Uptake of unnatural trehalose analogs as a reporter for Mycobacterium tuberculosis

The detection of tuberculosis currently relies upon insensitive and unspecific techniques; newer diagnostics would ideally co-opt specific bacterial processes to provide real-time readouts. The trehalose mycolyltransesterase enzymes (antigens 85A, 85B and 85C (Ag85A, Ag85B, Ag85C)) serve as essential mediators of cell envelope function and biogenesis in Mycobacterium tuberculosis. Through the construction of a systematically varied sugar library, we show here that Ag85 enzymes have exceptionally broad substrate specificity. This allowed exogenously added synthetic probes to be specifically incorporated into M. tuberculosis growing in vitro and within macrophages. Even bulky substituents, such as a fluorescein-containing trehalose probe (FITC-trehalose), were incorporated by growing bacilli, thereby producing fluorescent bacteria; microscopy revealed selective labeling of poles and membrane. Addition of FITC-trehalose to M. tuberculosis-infected macrophages allowed selective, sensitive detection of M. tuberculosis within infected mammalian macrophages. These studies suggest that analogs of trehalose may prove useful as probes of function and for other imaging modalities.     

Crystal structure of a substrate complex of Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III (FabH) with lauroyl-coenzyme A

Beta-ketoacyl-acyl carrier protein synthase III (FabH) catalyzes a two step reaction that initiates the pathway of fatty acid biosynthesis in plants and bacteria. In Mycobacterium tuberculosis, FabH catalyzes extension of lauroyl, myristoyl and palmitoyl groups from which cell wall mycolic acids of the bacterium are formed. The first step of the reaction is an acyl group transfer from acyl-coenzyme A to the active-site cysteine of the enzyme; the second step is acyl chain extension by two carbon atoms through Claisen condensation with malonyl-acyl carrier protein. We have previously determined the crystal structure of a type II, dissociated M.tuberculosis FabH, which catalyzes extension of lauroyl, myristoyl and palmitoyl groups. Here we describe the first long-chain Michaelis substrate complex of a FabH, that of lauroyl-coenzyme A with a catalytically disabled Cys-->Ala mutant of M.tuberculosis FabH. An elongated channel extending from the mutated active-site cysteine defines the acyl group binding locus that confers unique acyl substrate specificity on M.tuberculosis FabH. CoA lies in a second channel, bound primarily through interactions of its nucleotide group at the enzyme surface. The apparent weak association of CoA in this complex may play a role in the binding and dissociation of long chain acyl-CoA substrates and products and poses questions pertinent to the mechanism of this enzyme.     

Mucosal administration of Ag85B-ESAT-6 protects against infection with Mycobacterium tuberculosis and boosts prior bacillus Calmette-Guerin immunity

We have examined the intranasal administration of a vaccine against Mycobacterium tuberculosis (M.tb) consisting of the mucosal adjuvant LTK63 and the Ag Ag85B-ESAT-6. Vaccination with LTK63/Ag85B-ESAT-6 gave a strong and sustained Th1 response mediated by IFN-gamma-secreting CD4 cells, which led to long-lasting protection against tuberculosis, equivalent to that observed with bacillus Calmette-Guérin (BCG) or Ag85B-ESAT-6 in dimethyldioctadecylammonium bromide/monophosphoryl lipid A. Because a crucial element of novel vaccine strategies is the ability to boost BCG-derived immunity, we also tested whether LTK63/Ag85B-ESAT-6 could act as a BCG booster vaccine in BCG-vaccinated mice. We found that vaccinating with LTK63/Ag85B-ESAT-6 strongly boosted prior BCG-stimulated immunity. Compared with BCG-vaccinated nonboosted mice, we observed that infection with M.tb led to a significant increase in anti-M.tb-specific CD4 T cells in the lungs of LTK63/Ag85B-ESAT-6-boosted animals. This correlated with a significant increase in the protection against M.tb in LTK63/Ag85B-ESAT-6-boosted mice, compared with BCG-vaccinated animals. Thus, LTK63/Ag85B-ESAT-6 represents an efficient preventive vaccine against tuberculosis with a strong ability to boost prior BCG immunity.     

The immunity and protective effects of antigen 85A and heat-shock protein X against progressive tuberculosis

The anti-tuberculosis vaccine, Mycobacterium bovis BCG, has been used worldwide, but its protective efficacy is variable against adult pulmonary tuberculosis. In this study, immune responses of antigen 85A (Ag85A) and heat-shock protein X (HspX) antigen of Mycobacterium tuberculosis were investigated during acute and stationary stage of infection in the murine aerosol TB challenge model and their protective effects were evaluated against progressive tuberculosis. A high level of Ag85A-specific IFN-γ production was induced from the early stage of the infection, whereas HspX-specific IFN-γ production was increased in the later stationary stage. As a subunit vaccine, Ag85A and HspX antigen vaccine induced high levels of IFN-γ, and a vaccine comprising both antigens induced the highest level of IFN-γ. At 30 days post-challenge, the Ag85A subunit vaccine was protective against M. tuberculosis challenge, but the HspX subunit vaccine was not. Interestingly, the HspX antigen vaccine induced significant protective efficacy at 90 days post-challenge. Moreover, the combined antigen vaccine induced the highest protective efficacy against M. tuberculosis challenge both at 30 days and 90 days post-challenge. These results suggest that the vaccine comprising Ag85A and HspX antigen which react in different stages of infection is highly protective against progressive tuberculosis.     

Key enzymes for biosynthesis of neutral lipid storage compounds in prokaryotes: properties, function and occurrence of wax ester synthases/acyl-CoA: diacylglycerol acyltransferases

Triacylglycerols (TAGs) and wax esters (WEs) are beside polyhydroxyalkanoates (PHAs) important storage lipids in some groups of prokaryotes. Accumulation of these lipids occurs in cells when they are cultivated under conditions of unbalanced growth in the presence of high concentrations of a suitable carbon source, which can be used for fatty acid and storage lipid biosyntheses. The key enzymes, which mediate both WE and TAG formations from long-chain acyl-coenzyme A (CoA) as acyl donor and long-chain fatty alcohols or diacylglycerols as respective acyl acceptors in bacteria, are WE synthases/acyl-CoA:diacylglycerol acyltransferases (WS/DGATs). The WS/DGATs identified so far represent rather unspecific enzymes with broad spectra of possible substrates; this makes them interesting for many biotechnological applications. This review traces the molecular structure and biochemical properties including the probable regions responsible for acyltransferase properties, enzymatic activity and substrate specifities. The phylogenetic relationships based on amino acid sequence similarities of this unique class of enzymes were revealed. Furthermore, recent advances in understanding the physiological functions of WS/DGATs in their natural hosts including pathogenic Mycobacterium tuberculosis were discussed.     

An interfacial mechanism and a class of inhibitors inferred from two crystal structures of the Mycobacterium tuberculosis 30 kDa major secretory protein (Antigen 85B), a mycolyl transferase

The Mycobacterium tuberculosis 30 kDa major secretory protein (antigen 85B) is the most abundant protein exported by M. tuberculosis, as well as a potent immunoprotective antigen and a leading drug target. A mycolyl transferase of 285 residues, it is closely related to two other mycolyl transferases, each of molecular mass 32 kDa: antigen 85A and antigen 85C. All three catalyze transfer of the fatty acid mycolate from one trehalose monomycolate to another, resulting in trehalose dimycolate and free trehalose, thus helping to build the bacterial cell wall. We have determined two crystal structures of M. tuberculosis antigen 85B (ag85B), initially by molecular replacement using antigen 85C as a probe. The apo ag85B model is refined against 1.8 A data, to an R-factor of 0.196 (R(free) is 0.276), and includes all residues except the N-terminal Phe. The active site immobilizes a molecule of the cryoprotectant 2-methyl-2,4-pentanediol. Crystal growth with addition of trehalose resulted in a second ag85B crystal structure (1.9 A resolution; R-factor is 0.195; R(free) is 0.285). Trehalose binds in two sites at opposite ends of the active-site cleft. In our proposed mechanism model, the trehalose at the active site Ser126 represents the trehalose liberated by temporary esterification of Ser126, while the other trehalose represents the incoming trehalose monomycolate just prior to swinging over to the first trehalose site to displace the mycolate from its serine ester. Our proposed interfacial mechanism minimizes aqueous exposure of the apolar mycolates. Based on the trehalose-bound structure, we suggest a new class of antituberculous drugs, made by connecting two trehalose molecules by an amphipathic linker.     

A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1

Triacylglycerols (TAGs) and wax esters are neutral lipids with considerable importance for dietetic, technical, cosmetic, and pharmaceutical applications. Acinetobacter calcoaceticus ADP1 accumulates wax esters and TAGs as intracellular storage lipids. We describe here the identification of a bifunctional enzyme from this bacterium exhibiting acyl-CoA:fatty alcohol acyltransferase (wax ester synthase, WS) as well as acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. Experiments with a knock-out mutant demonstrated the key role of the bifunctional WS/DGAT for biosynthesis of both storage lipids in A. calcoaceticus. This novel type of long-chain acyl-CoA acyltransferase is not related to known acyltransferases including the WS from jojoba (Simmondsia chinensis), the DGAT1 or DGAT2 families present in yeast, plants, and animals, and the phospholipid:diacylglycerol acyltransferase catalyzing TAG formation in yeast and plants. A large number of WS/DGAT-related proteins were identified in Mycobacterium and Arabidopsis thaliana indicating an important function of these proteins. WS and DGAT activity was demonstrated for the translational product of one WS/DGAT homologous gene from M. smegmatis mc(2)155. The potential of WS/DGAT to establish novel processes for biotechnological production of jojoba-like wax esters was demonstrated by heterologous expression in recombinant Pseudomonas citronellolis. The potential of WS/DGAT as a selective therapeutic target of mycobacterial infections is discussed.     

Diglycosyl diacylglycerol of Mycobacterium tuberculosis.

A diglycosyl diacylglycerol was isolated from Mycobacterium tuberculosis, and its structure was established by a combination of methylation analysis, 1H nuclear magnetic resonance, and fast atom bombardment-mass spectrometry. It is a 1,2-diacyl-[beta-D-glucopyranosyl(1"----6')-beta-D-glucopyranosyl(1'---- 3)]- sn-glycerol and exists in at least five molecular species differing in fatty acyl substituents. The major constituent fatty acids were identified as iso- and anteisopentadecanoate, iso- and n-hexadecanoate, and iso- and anteisoheptadecanoate. Although glycosyl diacylglycerols are common membrane components of gram-positive bacteria, this report represents the first substantial evidence for the presence of a glycosyl diacylglycerol within a member of the Mycobacterium genus. Although the glycolipid is not a major component of M. tuberculosis, it reacts readily in enzyme-linked immunosorbent assay against rabbit antibodies raised against whole bacteria and thus may be useful for the serodiagnosis of tuberculosis.     

Polyphthienoyl trehalose, glycolipids specific for virulent strains of the tubercle bacillus

Phthienoic acids constitute a family of dextro-rotary odd-numbered unsaturated fatty acids isolated exclusively from virulent strains of human and bovine tubercle bacilli. In the bacterial cell they are not free and a search for their linked form in complex wall lipids of Mycobacterium tuberculosis (strain Canetti) showed that they esterified trehalose. Structural elucidation of the major phthienoyl trehalose showed the occurrence of five acyl residues located at 2, 2', 3', 4 and 6' positions of trehalose. The acyl substituents were mainly 2,4,6-trimethyl tetracos-2-enoic acid (C27 phthienoic acid) accompanied by its homologs. In addition to these branched fatty acids, straight-chain C16 and C18 acyls composed about 20% of the substituents. The proposed structure is a new one, both for the mycobacterial-specific glycolipid and for the substituted positions on trehalose. Other minor acyl trehaloses were detected in M. tuberculosis (strain Canetti), differing from the major component by the occurrence of an additional hydroxy fatty acid (3-hydroxy-2,4,6-trimethyl tetracosanoic acid) or by the number of acyl substituents. The major glycolipid presented a weak activity in vitro on mitochondrial oxidative phosphorylation. These glycolipids and phthienoic acids could serve as virulence indicators.     

Docking of phosphonate and trehalose analog inhibitors into M. tuberculosis mycolyltransferase Ag85C: Comparison of the two scoring fitness functions GoldScore and ChemScore, in the GOLD software

The Ag85 family enzymes are responsible for the synthesis of cell wall components in mycobacterial species. Inhibitors to these enzymes are potential antimycobacterial agents. We have carried out the docking of phoshonate and trehalose analog inhibitors into the three dimensional structure of mycolyltransferase enzyme, Ag85C of M. tuberculosis using the GOLD software. The inhibitor binding positions and affinity were evaluated using both the scoring fitness functions- GoldScore and ChemScore. We observed that the inhibitor binding position identified using the GoldScore was marginally better than the ChemScore. A qualitative agreement between the reported experimental biological activities (IC50) and the GoldScore was observed. We identified that amino acid residues Arg541, Trp762 are important for inhibitor recognition via hydrogen bonding interactions. This information can be exploited to design Ag85C specific inhibitors.     

Identification, function and structure of the mycobacterial sulfotransferase that initiates sulfolipid-1 biosynthesis

Sulfolipid-1 (SL-1) is an abundant sulfated glycolipid and potential virulence factor found in Mycobacterium tuberculosis. SL-1 consists of a trehalose-2-sulfate (T2S) disaccharide elaborated with four lipids. We identified and characterized a conserved mycobacterial sulfotransferase, Stf0, which generates the T2S moiety of SL-1. Biochemical studies demonstrated that the enzyme requires unmodified trehalose as substrate and is sensitive to small structural perturbations of the disaccharide. Disruption of stf0 in Mycobacterium smegmatis and M. tuberculosis resulted in the loss of T2S and SL-1 formation, respectively. The structure of Stf0 at a resolution of 2.6 A reveals the molecular basis of trehalose recognition and a unique dimer configuration that encloses the substrate into a bipartite active site. These data provide strong evidence that Stf0 carries out the first committed step in the biosynthesis of SL-1 and establish a system for probing the role of SL-1 in M. tuberculosis infection.     

Storage lipid synthesis is non-essential in yeast.

Steryl esters and triacylglycerol (TAG) are the main storage lipids in eukaryotic cells. In the yeast Saccharomyces cerevisiae, these storage lipids accumulate during stationary growth phase within organelles known as lipid bodies. We have used single and multiple gene disruptions to study storage lipid synthesis in yeast. Four genes, ARE1, ARE2, DGA1, and LRO1, were found to contribute to TAG synthesis. The most significant contribution is made by DGA1, which encodes a novel acyl-CoA:diacylglycerol acyltransferase. Two of the genes, ARE1 and ARE2, are also involved in steryl ester synthesis. A yeast strain that lacks all four genes is viable and has no apparent growth defects under standard conditions. The strain is devoid of both TAG and steryl esters, and fluorescence microscopy revealed that it also lacks lipid bodies. We conclude that neither storage lipids nor lipid bodies are essential for growth in yeast.     

Mechanism of lipid-body formation in prokaryotes: how bacteria fatten up

Neutral lipid accumulation is frequently observed in some Gram-negative prokaryotes like Acinetobacter sp. and most actinomycetes, including the pathogenic Mycobacterium tuberculosis and antibiotic producing streptomycetes. We examined the formation of wax ester- and triacylglycerol (TAG)-bodies in Acinetobacter calcoaceticus and Rhodococcus opacus using microscopic, immunological and biophysical methods. A general model for prokaryotic lipid-body formation is proposed, clearly differing from the current models for the formation of lipid inclusions in eukaryotes and of poly(hydroxyalkanoic acid) (PHA) inclusions in prokaryotes. Formation of lipid-bodies starts with the docking of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) to the cytoplasm membrane. Both, analyses of in vivo and in vitro lipid-body synthesis, demonstrated the formation of small lipid droplets (SLDs), which remain bound to the membrane-associated enzyme. SLDs conglomerated subsequently to membrane-bound lipid-prebodies which are then released into the cytoplasm. The formation of matured lipid-bodies in the cytoplasm occurred by means of coalescence of SLDs inside the lipid prebodies, which are surrounded by a half-unit membrane of phospholipids.     

Enhanced immunogenicity and protective efficacy against Mycobacterium tuberculosis of bacille Calmette-Guérin vaccine using mucosal administration and boosting with a recombinant modified vaccinia virus Ankara

Heterologous prime-boost immunization strategies can evoke powerful T cell immune responses and may be of value in developing an improved tuberculosis vaccine. We show that recombinant modified vaccinia virus Ankara, expressing Mycobacterium tuberculosis Ag 85A (M.85A), strongly boosts bacille Calmette-Guérin (BCG)-induced Ag 85A specific CD4(+) and CD8(+) T cell responses in mice. A comparison of intranasal (i.n.) and parenteral immunization of BCG showed that while both routes elicited comparable T cell responses in the spleen, only i.n. delivery elicited specific T cell responses in the lung lymph nodes, and these responses were further boosted by i.n. delivery of M.85A. Following aerosol challenge with M. tuberculosis, i.n. boosting of BCG with either BCG or M.85A afforded unprecedented levels of protection in both the lungs (2.5 log) and spleens (1.5 log) compared with naive controls. Protection in the lung correlated with the induction of Ag 85A-specific, IFN-gamma-secreting T cells in lung lymph nodes. These findings support further evaluation of mucosally targeted prime-boost vaccination approaches for tuberculosis.     

Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans

CD8(+) T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8(+) CTLs, epitopes for classical MHC class I-restricted CD8(+) T cells, which most likely represent a majority among CD8(+) T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K(b) transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4(+) T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8(+) T cells against Ag85B of M. tuberculosis in HLA-A2/K(b) transgenic mice and HLA-A*0201(+) humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8(+) CTLs were identified. These CD8(+) T cells produced IFN-gamma and TNF-alpha and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8(+) T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201(+) individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8(+) CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8(+) T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.     

A novel trehalase from Mycobacterium smegmatis - purification, properties, requirements

Trehalose is a nonreducing disaccharide of glucose (alpha,alpha-1,1-glucosyl-glucose) that is essential for growth and survival of mycobacteria. These organisms have three different biosynthetic pathways to produce trehalose, and mutants devoid of all three pathways require exogenous trehalose in the medium in order to grow. Mycobacterium smegmatis and Mycobacterium tuberculosis also have a trehalase that may be important in controlling the levels of intracellular trehalose. In this study, we report on the purification and characterization of the trehalase from M. smegmatis, and its comparison to the trehalase from M. tuberculosis. Although these two enzymes have over 85% identity throughout their amino acid sequences, and both show an absolute requirement for inorganic phosphate for activity, the enzyme from M. smegmatis also requires Mg(2+) for activity, whereas the M. tuberculosis trehalase does not require Mg(2+). The requirement for phosphate is unusual among glycosyl hydrolases, but we could find no evidence for a phosphorolytic cleavage, or for any phosphorylated intermediates in the reaction. However, as inorganic phosphate appears to bind to, and also to greatly increase the heat stability of, the trehalase, the function of the phosphate may involve stabilizing the protein conformation and/or initiating protein aggregation. Sodium arsenate was able to substitute to some extent for the sodium phosphate requirement, whereas inorganic pyrophosphate and polyphosphates were inhibitory. The purified trehalase showed a single 71 kDa band on SDS gels, but active enzyme eluted in the void volume of a Sephracryl S-300 column, suggesting a molecular mass of about 1500 kDa or a multimer of 20 or more subunits. The trehalase is highly specific for alpha,alpha-trehalose and did not hydrolyze alpha,beta-trelalose or beta,beta-trehalose, trehalose dimycolate, or any other alpha-glucoside or beta-glucoside. Attempts to obtain a trehalase-negative mutant of M. smegmatis have been unsuccessful, although deletions of other trehalose metabolic enzymes have yielded viable mutants. This suggests that trehalase is an essential enzyme for these organisms. The enzyme has a pH optimum of 7.1, and is active in various buffers, as long as inorganic phosphate and Mg(2+) are present. Glucose was the only product produced by the trehalase in the presence of either phosphate or arsenate.     

Pulmonary mononuclear cell responses to antigens of Mycobacterium tuberculosis in healthy household contacts of patients with active tuberculosis and healthy controls from the community

Protective immunity against Mycobacterium tuberculosis requires CD4+ lymphocyte-mediated immune responses and IFN-gamma activity. As the primary portal of entry of M. tuberculosis is the lung, pulmonary immune responses against multiple M. tuberculosis Ags were compared between both M. tuberculosis-exposed tuberculin skin test-positive healthy household contacts (HHC) of patients with active sputum smear and culture-positive tuberculosis and tuberculin skin test-positive healthy control individuals from the community (CC). Frequencies of M. tuberculosis Ag-specific IFN-gamma-producing cells, IFN-gamma concentrations in culture supernatants, and DNA synthesis in bronchoalveolar cells (BAC) and PBMC were studied in HHC (n = 10) and CC (n = 15). Using enzyme-linked immunospot assay we found higher frequencies of IFN-gamma-producing cells with specificity to M. tuberculosis-secreted Ag 85 (Ag 85) in BAC from HHC than in BAC from CC (p < 0.022) and relative to autologous PBMC, indicating compartmentalization of Ag 85-specific cells to the lungs. Further, IFN-gamma-producing cells with specificity to components A and B of Ag 85 were specifically compartmentalized to the lungs in HHC (p < 0. 05). IFN-gamma concentrations in culture supernatants of BAC and Ag-specific DNA synthesis were low and comparable in the two subject groups. Increased immune responses to Ag 85 at the site of repeated exposure to M. tuberculosis (the lung) may represent an important component of protective immunity against M. tuberculosis. Correlates of protective immunity against M. tuberculosis are required for assessment of the efficiency of anti-tuberculous vaccines.     

Functional characterization of the phospholipase C activity of Rv3487c and its localization on the cell wall of Mycobacterium tuberculosis

Mycobacterium tuberculosis survives and persists for prolonged periods within its host in an asymptomatic,latent state and can reactivate years later if the host's immune system weakens. The dormant bacilli synthesize and accumulate triacylglycerol, reputed to be an energy source during latency. Among the phospholipases, phospholipase C plays an important role in the pathogenesis. Mutations in a known phospholipase C, plcC, of M.tuberculosis attenuate its growth during the late phase of infection in mice. Hydrolysis of phospholipids by phospholipase C generates diacylglycerol, a well-known signalling molecule that participates in the activation of extracellular signal-regulated kinases (ERK) through protein kinase C leading to macrophage activation. In the present study, we show that M.tuberculosis possesses an additional cell wall-associated protein, Rv3487c, with phospholipase C activity. The recombinant Rv3487c hydrolyses the substrate phosphatidylcholine and generates diacylglycerol by removing the phosphocholine. Furthermore, Rv3487c is expressed during infection as it exhibits significant humoral immunoreactivity with sera from children with tuberculosis, but not with that from adult patients.     

Crystal structure of the secreted form of antigen 85C reveals potential targets for mycobacterial drugs and vaccines

The antigen 85 (ag85) complex, composed of three proteins (ag85A, B and C), is a major protein component of the Mycobacterium tuberculosis cell wall. Each protein possesses a mycolyltransferase activity required for the biogenesis of trehalose dimycolate (cord factor), a dominant structure necessary for maintaining cell wall integrity. The crystal structure of recombinant ag85C from M. tuberculosis, refined to a resolution of 1.5 A, reveals an alpha/beta-hydrolase polypeptide fold, and a catalytic triad formed by Ser 124, Glu 228 and His 260. ag85C complexed with a covalent inhibitor implicates residues Leu 40 and Met 125 as components of the oxyanion hole. A hydrophobic pocket and tunnel extending 21 A into the core of the protein indicates the location of a probable trehalose monomycolate binding site. Also, a large region of conserved surface residues among ag85A, B and C is a probable site for the interaction of ag85 proteins with human fibronectin.     

The DGA1 gene determines a second triglyceride synthetic pathway in yeast.

Diacylglycerol esterification provides an excellent target for the pharmacological reduction of triglyceride accumulation in several human disease states. We have used Saccharomyces cerevisiae as a model system to study this critical component of triglyceride synthesis. Recent studies of an oleaginous fungus, Mortierella ramanniana, identified a new family of enzymes with in vitro acyl-CoA:diacylglycerol acyltransferase activity. We show here that DGA1, the sole member of this gene family in yeast, has a physiological role in triglyceride synthesis. Metabolic labeling of DGA1 deletion strains with triglyceride precursors detected significant reductions in triglyceride synthesis. Triglyceride synthesis was virtually abolished in four different growth conditions when DGA1 was deleted in concert with LRO1, an enzyme that esterifies diacylglycerol from a phospholipid acyl donor. The relative contributions of the two enzymes depended on growth conditions. The residual synthesis was lost when ARE2, encoding an acyl-CoA:sterol acyltransferase, was deleted. In vitro microsomal assays verified that DGA1 and ARE2 mediate acyl-CoA:diacylglycerol acyltransferase reactions. Three enzymes can thus account for diacylglycerol esterification in yeast. Yeast strains deficient in both diacylglycerol and sterol esterification showed only a slight growth defect indicating that neutral lipid synthesis is dispensable under common laboratory conditions.