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Mycobacterium tuberculosisis the causative agent of tuberculosis and remains one of the most widespread and deadliest bacterial pathogens in the world. A distinguishing feature of mycobacteria that sets them apart from other bacteria is the unique architecture of their cell wall, characterized by various species-specific lipids, most notably mycolic acids (MAs). Therefore, targeted inhibition of enzymes involved in MA biosynthesis, transport, and assembly has been extensively explored in drug discovery. Additionally, more recent evidence suggests that many enzymes in the MA biosynthesis pathway are regulated by kinase-mediated phosphorylation, thus opening additional drug-development opportunities. However, how phosphorylation regulates MA production remains unclear. Here, we used genetic strategies combined with lipidomics and phosphoproteomics approaches to investigate the role of protein phosphorylation in Mycobacterium. The results of this analysis revealed that the Ser/Thr protein kinase PknB regulates the export of MAs and promotes the remodeling of the mycobacterial cell envelope. In particular, we identified the essential MmpL3 as a substrate negatively regulated by PknB. Taken together, our findings add to the understanding of how PknB activity affects the mycobacterial MA biosynthesis pathway and reveal the essential role of protein phosphorylation/dephosphorylation in governing lipid metabolism, paving the way for novel antimycobacterial strategies.

ABSTRACTTuberculosis is still a cause of major concern, partly due to the emergence of multidrug-resistant strains. New drugs are therefore needed. Vancomycin can target mycobacteria with cell envelope deficiency. In this study, we used a vancomycin susceptibility assay to detect drugs hampering lipid synthesis in Mycobacterium bovisBCG and in Mycobacterium tuberculosis. We tested three drugs already used to treat human obesity: tetrahydrolipstatin (THL), simvastatin, and fenofibrate. Only vancomycin and THL were able to synergize on M. bovisBCG and on M. tuberculosis, although mycobacteria could also be inhibited by simvastatin alone. Lipid analysis allowed us to identify several lipid modifications in M. tuberculosisH37Rv treated with those drugs. THL treatment mainly reduced the phthiocerol dimycocerosate (PDIM) content in the mycobacterial cell wall, providing an explanation for the synergy, since PDIM deficiency has been related to vancomycin susceptibility. Proteomic analysis suggested that bacteria treated with THL, in contrast to bacteria treated with simvastatin, tried to recover, inducing, among other reactions, lipid synthesis. The combination of THL and vancomycin should be considered a promising solution in developing new strategies to treat multidrug-resistant tuberculosis.

ABSTRACTMycobacterium tuberculosisis wrapped in complex waxes, impermeable to most antibiotics. Comparing Mycobacterium bovisBCG and M. tuberculosismutants that lack phthiocerol dimycocerosates (PDIM) and/or phenolic glycolipids with wild-type strains, we observed that glycopeptides strongly inhibited PDIM-deprived mycobacteria. Vancomycin together with a drug targeting lipid synthesis inhibited multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates. Our study puts glycopeptides in the pipeline of potential antituberculosis (TB) agents and might provide a new antimycobacterial drug-screening strategy.

FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the biosynthesis of mycolic acids, major and specific lipid components of the mycobacterial cell envelope, is essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel antituberculous drugs. Here, we describe the optimization of the protein purification procedure and the development of a high-throughput screening assay for FadD32 activity. This spectrophotometric assay measuring the release of inorganic phosphate was optimized using the Mycobacterium smegmatisFadD32 as a surrogate enzyme. We describe the use of Tm(melting temperature) shift assay, which measures the modulation of FadD32 thermal stability, as a tool for the identification of potential ligands and for validation of compounds as inhibitors. Screening of a selected library of compounds led to the identification of five novel classes of inhibitors.

Mycobacterium tuberculosis, the causative agent of tuberculosis, initially contacts host cells with elements of its outer cell wall, or capsule. We have shown that capsular material from the surface of M. tuberculosis competitively inhibits the nonopsonic binding of whole M. tuberculosis bacilli to macrophages in a dose-dependent manner that is not acting through a global inhibition of macrophage binding. We have further demonstrated that isolated M. tuberculosis capsular proteins mediate a major part of this inhibition. Two-dimensional polyacrylamide gel electrophoresis analysis of the capsular proteins showed the presence of a wide variety of protein species, including proportionately high levels of the Cpn60.2 (Hsp65, GroEL2) and DnaK (Hsp70) molecular chaperones. Both of these proteins were subsequently detected on the bacterial surface. To determine whether these molecular chaperones play a role in bacterial binding, recombinant Cpn60.2 and DnaK were tested for their ability to inhibit the association of M. tuberculosis bacilli with macrophages. We found that recombinant Cpn60.2 can inhibit ∼57% of bacterial association with macrophages, while DnaK was not inhibitory at comparable concentrations. Additionally, when polyclonal F(ab')2fragments of anti-Cpn60.2 and anti-DnaK were used to mask the surface presentation of these molecular chaperones, a binding reduction of ∼34% was seen for anti-Cpn60.2 F(ab')2, while anti-DnaK F(ab')2did not significantly reduce bacterial association with macrophages. Thus, our findings suggest that while M. tuberculosis displays both surface-associated Cpn60.2 and DnaK, only Cpn60.2 demonstrates adhesin functionality with regard to macrophage interaction.

ABSTRACTMycobacterium tuberculosis, the causative agent of tuberculosis, initially contacts host cells with elements of its outer cell wall, or capsule. We have shown that capsular material from the surface of M. tuberculosiscompetitively inhibits the nonopsonic binding of whole M. tuberculosisbacilli to macrophages in a dose-dependent manner that is not acting through a global inhibition of macrophage binding. We have further demonstrated that isolated M. tuberculosiscapsular proteins mediate a major part of this inhibition. Two-dimensional polyacrylamide gel electrophoresis analysis of the capsular proteins showed the presence of a wide variety of protein species, including proportionately high levels of the Cpn60.2 (Hsp65, GroEL2) and DnaK (Hsp70) molecular chaperones. Both of these proteins were subsequently detected on the bacterial surface. To determine whether these molecular chaperones play a role in bacterial binding, recombinant Cpn60.2 and DnaK were tested for their ability to inhibit the association of M. tuberculosisbacilli with macrophages. We found that recombinant Cpn60.2 can inhibit ∼57% of bacterial association with macrophages, while DnaK was not inhibitory at comparable concentrations. Additionally, when polyclonal F(ab′)2fragments of anti-Cpn60.2 and anti-DnaK were used to mask the surface presentation of these molecular chaperones, a binding reduction of ∼34% was seen for anti-Cpn60.2 F(ab′)2, while anti-DnaK F(ab′)2did not significantly reduce bacterial association with macrophages. Thus, our findings suggest that while M. tuberculosisdisplays both surface-associated Cpn60.2 and DnaK, only Cpn60.2 demonstrates adhesin functionality with regard to macrophage interaction.

Mycolic acids, the hallmark of mycobacteria and related bacteria, are major and specific components of their cell envelope and essential for the mycobacterial survival. Mycobacteria contain structurally related long-chain lipids, but the metabolic relationships between these various classes of compounds remain obscure. To address this question a series of C35to C54nonhydroxylated fatty acids (mycobacteric acids), ketones, and alcohols structurally related to the C70–80dicyclopropanated or diethylenic mycolic acids were characterized in three mycobacterial strains and their structures compared. The relationships between these long-chain acids and mycolic acids were established by following the in vivo traffic of 14C labeled α-mycolic acids purified from the same mycobacterial species. The labeling was exclusively found in mycobacteric acids. The mechanism of this degradation was established by incorporation of 18O2into long-chain lipids and shown to consist in the rupture of mycolic acids between carbon 3 and 4 by a Baeyer-Villiger-like reaction. We also demonstrated that mycobacteric acids occur exclusively in the triacylglycerol (TAG) fraction where one molecule of these acids esterifies one of the three hydroxyl groups of glycerol. Altogether, these data suggest that these compounds represent a pathway of metabolic energy that would be used by mycobacteria in particular circumstances.

Mycolic acids, the hallmark of mycobacteria and related bacteria, are major and specific components of their cell envelope and essential for the mycobacterial survival. Mycobacteria contain structurally related long-chain lipids, but the metabolic relationships between these various classes of compounds remain obscure. To address this question a series of C35to C54nonhydroxylated fatty acids (mycobacteric acids), ketones, and alcohols structurally related to the C70–80dicyclopropanated or diethylenic mycolic acids were characterized in three mycobacterial strains and their structures compared. The relationships between these long-chain acids and mycolic acids were established by following the in vivo traffic of 14C labeled α-mycolic acids purified from the same mycobacterial species. The labeling was exclusively found in mycobacteric acids. The mechanism of this degradation was established by incorporation of 18O2into long-chain lipids and shown to consist in the rupture of mycolic acids between carbon 3 and 4 by a Baeyer-Villiger-like reaction. We also demonstrated that mycobacteric acids occur exclusively in the triacylglycerol (TAG) fraction where one molecule of these acids esterifies one of the three hydroxyl groups of glycerol. Altogether, these data suggest that these compounds represent a pathway of metabolic energy that would be used by mycobacteria in particular circumstances.

Two subfamilies of the polar glycopeptidolipids (GPLs) located on the surface of Mycobacterium smegmatis, along with unknown phospholipids, were recently shown to participate in the nonopsonic phagocytosis of mycobacteria by human macrophages (Villeneuve, C., G. Etienne, V. Abadie, H. Montrozier, C. Bordier, F. Laval, M. Daffe, I. Maridonneau-Parini, and C. Astarie-Dequeker. 2003. Surface-exposed glycopeptidolipids of Mycobacterium smegmatisspecifically inhibit the phagocytosis of mycobacteria by human macrophages. Identification of a novel family of glycopeptidolipids. J. Biol. Chem.278: 51291–51300). As demonstrated herein, a phospholipid mixture that derived from the methanol-insoluble fraction inhibited the phagocytosis of M. smegmatis. Inhibition was essentially attributable to phosphatidylinositol mannosides (PIMs), namely PIM2and PIM6, because the purified phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol were inactive. This was further confirmed using purified PIM2and PIM6from M. bovisBCG that decreased by half the internalization of M. smegmatis. Both compounds also inhibited the uptake of M. tuberculosisand M. aviumbut had no effect on the internalization of zymosan used as a control particle of the phagocytic process. When coated on latex beads, PIM2and polar GPL (GPL III) favored the particle entry through complement receptor 3. GPL III, but not PIM2,also directed particle entry through the mannose receptor.

ABSTRACTMycobacteria contain an outer membrane of unusually low permeability which contributes to their intrinsic resistance to many agents. It is assumed that small and hydrophilic antibiotics cross the outer membrane via porins, whereas hydrophobic antibiotics may diffuse through the membrane directly. A mutant of Mycobacterium smegmatislacking the major porin MspA was used to examine the role of the porin pathway in antibiotic sensitivity. Deletion of the mspAgene caused high-level resistance of M. smegmatisto 256 μg of ampicillin/ml by increasing the MIC 16-fold. The permeation of cephaloridine in the mspAmutant was reduced ninefold, and the resistance increased eightfold. This established a clear relationship between the activity and the outer membrane permeation of cephaloridine. Surprisingly, the MICs of the large and/or hydrophobic antibiotics vancomycin, erythromycin, and rifampin for the mspAmutant were increased 2- to 10-fold. This is in contrast to those for Escherichia coli, whose sensitivity to these agents was not affected by deletion of porin genes. Uptake of the very hydrophobic steroid chenodeoxycholate by the mspAmutant was retarded threefold, which supports the hypothesis that loss of MspA indirectly reduces the permeability by the lipid pathway. The multidrug resistance of the mspAmutant highlights the prominent role of outer membrane permeability for the sensitivity of M. smegmatisto antibiotics. An understanding of the pathways across the outer membrane is essential to the successful design of chemotherapeutic agents with activities against mycobacteria.

ABSTRACTThe first-line specific antituberculous drug isoniazid inhibits the fatty acid elongation system (FAS) FAS-II involved in the biosynthesis of mycolic acids, which are major lipids of the mycobacterial envelope. The MabA protein that catalyzes the second step of the FAS-II elongation cycle is structurally and functionally related to the in vivo target of isoniazid, InhA, an NADH-dependent enoyl-acyl carrier protein reductase. The present work shows that the NADPH-dependent β-ketoacyl reduction activity of MabA is efficiently inhibited by isoniazid in vitro by a mechanism similar to that by which isoniazid inhibits InhA activity. It involves the formation of a covalent adduct between MnIII-activated isoniazid and the MabA cofactor. Liquid chromatography-mass spectrometry analyses revealed that the isonicotinoyl-NADP adduct has multiple chemical forms in dynamic equilibrium. Both kinetic experiments with isolated forms and purification of the enzyme-ligand complex strongly suggested that the molecules active against MabA activity are the oxidized derivative and a major cyclic form. Spectrofluorimetry showed that the adduct binds to the MabA active site. Modeling of the MabA-adduct complex predicted an interaction between the isonicotinoyl moiety of the inhibitor and Tyr185. This hypothesis was supported by the fact that a higher 50% inhibitory concentration of the adduct was measured for MabA Y185L than for the wild-type enzyme, while both proteins presented similar affinities for NADP+. The crystal structure of MabA Y185L that was solved showed that the substitution of Tyr185 induced no significant conformational change. The description of the first inhibitor of the β-ketoacyl reduction step of fatty acid biosynthesis should help in the design of new antituberculous drugs efficient against multidrug-resistant tubercle bacilli.

Diesters of phthiocerol and phenolphthiocerol are important virulence factors ofMycobacterium tuberculosisand Mycobacterium leprae, the two main mycobacterial pathogens in humans. They are both long-chain β-diols, and their biosynthetic pathway is beginning to be elucidated. Although the two classes of molecules share a common lipid core, phthiocerol diesters have been found in all the strains of the M. tuberculosiscomplex examined although phenolphthiocerol diesters are produced by only a few groups of strains. To address the question of the origin of this diversity 8 reference strains and 10 clinical isolates of M. tuberculosiswere analyzed. We report the presence of glycosylated p-hydroxybenzoic acid methyl esters, structurally related to the type-specific phenolphthiocerol glycolipids, in the culture media of all reference strains of M. tuberculosis, suggesting that the strains devoid of phenolphthiocerol derivatives are unable to elongate the putativep-hydroxybenzoic acid precursor. We also show that all the strains of M. tuberculosisexamined and deficient in the production of phenolphthiocerol derivatives are natural mutants with a frameshift mutation in pks15/1whereas a single open reading frame for pks15/1is found in Mycobacterium bovisBCG, M. leprae,and strains of M. tuberculosisthat produce phenolphthiocerol derivatives. Complementation of the H37Rv strain of M. tuberculosis,which is devoid of phenolphthiocerol derivatives, with the fusedpks15/1gene from M. bovisBCG restored phenolphthiocerol glycolipids production. Conversely, disruption of thepks15/1gene in M. bovisBCG led to the abolition of the synthesis of type-specific phenolphthiocerol glycolipid. These data indicate that Pks15/1 is involved in the elongation of p-hydroxybenzoic acid to givep-hydroxyphenylalkanoates, which in turn are converted, presumably by the PpsA-E synthase, to phenolphthiocerol derivatives.

Biogenesis of phagolysosomes is a very rapid event in neutrophils which takes place with nascent unclosed phagosomes, leading to the release of lysosomal enzymes such as beta-glucuronidase in the extracellular medium. We have previously shown that, under nonopsonic conditions, both pathogenic and nonpathogenic mycobacteria uncouple phagocytosis from fusion of azurophil granules (specialized secretory lysosomes) with phagosomes. In the present study we questioned whether they actively act on neutrophils to block this process or use phagocytic receptors that negatively control the biogenesis of phagolysosomes. As for live unicellular Mycobacterium smegmatis, we observed that nonopsonic phagocytosis of heat-killed mycobacteria did not induce the release of beta-glucuronidase, indicating that M. smegmatis does not actively act on the fusion process in neutrophils. In contrast, phagocytosis of unicellular M. smegmatis opsonized in immune serum or that of small nonopsonized mycobacterial aggregates restored the biogenesis of phagolysosomes. Aggregates were internalized in a CR3- and cholesterol-dependent manner as unicellular mycobacteria. However, aggregates but not unicellular bacteria triggered F-actin and Hck recruitment at the phagosomes, events that have been associated with lysosome fusion. Thus, we propose that M. smegmatis does not actively control the fusion of azurophil granules at early time points postinfection and that mycobacterial aggregates recruit large clusters of receptors at the neutrophil surface which could trap proteins implicated in the biogenesis of phagolysosomes.

ABSTRACTBiogenesis of phagolysosomes is a very rapid event in neutrophils which takes place with nascent unclosed phagosomes, leading to the release of lysosomal enzymes such as β-glucuronidase in the extracellular medium. We have previously shown that, under nonopsonic conditions, both pathogenic and nonpathogenic mycobacteria uncouple phagocytosis from fusion of azurophil granules (specialized secretory lysosomes) with phagosomes. In the present study we questioned whether they actively act on neutrophils to block this process or use phagocytic receptors that negatively control the biogenesis of phagolysosomes. As for live unicellular Mycobacterium smegmatis, we observed that nonopsonic phagocytosis of heat-killed mycobacteria did not induce the release of β-glucuronidase, indicating that M. smegmatisdoes not actively act on the fusion process in neutrophils. In contrast, phagocytosis of unicellular M. smegmatisopsonized in immune serum or that of small nonopsonized mycobacterial aggregates restored the biogenesis of phagolysosomes. Aggregates were internalized in a CR3- and cholesterol-dependent manner as unicellular mycobacteria. However, aggregates but not unicellular bacteria triggered F-actin and Hck recruitment at the phagosomes, events that have been associated with lysosome fusion. Thus, we propose that M. smegmatisdoes not actively control the fusion of azurophil granules at early time points postinfection and that mycobacterial aggregates recruit large clusters of receptors at the neutrophil surface which could trap proteins implicated in the biogenesis of phagolysosomes.

Among the few characterized genes that have products involved in the pathogenicity of Mycobacterium tuberculosis, the etiological agent of tuberculosis, are those of the phthiocerol dimycocerosate (DIM) locus. Genes involved in biosynthesis of these compounds are grouped on a 50-kilobase fragment of the chromosome containing 13 genes. Analysis of mRNA produced from this 50-kilobase fragment in the wild type strain showed that this region is subdivided into three transcriptional units. Biochemical characterization of five mutants with transposon insertions in this region demonstrated that (i) the complete DIM molecules are synthesized in the cytoplasm of M. tuberculosisbefore being translocated into the cell wall; (ii) the genesfadD26and fadD28are directly involved in their biosynthesis; and (iii) both the drrCandmmpL7genes are necessary for the proper localization of DIMs. Insertional mutants unable to synthesize or translocate DIMs exhibit higher cell wall permeability and are more sensitive to detergent than the wild type strain, indicating for the first time that, in addition to being important virulence factors, extractable lipids of M. tuberculosisplay a role in the cell envelope architecture and permeability. This function may represent one of the molecular mechanisms by which DIMs are involved in the virulence ofM. tuberculosis.

The subcomponents of bacille Calmette-Guérin (BCG) involved in the mechanism of action of intravesical BCG immunotherapy used for prophylaxis of superficial bladder cancer recurrences have been poorly investigated. We purified various BCG subcomponents and analyzed in vitro their ability to enhance a Th1 polarized immune response as well as to increase lymphocyte-mediated cytotoxicity against bladder tumors. Human peripheral blood mononuclear cells (PBMCs) from healthy purified protein derivative–positive subjects were incubated for 7 days with whole BCG and various fractions (BCG cell wall, plasma membrane, cytosol, purified polysaccharides as glucan or arabinomannan, purified native proteins from BCG culture filtrate, recombinant 22 kDa protein, phosphate transporter PstS-2 and -3 proteins). IFN-γ, IL-12, IL-2, and IL-6 production by stimulated PBMCs was compared to unstimulated controls and the phenotype of expanded cells analyzed by flow cytometry (FACS analysis). A ⁵¹Cr-release assay monitored the cytotoxicity of amplified effector cells against T24 bladder tumor cells. Live BCG and most of its subcomponents (with the exception of cytosol, PstS-2 and -3) significantly enhanced IFN-γ and IL-12 secretion, expanded CD3^–CD56⁺ cells and the non-MHC-restricted cytotoxicity against bladder tumor cells compared to unstimulated controls (all P < 0.001, t-test). IL-2 receptor blockage resulted in a clear reduction in the cytotoxic activity of stimulated PBMCs. Numerous BCG subcomponents thus provide positive stimuli for Th1 cell differentiation and enhance in vitro, non-MHC-restricted cytotoxicity against bladder tumor cells. Our findings provide the basis for the therapeutic use of several of these subfractions in experimental animal models bearing bladder tumors. Int. J. Cancer 87:844–852, 2000. © 2000 Wiley-Liss, Inc.

Five new inteins were discovered in a survey of 39 mycobacterial strains that was undertaken to clarify the role of RecA inteins in mycobacteria. They are all inserted at the RecA‐b site of the recAgene of Mycobacterium chitae, M. fallax, M. gastri, M. shimodeiand M. thermoresistibileand belong to the MleRecA allelic family. Sequence analysis showed that although only M. tuberculosisharbours an intein at the RecA‐a site the sequence of the RecA‐b site is well conserved between species. Furthermore, the presence of inteins does not correlate with specific characteristics of the species such as pathogenicity or growth rate.

Characterization of proteins with unknown functions is a critical research priority as the intracellular growth and metabolic state of Mycobacterium tuberculosis, the causative agent of tuberculosis, remain poorly understood. Mycofactocin is a peptide-derived redox cofactor present in almost all mycobacterial species; however, its functional relevance in M. tuberculosispathogenesis and host survival has never been studied experimentally.

A careful re‐examination of the glycolipid content of clinical isolates and reference strains of the tubercle bacillus, Mycobacterium tuberculosis, led to the identification of a glycoconjugate that passed unnoticed in earlier studies. Nuclear magnetic resonance spectroscopy, gas chromatography‐mass spectrometry and chemical degradations were used to identify the glycolipid as a 2,3,6‐triacyl trehalose. The glycolipid contains a phthienoic acyl substituent, a family of multimethyl‐branched, α,β‐unsaturated fatty acids specific for virulent strains of the tubercle bacillus. It reacted with sera from tuberculosis patients with a specificity and sensitivity of 96.2% and 76%, respectively. Comparable data were obtained with the 2,3‐diacyl trehaloses of M. tuberculosisand M. fortuitumand with the triacyl trehaloses of M. fortuitum,suggesting that the antigens from the latter species may be used for the serodiagnosis of tuberculosis.

Essential oil was obtained in a yield 1.1%, w/w, by steam distillation of Elionurus tristisleaves from Madagascar. The chemical composition was analyzed qualitatively and quantitatively by GC-MS and GC-FID, respectively. To the best of our knowledge, this is the first chemical analysis of this essential oil. Seventy-three compounds were identified, corresponding to 94.9% of the total essential oil. The principal compounds were sesquiterpenes and the more represented were β-gurjunene (18.4%), neoclovene (15.8%) and nootkatone (10.4%). Through a comparative study, we observed a large variability between the components of E. tristisessential oil and those from others species of the same genus. Evaluation of the antioxidant (ABTS and DPPH assays) and anti-tuberculosis activities of the essential oil showed weak antioxidant potency but an interesting anti-tuberculosis activity with a MIC of 32 mg/L. This activity prompted us to evaluate individually the major components for the treatment of tuberculosis.

Tsukamurella paurometabolum and Mycobacterium fallax are members of the suprageneric actinomycete group Corynebacterineae that possesses a cell wall skeleton composed of a peptidoglycan to which an arabinogalactan is covalently attached. This polysaccharide is further modified by esterification with C60–C80 mycolic acid residues in mycobacteria and T. paurometabolum. However, M. fallax and T. paurometabolum produce polyenoic (up to six double bonds) mycolic acids whereas the most common type of mycobacterial mycolates, called α-mycolates, are mono- and di-enoic or -cyclopropanated mycolic acids. To determine whether this difference also applied to the structures of cell wall arabinogalactans, competitive inhibition experiments using antibodies raised against the cell wall from Mycobacterium bovis and the arabinogalactans from T. paurometabolum and M. fallax were performed. They demonstrated the structural identity between the polysaccharide of M. fallax and those of mycobacteria and showed a strong similarity between the latter polysaccharides and that of T. paurometabolum. Structural analyses of the per-O-alkylated alditol fragments derived from the polysaccharides by gas chromatography–mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR) spectroscopy of the intact solubilized polysaccharides demonstrated that the polysaccharides from the two species analyzed contained all the major structural features previously characterized in mycobacterial arabinogalactans. These include (1) the homogalactan of alterning 5-linked galactofuranosyl (Galf) and 6-linked Galf residues, (2) a linear 5-linked arabino furanosyl (Araf), (3) a β-Araf-(1→2)-α-Araf disaccharide branched on both position 3 and position 5 of an α-Araf unit, and (4) a 5-linked-α-Araf unit branched on both position 3 and position 5 of an α-Araf residue. The polysaccharide from T. paurometabolum possesses additional structural domains composed of a terminal (t) Araf directly linked to either a 5-linked-α-Araf or to both position 3 and position 5 of a 3,5-linked α-Araf unit. Both the remarkable similarity of arabinogalactans from Corynebacterineae and their genus- and/or species-specificities are reflected in their 13C NMR spectra that may be used as a valuable help in the identification of members of the actinomycete group.

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 M. fortuitum, was due to the nature of the fatty acyl substituents 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.

Incubation of Mycobacterium tuberculosis and M. smegmatis cells with the sugar components of their surface-exposed glycans demonstrated that d-arabinose, but not α-d-glucose or d-mannose, led to the dispersion of the large clumps formed by the bacilli in stationary liquid cultures. These results confirm the presence of arabinose-containing glycans on the mycobacterial cell surface and demonstrate the implication of selective sugars in cell aggregation, suggesting that the clumping of mycobacterial cells is probably mediated by lectin-carbohydrate interactions.

A careful re-examination of the glycolipid content of clinical isolates and reference strains of the tubercle bacillus, Mycobacterium tuberculosis, led to the identification of a glycoconjugate that passed unnoticed in earlier studies. Nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry and chemical degradations were used to identify the glycolipid as a 2,3,6-triacyl trehalose. The glycolipid contains a phthienoic acyl substituent, a family of multimethyl-branched, α,β-unsaturated fatty acids specific for virulent strains of the tubercle bacillus. It reacted with sera from tuberculosis patients with a specificity and sensitivity of 96.2% and 76%, respectively. Comparable data were obtained with the 2,3-diacyl trehaloses of M. tuberculosis and M. fortuitum and with the triacyl trehaloses of M. fortuitum, suggesting that the antigens from the latter species may be used for the serodiagnosis of tuberculosis.

Nuclear magnetic resonance spectroscopy, fast-atom bombardment mass spectrometry as well as various chemical degradations and chromatographic techniques were used to re-examine the structure of a highly immunoreactive glycolipid previously described in Mycobacterium tuberculosis (strain Canetti) as a 2,3-diacyl trehalose 2′-sulfate (labelled SL-IV). Ion exchange chromatography allowed the recognition of a neutral and an acidic glycolipid, indistinguishable on conventional silica gel. The neutral glycolipid was shown to be serologically identical to SL-IV and its structure was established as 2,3-diacyl trehalose. It corresponded to the non-chemically defined highly observed immunoreactive lipid previously recognized by other in M. tuberculosis (H37Rv).