Th1-skewed tissue responses to a mycolyl glycolipid in mycobacteria-infected rhesus macaques

Trehalose 6,6′-dimycolate (TDM) is a major glycolipid of the cell wall of mycobacteria with remarkable adjuvant functions. To avoid detection by the host innate immune system, invading mycobacteria down-regulate the expression of TDM by utilizing host-derived glucose as a competitive substrate for their mycolyltransferases; however, this enzymatic reaction results in the concomitant biosynthesis of glucose monomycolate (GMM) which is recognized by the acquired immune system. GMM-specific, CD1-restricted T cell responses have been detected in the peripheral blood of infected human subjects and monkeys as well as in secondary lymphoid organs of small animals, such as guinea pigs and human CD1-transgenic mice. Nevertheless, it remains to be determined how tissues respond at the site where GMM is produced. Here we found that rhesus macaques vaccinated with Mycobacterium bovis bacillus Calmette–Guerin mounted a chemokine response in GMM-challenged skin that was favorable for recruiting T helper (Th)1 T cells. Indeed, the expression of interferon-γ, but not Th2 or Th17 cytokines, was prominent in the GMM-injected tissue. The GMM-elicited tissue response was also associated with the expression of monocyte/macrophage-attracting CC chemokines, such as CCL2, CCL4 and CCL8. Furthermore, the skin response to GMM involved the up-regulated expression of granulysin and perforin. Given that GMM is produced primarily by pathogenic mycobacteria proliferating within the host, the Th1-skewed tissue response to GMM may function efficiently at the site of infection.     

Temperature-dependent biosynthesis of glucose monomycolate and its recognition by CD1-restricted T cells

Mycolic acids are long chain fatty acids that constitute the lipid-rich cell wall framework of mycobacteria. Upon infection, mycobacteria begin to synthesize glucose monomycolate (GMM), a glucosylated species of mycolic acids, by utilizing host-derived glucose as sugar source. Accordingly, GMM production serves as a good indicator for local invasion of mycobacteria, and its detection by the host immune system would favor efficient monitoring of mycobacterial infection. Here, we found that GMM was produced abundantly at 30 degrees C rather than at 37 degrees C and recognized by a GMM-specific, CD1-restricted T cell line that was isolated from mycobacteria-infected human skin. Since the common portal sites for mycobacterial infection include ventilating alveoli of the lung and the externally exposed skin that often render invading microbes survive at reduced temperature, sampling GMM by CD1 lipid antigen-presenting molecules may allow the host to detect mycobacterial infection at its early phases.     

Control of cell wall assembly by a histone-like protein in Mycobacteria

Bacteria coordinate assembly of the cell wall as well as synthesis of cellular components depending on the growth state. The mycobacterial cell wall is dominated by mycolic acids covalently linked to sugars, such as trehalose and arabinose, and is critical for pathogenesis of mycobacteria. Transfer of mycolic acids to sugars is necessary for cell wall biogenesis and is mediated by mycolyltransferases, which have been previously identified as three antigen 85 (Ag85) complex proteins. However, the regulation mechanism which links cell wall biogenesis and the growth state has not been elucidated. Here we found that a histone-like protein has a dual concentration-dependent regulatory effect on mycolyltransferase functions of the Ag85 complex through direct binding to both the Ag85 complex and the substrate, trehalose-6-monomycolate, in the cell wall. A histone-like protein-deficient Mycobacterium smegmatis strain has an unusual crenellated cell wall structure and exhibits impaired cessation of glycolipid biosynthesis in the growth-retarded phase. Furthermore, we found that artificial alteration of the amount of the extracellular histone-like protein and the Ag85 complex changes the growth rate of mycobacteria, perhaps due to impaired down-regulation of glycolipid biosynthesis. Our results demonstrate novel regulation of cell wall assembly which has an impact on bacterial growth.     

Glycerol Monomycolate Is a Novel Ligand for the Human,but Not Mouse Macrophage Inducible C-type Lectin,Mincle

An array of lipidic compounds that constitute the cell wall of mycobacteria is recognized by host receptors. Examples include trehalose dimycolate (TDM), which is a major surface-exposed glycolipid of mycobacteria, that interacts with the macrophage inducible C-type lectin, Mincle, and exerts its highly potent adjuvant functions. Recent evidence has suggested that glycerol monomycolate (GroMM), another mycolate-containing lipid species produced by mycobacteria, can stimulate innate immune cells; however, its specific host receptors have yet to be identified. We here demonstrated that cell transfectants expressing human Mincle (hMincle) reacted to both TDM and GroMM, while those expressing mouse Mincle (mMincle) only reacted to TDM and failed to recognize GroMM. Studies using domain swap chimeras confirmed that the ectodomain of hMincle, but not that of mMincle, interacted with GroMM, and site-directed mutagenesis analyses revealed that short stretches of amino acid residues at positions 174–176 and 195–196 were involved in GroMM recognition. To further substantiate the differential recognition of GroMM by hMincle and mMincle, hMincle transgenic/mMincle knock-out mice (i.e. hMincle⁺ mice) were established and compared with non-transgenic mice (i.e. mMincle⁺ mice). We showed that macrophages derived from hMincle⁺ mice were activated by GroMM and produced inflammatory cytokines, whereas those derived from mMincle⁺ mice did not exhibit any reactivity to GroMM. Furthermore, local inflammatory responses were elicited in the GroMM-injected skin of hMincle⁺, but not mMincle⁺ mice. These results demonstrated that GroMM is a unique ligand for hMincle that is not recognized by mMincle.     

Biosynthetic origin of mycobacterial cell wall arabinosyl residues.

Designing new drugs that inhibit the biosynthesis of the D-arabinan moiety of the mycobacterial cell wall arabinogalactan is one important basic approach for treatment of mycobacterial diseases. However, the biosynthetic origin of the D-arabinosyl monosaccharide residues themselves is not known. To obtain information on this issue, mycobacteria growing in culture were fed glucose labeled with 14C or 3H in specific positions. The resulting radiolabeled cell walls were isolated and hydrolyzed, the arabinose and galactose were separated by high-pressure liquid chromatography, and the radioactivity in each sugar was determined. [U-14C]glucose, [6-3H]glucose, [6-14C]glucose, and [1-14C]glucose were all converted to cell wall arabinosyl residues with equal retention of radioactivity. The positions of the labeled atoms in the arabinose made from [1-14C]glucose and [6-3H]glucose were shown to be C-1 and H-5, respectively. These results demonstrated that the arabinose carbon skeleton is formed via the nonoxidative pentose shunt and not via hexose decarboxylation or via triose condensations. Since the pentose shunt product, ribulose-5-phosphate, is converted to arabinose-5-phosphate as the first step in 3-keto-D-manno-octulosonic acid biosynthesis by gram-negative bacteria, such a conversion was then searched for in mycobacteria. However, cell-free enzymatic analysis using both phosphorous nuclear magnetic resonance spectrometry and colorimetric methods failed to detect the conversion. Thus, the conversion of the pentose shunt intermediates to the D-arabino stereochemistry is not via the expected isomerase but rather must occur via novel metabolic transformations.     

Isolation and characterization of arabinose mycolate from firmly bound lipids of mycobacteria

The glycolipid, d-arabinose-5-mycolate, was purified from bound lipids of mycobacteria. The significance of this glycolipid in the chemical structure of cell wall and wax D is discussed. The hypothetical chemical structure of mycolic acid-arabinogalactan portion of cell wall and wax D is proposed.     

Negative regulation by Ser/Thr phosphorylation of HadAB and HadBC dehydratases from Mycobacterium tuberculosis type II fatty acid synthase system

The type II fatty acid synthase system of mycobacteria is involved in the biosynthesis of major and essential lipids, mycolic acids, key-factors of Mycobacterium tuberculosis pathogenicity. One reason of the remarkable survival ability of M. tuberculosis in infected hosts is partly related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate synthesis of these lipids in response to environmental changes are unknown. We demonstrate here that HadAB and HadBC dehydratases of this system are phosphorylated by Ser/Thr protein kinases, which negatively affects their enzymatic activity. The phosphorylation of HadAB/BC is growth phase-dependent, suggesting that it represents a mechanism by which mycobacteria might tightly control mycolic acid biosynthesis under non-replicating condition.     

Hijacking of a substrate-binding protein scaffold for use in mycobacterial cell wall biosynthesis

The waxy cell wall is crucial to the survival of mycobacteria within the infected host. The cell wall is a complex structure rich in unusual molecules that includes two related lipoglycans, the phosphatidylinositol mannosides (PIMs) and lipoarabinomannans (LAMs). Many proteins implicated in the PIM/LAM biosynthetic pathway, while attractive therapeutic targets, are poorly defined. The 2.4A resolution crystal structure of an essential lipoprotein, LpqW, implicated in LAM biosynthesis is reported here. LpqW adopts a scaffold reminiscent of the distantly related, promiscuous substrate-binding proteins of the ATP-binding cassette import system. Nevertheless, the unique closed conformation of LpqW suggests that mycobacteria and other closely related pathogens have hijacked this scaffold for use in key processes of cell wall biosynthesis. In silico docking provided a plausible model in which the candidate PIM ligand binds within a marked electronegative region located on the surface of LpqW. We suggest that LpqW represents an archetypal lipoprotein that channels intermediates from a pathway for mature PIM production into a pathway for LAM biosynthesis, thus controlling the relative abundance of these two important components of the cell wall.     

PimE is a polyprenol-phosphate-mannose-dependent mannosyltransferase that transfers the fifth mannose of phosphatidylinositol mannoside in mycobacteria

Phosphatidylinositol mannosides (PIMs) are a major class of glycolipids in all mycobacteria. AcPIM2, a dimannosyl PIM, is both an end product and a precursor for polar PIMs, such as hexamannosyl PIM (AcPIM6) and the major cell wall lipoglycan, lipoarabinomannan (LAM). The mannosyltransferases that convert AcPIM2 to AcPIM6 or LAM are dependent on polyprenol-phosphate-mannose (PPM), but have not yet been characterized. Here, we identified a gene, termed pimE that is present in all mycobacteria, and is required for AcPIM6 biosynthesis. PimE was initially identified based on homology with eukaryotic PIG-M mannosyltransferases. PimE-deleted Mycobacterium smegmatis was defective in AcPIM6 synthesis, and accumulated the tetramannosyl PIM, AcPIM4. Loss of PimE had no affect on cell growth or viability, or the biosynthesis of other intracellular and cell wall glycans. However, changes in cell wall hydrophobicity and plasma membrane organization were detected, suggesting a role for AcPIM6 in the structural integrity of the cell wall and plasma membrane. These defects were corrected by ectopic expression of the pimE gene. Metabolic pulse-chase radiolabeling and cell-free PIM biosynthesis assays indicated that PimE catalyzes the alpha1,2-mannosyl transfer for the AcPIM5 synthesis. Mutation of an Asp residue in PimE that is conserved in and required for the activity of human PIG-M resulted in loss of PIM-biosynthetic activity, indicating that PimE is the catalytic component. Finally, PimE was localized to a distinct membrane fraction enriched in AcPIM4-6 biosynthesis. Taken together, PimE represents the first PPM-dependent mannosyl-transferase shown to be involved in PIM biosynthesis, where it mediates the fifth mannose transfer.     

Effects of glucanase treatment on the structure and mechanical properties of cell walls in the giant‐celled xanthophycean alga Vaucheria frigida

The cell wall of the tip‐growing cells of the giant‐cellular xanthophycean alga Vaucheria frigida is mainly composed of cellulose microfibrils (CMFs) arranged in random directions and the major matrix component into which the CMFs are embedded throughout the cell. The mechanical properties of a cell‐wall fragment isolated from the tip‐growing region, which was inflated by artificially applied pressure, were measured after enzymatic removal of the matrix component by using a protease; the results showed that the matrix component is involved in the maintenance of cell wall strength. Since glucose and uronic acid are present in the matrix component of Vaucheria cell walls, we measured the mechanical properties of the cell wall after treatment with endo‐1,3‐ß‐glucanase and observed the fine structures of its surfaces by atomic force microscopy. The major matrix component was partially removed from the cell wall by glucanase, and the enzyme treatment significantly weakened the cell wall strength without affecting the pH dependence of cell wall extensibility. The enzymatic removal of the major matrix component by using a protease released polysaccharide containing glucose and glucuronic acid. This suggests that the major matrix component of the algal cell walls contains both proteins (or polypeptides) and polysaccharides consisting of glucose and glucuronic acid as the main constituents.     

Mechanisms involved in the intrinsic isoniazid resistance of Mycobacterium avium

Isoniazid (INH), which acts by inhibiting mycolic acid biosynthesis, is very potent against the tuberculous mycobacteria. It is about 100-fold less effective against Mycobacterium avium . This difference has often been attributed to a decreased permeability of the cell wall. We measured the rate of conversion of radiolabelled INH to 4-pyridylmethanol by whole cells and cell-free extracts and estimated the permeability barrier imposed by the cell wall to INH influx in Mycobacterium tuberculosis and M . avium . There was no significant difference in the relative permeability to INH between these two species. However, the total conversion rate in M . tuberculosis was found to be four times greater. Examination of in vitro -generated mutants revealed that the major resistance mechanism for both species is loss of the catalase-peroxidase KatG. Analysis of lipid and protein biosynthetic profiles demonstrated that the molecular target of activated INH was identical for both species. M . avium , however, formed colonies at INH concentrations inhibitory for mycolic acid biosynthesis. These mycolate-deficient M . avium exhibited altered colony morphologies, modified cell wall ultrastructure and were 10-fold more sensitive to treatment with hydrophobic antibiotics, such as rifampin. These findings may significantly impact the design of new therapeutic regimens for the treatment of infections with atypical mycobacteria.     

The OtsAB pathway is essential for trehalose biosynthesis in Mycobacterium tuberculosis

The disaccharide trehalose is the major free sugar in the cytoplasm of mycobacteria; it is a constituent of cell wall glycolipids, and it plays a role in mycolic acid transport during cell wall biogenesis. The pleiotropic role of trehalose in the biology of Mycobacterium tuberculosis and its absence from mammalian cells suggests that its biosynthesis may provide a useful target for novel drugs. However, there are three potential pathways for trehalose biosynthesis in M. tuberculosis, and the aim of the present study was to introduce mutations into each of the pathways to determine whether or not they are functionally redundant. The results show that the OtsAB pathway, which generates trehalose from glucose and glucose-6-phosphate, is the dominant pathway required for M. tuberculosis growth in laboratory culture and for virulence in a mouse model. Of the two otsB homologues annotated in the genome sequence of M. tuberculosis, only OtsB2 (Rv3372) has a functional role in the pathway. OtsB2, trehalose-6-phosphate phosphatase, is strictly essential for growth and provides a tractable target for high throughput screening. Inactivation of the TreYZ pathway, which can generate trehalose from alpha-1,4-linked glucose polymers, had no effect on the growth of M. tuberculosis in vitro or in mice. Deletion of the treS gene altered the late stages of pathogenesis of M. tuberculosis in mice, significantly increasing the time to death in a chronic infection model. Because the TreS enzyme catalyzes the interconversion of trehalose and maltose, the mouse phenotype could reflect either a requirement for synthesis of additional trehalose or, conversely, a requirement for breakdown of stored trehalose to liberate free glucose.     

The A' and F' pockets of human CD1b are both required for optimal presentation of lipid antigens to T cells

CD1 proteins are unique in their ability to present lipid Ags to T cells. Human CD1b shares significant amino acid homology with mouse CD1d1, which contains an unusual putative Ag-binding groove formed by two large hydrophobic pockets, A' and F'. We investigated the function of the amino acid residues that line the A' and F' pockets of CD1b by engineering 36 alanine-substitution mutants and analyzing their ability to present mycobacterial glycolipid Ags. Two lipid Ags presented by CD1b were studied, a naturally occurring glucose monomycolate (GMM) isolated from mycobacteria, which contains two long alkyl chains (C54-C62 and C22-C24) and synthetic GMM (sGMM), which includes two short alkyl chains (C18 and C14). We identified eight residues in both the A' and F' pockets that were involved in the presentation of both GMM and sGMM to T cells. Interestingly, four additional residues located in the distal portion of the A' pocket were required for the optimal presentation of GMM, but not sGMM. Conversely, nine residues located between the center of the groove and the F' pocket were necessary for the optimal presentation of sGMM, but not GMM. These data indicate that both the A' and F' pockets of human CD1b are required for the presentation of lipid Ags to T cells.     

Molecular packing of cord factor and its interaction with phosphatidylinositol in mixed monolayers.

Cord factor (trehalose 6,6'-dimycolate, CF) is a glycolipid located in the outer mycobacterial cell wall that is implicated in the pathogenesis of mycobacteria. Furthermore, CF is a convenient model for studying mycolic acid residues, the major lipid constituents of the mycobacterial cell wall that are believed to form a barrier against drug penetration. The surface properties of CF and its interactions with phosphatidylinositol (PI) have been investigated using the monolayer technique. During compression/expansion/recompression cycles, CF monolayers switch from a loosely packed to a more tightly packed structure. The change in surface properties suggests a molecular rearrangement, perhaps involving interdigitation of long and short chains of the CF molecules. In CF-PI monolayers, maximal lateral packing density occurs between 0.5 and 0.7 mole fraction CF, which is close to the relative composition of mycolic acid residues and shorter-chain lipids in the mycobacterial cell wall. Low concentrations of CF increase the order in PI monolayers, consistent with CF toxicity involving rigidification of cell membranes.     

Biochemical effects of sparfloxacin on cell envelope of mycobacteria.

Sparfloxacin, a difluorinated quinolone is a potent anti-mycobacterial agent used in the treatment of mycobacterial infections. We have investigated whether sparfloxacin had other, more subtle effects on mycobacteria besides its interaction with DNA gyrase that could contribute to its therapeutic efficacy. Mycobacterium smegmatis cells grown in media with sub-inhibitory concentration of sparfloxacin were observed to have significant reduction in the biosynthesis of vital macromolecules, as shown by the incorporation of various radiolabelled precursors. The analysis of subcellular distribution of phospholipids of sparfloxacin-treated cells demonstrated an increase in the cell membrane and reduction in the cell wall, suggesting changes in the cell envelope architecture by sparfloxacin. Significant changes were also observed in other chemical constituents of the cell wall, especially in the arabinose and glucosamine contents. Mycolic acids, the major component of mycobacterial cell wall were reduced in the presence of MIC50 of sparfloxacin. There was a decrease in the limiting fluorescence intensity (Fmax) of 1-anilinonaphthalene 8-sulfonate (ANS) indicating alterations in the organization and conformation of mycobacterial cell surface. These results suggest that the mechanism of action of anti-mycobacterial action of sparfloxacin involves mycobacterial cell envelope.     

Cell wall analysis

The cell wall is a rigid structure essential for survival of the fungal cell. Because of its absence in mammalian cells, the cell wall is an attractive target for antifungal agents. Thus, for different reasons, it is important to know how the cell wall is synthesized and how different molecules regulate that synthesis. The Schizosaccharomyces pombe cell wall is mainly formed by glucose polysaccharides and some galactomannoproteins. Here, we describe a fast and reliable method to analyze changes in S. pombe cell wall composition by using specific enzymatic degradation and chemical treatment of purified cell walls. This approach provides a powerful means to analyze changes in (1,3)beta-glucan and (1,3)alpha-glucan, two main polysaccharides present in fungal cell walls. Analysis of cell wall polymers will be useful to search for new antifungal drugs that may inhibit cell wall biosynthesis and/or alter cell wall structure.     

Fatty acid remodeling: a novel reaction sequence in the biosynthesis of trypanosome glycosyl phosphatidylinositol membrane anchors.

The trypanosome variant surface glycoprotein (VSG) is anchored to the plasma membrane via a glycosyl phosphatidylinositol (GPI). The GPI is synthesized as a precursor, glycolipid A, that is subsequently linked to the VSG polypeptide. The VSG anchor is unusual, compared with anchors in other cell types, in that its fatty acid moieties are exclusively myristic acid. To investigate the mechanism for myristate specificity we used a cell-free system for GPI biosynthesis. One product of this system, glycolipid A', is indistinguishable from glycolipid A except that its fatty acids are more hydrophobic than myristate. Glycolipid A' is converted to glycolipid A through highly specific fatty acid remodeling reactions involving deacylation and subsequent reacylation with myristate. Therefore, myristoylation occurs in the final phase of trypanosome GPI biosynthesis.     

Mechanisms of augmented resistance of cyclosporin A-treated mice to influenza virus infection by trehalose-6,6'-dimycolate.

Cyclosporin A (CsA), which is an immunosuppressive drug of helper T lymphocytes, diminished a resistance of mice to influenza virus infection. Mice inoculated intravenously with trehalose-6,6'-dimycolate (TDM, a glycolipid component of the cell wall of Mycobacterium) in an oil-in-water emulsion (TDM emulsion) recovered the resistance to influenza virus infection impaired by CsA. Number of antibody-producing cells was markedly reduced in CsA- and/or TDM-treated mice. Interferon production in lung of TDM-treated mice was augmented; however, it was extremely reduced not only in CsA-treated mice, but also in CsA- and TDM-treated mice. Activities of natural killer cells of CsA- and/or TDM-treated mice were not different from that of control mice. Numbers of lymphocytes in lung of TDM-treated mice and CsA- and TDM-treated mice were more predominantly increased than that of control mice. Analysis of lung lymphocytes by flow cytometry revealed no difference between the populations of L3T4+ T lymphocytes and Lyt-2.2+ T lymphocytes in CsA- and/or TDM-treated mice and the populations in control mice. However, the population of gamma delta T cell receptor positive (gamma delta TCR+) lymphocytes increased markedly in lung of TDM-treated mice and also CsA- and TDM-treated mice. In vitro experiments showed that macrophage cultures treated with TDM emulsion released a mediator(s) which activates T lymphocytes, but not B lymphocytes. These and our earlier results suggest that the recovered anti-influenza virus resistance of CsA-treated mice by treatment with TDM emulsion was caused by elicitation of macrophages with TDM, then activation of T lymphocytes, especially gamma delta TCR+ lymphocytes.