Delphi Chatterjee

Trafficking and release of mycobacterial lipids from infected macrophages.

Analysis of infected macrophages revealed that lipid‐containing moieties of the mycobacterial cell wall are actively trafficked out of the mycobacterial vacuole. To facilitate the analysis of vesicular trafficking from mycobacteria‐containing phagosomes, surface‐exposed carbohydrates were labeled with hydrazide‐tagged markers. The distribution of labeled carbohydrate/lipid moieties and subsequent interaction with cellular compartments were analyzed by immunoelectron microscopy and by fluorescence microscopy of live cells. The released mycobacterial constituents were associated with several intracellular organelles and were enriched strikingly in tubular endocytic compartments. Subcellular fractionation of infected macrophages by density gradient electrophoresis showed temporal movement of labeled bacterial constituents through early and late endosomes. Thin layer chromatography analysis of these subcellular fractions confirmed their lipid nature and revealed five dominant bacteria‐derived species. These mycobacterial lipids were also found in extracellular vesicles isolated from the medium and could be observed in un‐infected ‘bystander’ cells. Their transfer to bystander cells could expand the bacterias sphere of influence beyond the immediate confines of the host cell.

Molecular Interaction of CD1b with Lipoglycan Antigens

The ability of human CD1b molecules to present nonpeptide antigens is suggested by the T cell recognition of microbial lipids and lipoglycans in the presence of CD1b-expressing antigen-presenting cells. We demonstrate the high-affinity interaction of CD1b molecules with the acyl side chains of known T cell antigens, lipoarabinomannan, phosphatidylinositol mannoside, and glucose monomycolate. Furthermore, CD1b-antigen binding was optimal at acidic pH, consistent with the known requirement for endosomal acidification in CD1b-restricted antigen presentation. The mechanism for CD1b-ligand interaction involves the partial unfolding of the alpha helices of CD1b at acidic pH, revealing a hydrophobic binding site that could accommodate lipid. These data provide direct evidence that the CD1b molecule has evolved unique biochemical properties that enable the binding of lipid-containing antigens from intracellular pathogens.

The mycobacterial cell wall: structure, biosynthesis and sites of drug action

Structural analysis has been successfully implemented recently to obtain valuable information on the mycobacterial cell wall components, many of which have formed the basis for biosynthesis and functional studies towards developing better drugs and possible vaccines. The highly complex and well organized structure unique to mycobacteria, represents the best target for novel antimycobacterial agents. Until recently, our knowledge of the enzymes responsible for the biogenesis of the cell wall components was almost negligible. The pathways are now being elucidated in several laboratories. Highlights of this review include significant advances in the structure and biochemistry of the major cell wall components and potential targets for generation of new drugs.

Structure and antigenicity of lipoarabinomannan from Mycobacterium bovis BCG

Lipoarabinomannan (LAM), a major lipoglycan of the mycobacterial cell envelope, was previously recognized as existing in two major forms: LAM with arabinofuranosyl (Araf)-containing termini (AraLAM) and a mannose-capped version (ManLAM) in which the majority of these termini are modified by additional mannose residues. Since ManLAM was first recognized in the virulent (Erdman) strain of Mycobacterium tuberculosis and the noncapped version in a rapidly growing, attenuated, H37Ra strain, it was thought that mannose capping may be a key factor in virulence. In the present study, LAM from M. bovis BCG was isolated and the non-reducing termini sequenced through differential O-alkylation, partial depolymerization and gas chromatography-mass spectrometric analyses of fragments. LAM from M. bovis BCG contains a short mannan backbone, highly branched arabinofuranosyl-containing side chains and several mannosyl residues capping the non-reducing termini of these side chains. Thus, LAM from M. bovis BCG is of the ManLAM type, showing no major structural differences at the non-reducing ends from the M. tuberculosis Erdman product. This observation led us to examine the earlier strain and to conclude that it showed little resemblance to conventional strains of M. tuberculosis. Thus, the absence of mannose caps may be more a feature of rapid growth than of avirulence. These results demonstrate that the relationship between mannose capping and disease induction is not a simple one. However, use of a panel of LAM-specific monoclonal antibodies showed antigenic differences between the BCG and the Erdman products, suggesting the presence of features specific to the different strains and pointing to LAM as a molecule within which further species and strain variations reside.

The Emb proteins of mycobacteria direct arabinosylation of lipoarabinomannan and arabinogalactan via an N‐terminal recognition region and a C‐terminal synthetic region

The arabinans of the mycobacterial cell wall are key structural and immunological polymers in the context of arabinogalactan (AG) and lipoarabinomannan (LAM) respectively. The three homologous membrane proteins EmbA, EmbB and EmbC are known to be involved in the synthesis of arabinan but their biochemical functions are not understood. Herein we show, that synthesis of LAM, but not AG, ceases after inactivation of embC in Mycobacterium smegmatis by insertional mutagenesis. LAM synthesis is restored upon complementation with the embC wild‐type gene. Previously we have shown that the synthesis of the arabinan of AG is affected by embA or embB disruption. Thus the Emb proteins are capable of differential recognition of the galactan or mannan acceptors prior to appropriate arabinosylation. In addition, a combination of genetic and biochemical approaches have allowed us to assign some specific functions to the regions of emb gene products. Complementation of the embC¯ mutant with a hybrid gene encoding the N‐terminus of EmbC and the C‐terminus of EmbB resulted in LAM with a lower molecular weight than the wild‐type LAM. Structural studies involving enzyme digestion, chromatography and mass spectrometry analyses revealed that the arabinan of the ‘LAM’ formed in the hybrid was of AG kind rather than LAM type of arabinan.

The pimB gene of Mycobacterium tuberculosis encodes a mannosyltransferase involved in lipoarabinomannan biosynthesis.

The biosynthesis of lipoarabinomannan (LAM), a key mycobacterial lipoglycan that has been implicated in numerous immunoregulatory functions, was examined utilizingd-mannosamine (ManN) as a tool to identify mannosyltransferase genes involved in LAM synthesis. Cell-free reactions utilizing cellular membranes of mycobacteria as the enzyme source indicated that ManN inhibited the synthesis of phosphatidylinositol mannosides, early precursors to LAM. A selection strategy was devised to screen a Mycobacterium tuberculosisgenomic library in Mycobacterium smegmatis for clones conferring conditional resistance to ManN, with the rationale that overexpression of the gene(s) encoding a target of ManN would impart a ManN-resistant phenotype under these conditions. This strategy led to the identification of pimB, whose deduced amino acid sequence shows similarity to mannosyltransferases and other glycosyltransferases. Partially purified recombinant PimB protein from Escherichia coli or membranes from M. smegmatis overexpressing the pimB gene were used in cell-free assays to show that PimB catalyzes the formation of triacylphosphatidylinositol dimannoside from GDP-mannose and triacylphosphatidylinositol monomannoside.

Truncated Structural Variants of Lipoarabinomannan in Ethambutol Drug-resistant Strains of Mycobacterium smegmatis INHIBITION OF ARABINAN BIOSYNTHESIS BY ETHAMBUTOL

The anti-tuberculosis drug, ethambutol (Emb), was previously shown to inhibit the synthesis of arabinans of both the cell wall arabinogalactan (AG) and lipoarabinomannan (LAM) of Mycobacterium tuberculosis and other mycobacteria. However, an Emb-resistant mutant, isolated by consecutive passage of the Mycobacterium smegmatis parent strain in media containing increasing concentrations of Emb, while synthesizing a normal version of AG, produced truncated forms of LAM when maintained on 10 μg/ml Emb (Mikušová, K., Slayden, R. A., Besra, G. S., and Brennan, P. J. (1995) Antimicrob. Agents Chemother. 39, 2482-2489). We have now isolated and characterized the truncated LAMs made by both the resistant mutant and a recombinant strain transfected with a plasmid containing the emb region from Mycobacterium avium which encodes for Emb resistance. By chemical analysis, endoarabinanase digestion, high pH anion exchange chromatography, and mass spectrometry analyses, truncation was demonstrated as primarily a consequence of selective and partial inhibition of the synthesis of the linear arabinan terminal motif, which constitutes a substantial portion of the arabinan termini in LAM but not of AG. However, at higher concentrations, Emb also affected the general biosynthesis of arabinan destined for both AG and LAM, resulting in severely truncated LAM as well as AG with a reduced Ara:Gal ratio. The results suggested that Emb exerts its antimycobacterial effect by inhibiting an array of arabinosyltransferases involved in the biosynthesis of arabinans unique to the mycobacterial cell wall. It was further concluded that the uniquely branched terminal Ara6 motif common to both AG and LAM is an essential structural entity for a functional cell wall and, consequently, that the biosynthetic machinery responsible for its synthesis is the effective target of Emb in its role as a potent anti-tuberculosis drug.

Evolution of high-level ethambutol-resistant tuberculosis through interacting mutations in decaprenylphosphoryl-β- D -arabinose biosynthetic and utilization pathway genes

To study the evolution of drug resistance, we genetically and biochemically characterized Mycobacterium tuberculosis strains selected in vitro for ethambutol resistance. Mutations in decaprenylphosphoryl-β-D-arabinose (DPA) biosynthetic and utilization pathway genes Rv3806c, Rv3792, embB and embC accumulated to produce a wide range of ethambutol minimal inhibitory concentrations (MICs) that depended on mutation type and number. Rv3806c mutations increased DPA synthesis, causing MICs to double from 2 to 4 μg/ml in a wild-type background and to increase from 16 to 32 μg/ml in an embB codon 306 mutant background. Synonymous mutations in Rv3792 increased the expression of downstream embC, an ethambutol target, resulting in MICs of 8 μg/ml. Multistep selection was required for high-level resistance. Mutations in embC or very high embC expression were observed at the highest resistance level. In clinical isolates, Rv3806c mutations were associated with high-level resistance and had multiplicative effects with embB mutations on MICs. Ethambutol resistance is acquired through the acquisition of mutations that interact in complex ways to produce a range of MICs, from those falling below breakpoint values to ones representing high-level resistance.

Identification and macrophage-activating activity of glycolipids released from intracellular Mycobacterium bovis BCG

Intracellular mycobacteria release cell wall glycolipids into the endosomal network of infected macrophages. Here, we characterize the glycolipids of Mycobacterium bovis BCG (BCG) that are released into murine bone marrow‐derived macrophages (BMMØ). Intracellularly released mycobacterial lipids were harvested from BMMØ that had been infected with 14C‐labelled BCG. Released BCG lipids were resolved by thin‐layer chromatography, and they migrated similarly to phosphatidylinositol dimannosides (PIM2), mono‐ and diphosphatidylglycerol, phosphatidylethanolamine, trehalose mono‐ and dimycolates and the phenolic glycolipid, mycoside B. Culture‐derived BCG lipids that co‐migrated with the intracellularly released lipids were purified and identified by electrospray ionization mass spectrometry. When delivered on polystyrene microspheres, fluorescently tagged BCG lipids were also released into the BMMØ, in a manner similar to release from viable or heat‐killed BCG bacilli. To determine whether the released lipids elicited macrophage responses, BCG lipid‐coated microspheres were delivered to interferon gamma‐primed macrophages (BMMØ or thioglycollate‐elicited peritoneal macrophages), and reactive nitrogen intermediates as well as tumour necrosis factor‐alpha and monocyte chemoattractant protein‐1 production were induced. When fractionated BCG lipids were delivered on the microspheres, PIM2 species reproduced the macrophage‐activating activity of total BCG lipids. These results demonstrate that intracellular mycobacteria release a heterogeneous mix of lipids, some of which elicit the production of proinflammatory cytokines from macrophages that could potentially contribute to the granulomatous response in tuberculous diseases.

Biosynthesis of mycobacterial lipoarabinomannan: Role of a branching mannosyltransferase

Lipoarabinomannan (LAM), one of the few known bacterial glycosylphosphoinositides (GPIs), occurs in various structural forms in Mycobacterium species. It has been implicated in key aspects of the physiology of Mycobacterium tuberculosis and the immunology and pathogenesis of tuberculosis. Yet, little is known of the biosynthesis of LAM. A bioinformatics approach identified putative integral membrane proteins, MSMEG4250 in Mycobacterium smegmatis and Rv2181 in M. tuberculosis, with 10 predicted transmembrane domains and a glycosyltransferase (GT) motif (DID), features that are common to eukaryotic mannosyltransferases (ManTs) of the GT-C superfamily that rely on polyprenyl-linked rather than nucleotide-linked sugar donors. Inactivation of M. smegmatis MSMEG4250 by allelic exchange resulted in altered growth and inability to synthesize lipomannan (LM) but accumulation of a previously uncharacterized, truncated LAM. MALDI-TOF/MS and NMR indicated a structure lower in molecular weight than the native molecule, a preponderance of 6-linked Manp residues, and the absence of 2,6-linked and terminal Manp. Complementation of the mutant with the corresponding ortholog of M. tuberculosis H37Rv restored normal LM/LAM synthesis. The data suggest that MSMEG4250 and Rv2181 are ManTs that are responsible for the addition of α(1→2) branches to the mannan core of LM/LAM and that arrest of this branching in the mutant deters formation of native LAM. The results allow for the presentation of a unique model of LM and LAM biosynthesis. The generation of mutants defective in the synthesis of LM/LAM will help define the role of these GPIs in the immunology and pathogenesis of mycobacterial infections and physiology of the organism.