fatty acyl substiuents identified primarily as 2-methyl-octadecen-2-oyl. The antigenic glycolipids typified the biovar. fortuitum, thus allowing its easy recognition from the C-mycoside glycopeptidolipid-containing biovar. peregrinum.

Fast atom bombardment mass spectrometry of Mycobacterial glycopeptidolipid antigens: Structural characterization by charge remote fragmentation.

Mycobacteria contain species- and type-specific antigens. Among them, glycopeptidolipids are present in medically relevant organisms belonging to Mycobacterium avium or M. fortuitum complexes. Fast-a tom bombardment mass spectrometry of glycopeptidolipids has proven to be difficult. In this article the cationization method with a metanitrobenzyl alcohol matrix, doped with sodium iodide, is described for analyzing these molecules. The molecular weight of the intact glycopeptidolipids was successfully determined and, using mass-analyzed ion kinetic energy spectrometry, the complete sequences of the peptide and saccharide moieties were elucidated. Moreover, the two structural variants present in these molecules were clearly differentiated. Application of the method showed that the same structural variant occurs in the glycopeptidolipids from two serologically related species of the M. fortuitum complex.

Ethylenic mycolic acid biosynthesis: extension of the biosynthetic model using cell-free extracts of Mycobacterium aurum and Mycobacterium smegmatis.

The hypothetical schemes proposed for the biosynthesis of unsaturated mycolic acids (R1-CH(OH)-CH(R2)-COOH) of Mycobacteria cell walls were experimentally tested by using cell-free extracts either of Mycobacterium aurum or of Mycobacterium smegmatis which produce two kinds of unsaturated mycolic acids (mono and dialkene), [1-14C]acetate being the precursor. Examination of specific radioactivities, in the presence or in the absence of isoniazid, an antituberculous drug inhibiting mycolic acid synthesis, showed that saturated C22 and C24 acids play a role as precursors of two distinct parts of the mycolic acids. Moreover, determination of labelling distribution into mycolic acid fragments obtained by oxidative and pyrolytic cleavages showed first that the side chain R2 and the methyl end R1 both have these C22 and C24 saturated fatty acids as common precursors. Secondly, it is thought that the fragments located between the methyl end R1 and the side chain R2 mainly result from elongation steps (one or two successive additions of seven or eight C2 units according to the mycolic acid type) and a biosynthetic model is proposed for unsaturated mycolic acids extending the published models and illustrating the missing step in monoalkene formation.

Glycopeptidolipids from Mycobacterium fortuitum: a variant in the structure of C-mycoside.

Strains from the Mycobacterium fortuitum complex contain surface species-specific lipids allowing their precise identification. In M. fortuitum biovar. peregrinum two major glycopeptidolipids, of the C-mycoside type, were characterized by a combination of chemical analyses, NMR, and FAB mass spectrometry. Important information was obtained by mass spectrometry both on their molecular weight and on the peptide and saccharide sequences without any derivatization. The basic structure of the two compounds was shown to be [formula: see text] The disaccharide part linked O-glycosidically to alaninol was either 3,4-di-O-methyl-alpha-L-rhamnopyranosyl (1----2) 3,4-di-O-methyl-alpha-L-rhamnopyranoside (mycoside I) or 3-O-methyl-alpha-L-rhamnopyranosyl (1----2) 3,4-di-O-methyl-alpha-L-rhamnopyranoside (mycoside II). This is an unusual structure of a C-mycoside since neither 6-deoxytalose nor its derivatives are present. Moreover, the oligosaccharide part is linked to the alaninol residue instead of the allo-threonine.