We report on the existence of two phosphatidic acid biosynthetic pathways in mycobacteria, a classical one wherein the acylation of the sn-1 position of glycerol-3-phosphate (G3P) precedes that of sn-2 and another wherein acylations proceed in the reverse order. Two unique acyltransferases, PlsM and PlsB2, participate in both pathways and hold the key to the unusual positional distribution of acyl chains typifying mycobacterial glycerolipids wherein unsaturated substituents principally esterify position sn-1 and palmitoyl principally occupies position sn-2. While PlsM selectively transfers a palmitoyl chain to the sn-2 position of G3P and sn-1-lysophosphatidic acid (LPA), PlsB2 preferentially transfers a stearoyl or oleoyl chain to the sn-1 position of G3P and an oleyl chain to sn-2-LPA. PlsM is the first example of an sn-2 G3P acyltransferase outside the plant kingdom and PlsB2 the first example of a 2-acyl-G3P acyltransferase. Both enzymes are unique in their ability to catalyze acyl transfer to both G3P and LPA.

Dectin-2 is a C-type lectin involved in the recognition of several pathogens such as Aspergillus fumigatus , Candida albicans , Schistosoma mansonii , and Mycobacterium tuberculosis that triggers Th17 immune responses. Identifying pathogen ligands and understanding the molecular basis of their recognition is one of the current challenges. Purified M . tuberculosis mannose-capped lipoarabinomannan (ManLAM) was shown to induce signaling via Dectin-2, an activity that requires the (α1 → 2)-linked mannosides forming the caps. Here, using isogenic M . tuberculosis mutant strains, we demonstrate that ManLAM is a bona fide and actually the sole ligand mediating bacilli recognition by Dectin-2, although M . tuberculosis produces a variety of cell envelope mannoconjugates, such as phosphatidyl- myo -inositol hexamannosides, lipomannan or manno(lipo)proteins, that bear (α1 → 2)-linked mannosides. In addition, we found that Dectin-2 can recognize lipoglycans from other bacterial species, such as Saccharotrix aerocolonigenes or the human opportunistic pathogen Tsukamurella paurometabola , suggesting that lipoglycans are prototypical Dectin-2 ligands. Finally, from a structure/function relationship perspective, we show, using lipoglycan variants and synthetic mannodendrimers, that dimannoside caps and multivalent interaction are required for ligand binding to and signaling via Dectin-2. Better understanding of the molecular basis of ligand recognition by Dectin-2 will pave the way for the rational design of potent adjuvants targeting this receptor.

Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the alpha-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5-7 unbranched alpha-6-linked-mannosyl residues followed by two or three alpha-6-linked mannosyl residues branched with single alpha-mannopyranose residues at O-2. After these branched mannosyl residues, the alpha-6-linked mannan chain is terminated with an alpha-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of arabinans attached to the mannan core of LM in two other mutants (Delta embC and Delta MSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with similar to 10-12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl "primer." Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.

Considerable progress has been made in recent years in our understanding of the structural basis of glycosyl transfer. Yet the nature and relevance of the conformational changes associated with substrate recognition and catalysis remain poorly understood. We have focused on the glucosyl-3-phosphoglycerate synthase (GpgS), a "retaining" enzyme, that initiates the biosynthetic pathway of methylglucose lipopolysaccharides in mycobacteria. Evidence is provided that GpgS displays an unusually broad metal ion specificity for a GT-A enzyme, with Mg(2+), Mn(2+), Ca(2+), Co(2+), and Fe(2+) assisting catalysis. In the crystal structure of the apo-form of GpgS, we have observed that a flexible loop adopts a double conformation L(A) and L(I) in the active site of both monomers of the protein dimer. Notably, the L(A) loop geometry corresponds to an active conformation and is conserved in two other relevant states of the enzyme, namely the GpgS·metal·nucleotide sugar donor and the GpgS·metal·nucleotide·acceptor-bound complexes, indicating that GpgS is intrinsically in a catalytically active conformation. The crystal structure of GpgS in the presence of Mn(2+)·UDP·phosphoglyceric acid revealed an alternate conformation for the nucleotide sugar β-phosphate, which likely occurs upon sugar transfer. Structural, biochemical, and biophysical data point to a crucial role of the β-phosphate in donor and acceptor substrate binding and catalysis. Altogether, our experimental data suggest a model wherein the catalytic site is essentially preformed, with a few conformational changes of lateral chain residues as the protein proceeds along the catalytic cycle. This model of action may be applicable to a broad range of GT-A glycosyltransferases.

Two genes from the "mycobacterial arabinogalactan biosynthetic cluster" spanning the region from Rv3779 to Rv3809c in the genome of Mycobacterium tuberculosis H37Rv were annotated as possible components of the ATP-binding cassette transporter. Rv3781 encodes a nucleotide-binding domain and Rv3783 determines production of a membrane-spanning domain. We have examined possible roles of these genes in mycobacterial cell wall biosynthesis through inactivation of their respective orthologs in Mycobacterium smegmatis mc(2)155, phenotypic characterization of the mutant strains via metabolic labeling with [U-(14)C]-glucose, cell-free reactions with UDP-[U-(14)C]-galactose monitoring galactan build-up and transcriptional analysis. Several lines of evidence suggest that this ABC transporter is involved in biosynthesis of arabinogalactan, although more investigation is needed to establish its precise role or the transported substrate.

Phosphatidyl-myo-inositol mannosides (PIMs) are key glycolipids of the mycobacterial cell envelope. They are considered not only essential structural components of the cell but also important molecules implicated in host-pathogen interactions. Although their chemical structures are well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still incomplete. Here we show for the first time that although both mannosyltransferases PimA and PimB' (MSMEG_4253) recognize phosphatidyl-myo-inositol (PI) as a lipid acceptor, PimA specifically catalyzes the transfer of a Manp residue to the 2-position of the myo-inositol ring of PI, whereas PimB' exclusively transfers to the 6-position. Moreover, whereas PimB' can catalyze the transfer of a Manp residue onto the PI-monomannoside (PIM1) product of PimA, PimA is unable in vitro to transfer Manp onto the PIM1 product of PimB'. Further assays using membranes from Mycobacterium smegmatis and purified PimA and PimB' indicated that the acylation of the Manp residue transferred by PimA preferentially occurs after the second Manp residue has been added by PimB'. Importantly, genetic evidence is provided that pimB' is an essential gene of M. smegmatis. Altogether, our results support a model wherein Ac1PIM2, a major form of PIMs produced by mycobacteria, arises from the consecutive action of PimA, followed by PimB', and finally the acyltransferase MSMEG_2934. The essentiality of these three enzymes emphasizes the interest of novel anti-tuberculosis drugs targeting the initial steps of PIM biosynthesis.

We report on the identification of a glycosyltransferase (GT) from Mycobacterium tuberculosis H37Rv, Rv3779, of the membranous GT-C superfamily responsible for the direct synthesis of polyprenyl-phosphomannopyranose and thus indirectly for lipoarabinomannan, lipomannan, and the higher-order phosphatidyl-myo-inositol mannosides.

The re-emergence of tuberculosis in its present-day manifestations - single, multiple and extensive drug-resistant forms and as HIV-TB coinfections - has resulted in renewed research on fundamental questions such as the nature of the organism itself, Mycobacterium tuberculosis, the molecular basis of its pathogenesis, definition of the immunological response in animal models and humans, and development of new intervention strategies such as vaccines and drugs. Foremost among these developments has been the precise chemical definition of the complex and distinctive cell wall of M. tuberculosis, elucidation of the relevant pathways and underlying genetics responsible for the synthesis of the hallmark moieties of the tubercle bacillus such as the mycolic acid-arabinogalactan-peptidoglycan complex, the phthiocerol- and trehalose-containing effector lipids, the phosphatidylinositol-containing mannosides, lipomannosides and lipoarabinomannosides, major immunomodulators, and others. In this review, the laboratory personnel who have been the focal point of some to these developments review recent progress towards a comprehensive understanding of the basic physiology and functions of the cell wall of M. tuberculosis.

Background Mycobacteria produce two unique families of cytoplasmic polymethylated polysaccharides - the methylglucose lipopolysaccharides (MGLPs) and the methylmannose polysaccharides (MMPs) - the physiological functions of which are still poorly defined. Towards defining the roles of these polysaccharides in mycobacterial physiology, we generated knock-out mutations of genes in their putative biosynthetic pathways. Methodology/Principal Findings We report here on the characterization of the Rv1208 protein of Mycobacterium tuberculosis and its ortholog in Mycobacterium smegmatis (MSMEG_5084) as the enzymes responsible for the transfer of the first glucose residue of MGLPs. Disruption of MSMEG_5084 in M. smegmatis resulted in a dramatic decrease in MGLP synthesis directly attributable to the almost complete abolition of glucosyl-3-phosphoglycerate synthase activity in this strain. Synthesis of MGLPs in the mutant was restored upon complementation with wild-type copies of the Rv1208 gene from M. tuberculosis or MSMEG_5084 from M. smegmatis. Conclusions/Significance This is the first evidence linking Rv1208 to MGLP biosynthesis. Thus, the first step in the initiation of MGLP biosynthesis in mycobacteria has been defined, and subsequent steps can be inferred.

Biosynthesis of phosphatidylinositol (PI)-containing lipoarabinomannan (LAM) and lipomannan (LM) of Mycobacterium spp. follows a conserved pathway involving multiple membrane-associated, substrate-specific mannosyltransferases (ManTs) responsible for the sequential addition of alpha-mannopyranosyl (Manp) units donated by decaprenyl-P-Manp on the periplasmic side of the plasma membrane. Because of their receptor-binding and immunomodulatory properties, the alpha(1-->2)-linked di- and tri-Manp motifs that functionalize the nonreducing arabinan termini of LAM (ManLAM) in Mycobacterium tuberculosis are of crucial importance. We now show that the M. tuberculosis ManT, Rv2181, is required for the addition of these alpha(1-->2)-linked Manp residues but also at other locations of the LAM molecule. Structural analyses of the LM and LAM variants produced by a M. tuberculosis Rv2181 knockout mutant revealed the presence of but a single Manp residue on the nonreducing arabinan termini of LAM and also a complete absence of alpha(1-->2)-linked Man branching on the mannan backbones of LM and LAM. A recombinant strain was constructed in ManLAM-deficient Mycobacterium smegmatis that coexpressed Rv2181 and Rv1635c-the ManT responsible for the addition of the first Manp capping residue of ManLAM. Analysis revealed LAM termini fully capped with di- and tri-Manp motifs in addition to alpha(1-->2)Man branching on the mannan backbones of LM and LAM, confirming the involvement of the alpha(1-->2)ManT Rv2181 in the dual role of Man capping and mannan-core branching, and in the process generated a rapidly growing, ManLAM-containing strain, a tool for the study of the role of ManLAM in the pathogenesis of tuberculosis.

Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharide biosynthesis in mycobacteria. These important molecules are believed to be involved in the regulation of fatty-acid and mycolic acid synthesis. The enzyme belongs to the recently defined GT81 family of retaining glycosyltransferases (CAZy, Carbohydrate-Active Enzymes Database; see http://www.cazy.org). Here, the purification, crystallization and preliminary crystallographic analysis are reported of GpgS from Mycobacterium tuberculosis and of its complex with UDP. GpgS crystals belonged to space group I4, with unit-cell parameters a = 98.85, b = 98.85, c = 127.64 A, and diffracted to 2.6 A resolution. GpgS-UDP complex crystals belonged to space group I4, with unit-cell parameters a = 98.32, b = 98.32, c = 127.96 A, and diffracted to 3.0 A resolution.

In Mycobacterium tuberculosis, two related Z-prenyl diphosphate synthases, E,Z-farnesyl diphosphate synthase (Rv1086) and decaprenyl diphosphate synthase (Rv2361c), work in series to synthesize decaprenyl phosphate (C(50)) from isopentenyl diphosphate and E-geranyl diphosphate. Decaprenyl phosphate plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan; thus, its synthesis has attracted considerable interest as a potential therapeutic target. Rv1086 is a unique prenyl diphosphate synthase in that it adds only one isoprene unit to geranyl diphosphate, generating the 15-carbon product (E,Z-farnesyl diphosphate). Rv2361c then adds a further seven isoprene units to E,Z-farnesyl diphosphate in a processive manner to generate the 50-carbon prenyl diphosphate, which is then dephosphorylated to generate a carrier for activated sugars. The molecular basis for chain-length discrimination by Rv1086 during synthesis is unknown. We also report the structure of apo Rv1086 with citronellyl diphosphate bound and with the product mimic E,E-farnesyl diphosphate bound. We report the structures of Rv2361c in the apo form, with isopentenyl diphosphate bound and with a substrate analogue, citronellyl diphosphate. The structures confirm the enzymes are very closely related. Detailed comparison reveals structural differences that account for chain-length control in Rv1086. We have tested this hypothesis and have identified a double mutant of Rv1086 that makes a range of longer lipid chains.

ABSTRACT Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli . In the MEP pathway, 4-diphosphocytidyl-2- C -methyl- d -erythritol is formed from 2- C -methyl- d -erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2- C -methyl- d -erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis : Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2- C -methyl- d -erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg 2+ being optimal, and replacement of CTP with other nucleotide 5′-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had K m values of 58.5 μM for MEP and 53.2 μM for CTP. Calculated k cat and k cat / K m values were 0.72 min −1 and 12.3 mM −1 min −1 for MEP and 1.0 min −1 and 18.8 mM −1 min −1 for CTP, respectively.

Several human pathogens are to be found within the bacterial genus Mycobacterium, notably Mycobacterium tuberculosis, the causative agent of tuberculosis, one of the most threatening of human infectious diseases, with an annual lethality of about two million people. The characteristic mycobacterial cell envelope is the dominant feature of the biology of M. tuberculosis and other mycobacterial pathogens, based on sugars and lipids of exceptional structure. The cell wall consists of a peptidoglycan-arabinogalactan-mycolic acid complex beyond the plasma membrane. Free-standing lipids, lipoglycans, and proteins intercalate within this complex, complement the mycolic acid monolayer and may also appear in a capsular-like arrangement. The consequences of these structural oddities are an extremely robust and impermeable cell envelope. This review reflects on these entities from the perspective of their synthesis, particularly the structural and functional aspects of the glycosyltransferases (GTs) of M. tuberculosis, the dominating group of enzymes responsible for the terminal stages of their biosynthesis. Besides the many nucleotide-sugar dependent GTs with orthologs in prokaryotes and eukaryotes, M. tuberculosis and related species of the order Actinomycetales, in light of the highly lipophilic environment prevailing within the cell envelope, carry a significant number of GTs of the GT-C class dependent on polyprenyl-phosphate-linked sugars. These are of special emphasis in this review.

Genetic construction of a mutant strain (designated MSMEG4245) of Mycobacterium smegmatis, defective in a broadly conserved gene for a putative glycosyltransferase of the glycosyltransferase-C superfamily, results in a phenotype marked by the virtual absence of the phosphatidylinositol-containing lipomannan and lipoarabinomannan, replaced instead by a novel truncated form of lipomannan. The normal spectrum of phosphatidylinositol mannosides, long presumed precursors of these lipoglycans, was retained. Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry of the mutated form of lipomannan shows a family of phosphatidylinositol-anchored lipomannans with from only 5 to 20 Manp residues as compared with lipomannan from the wild type strain consisting of 21-34 Manp residues but with few changes in the branching pattern. Thus, MSMEG4245 is apparently a key mannosyltransferase, required for the proper elongation of lipomannan to its normal state and subsequent synthesis of lipoarabinomannan. The corresponding ortholog in Mycobacterium tuberculosis H37Rv has been identified as Rv2174. This previously unrecognized feature of the biosynthesis of lipomannan/lipoarabinomannan allows a significant revision of structural and biosynthetic schemata and provides a molecular basis of selectivity in biosynthesis, as conferred by the MSMEG4245 gene.

Many eubacteria, including Mycobacterium tuberculosis, utilize the 2‐C‐methyl‐D‐erythritol (MEP) pathway for the biosynthesis of isopentenyl diphosphate, a precursor of all isoprenoid compounds. This pathway is of interest as a source of potential drug targets since it is absent in mammals and disruption of the genes involved in Escherichia coli are lethal. In the MEP pathway, 4‐diphosphocytidyl‐2‐C‐methyl‐D‐erythritol is formed from MEP and cytidine 5′‐triphosphate (CTP), which is catalyzed by a 4‐diphosphocytidyl‐2‐C‐methyl‐D‐erythritol (CDP‐ME) synthase (IspD in E. coli). Blast searches show that the protein encoded by Rv3582c in M. tuberculosis has significant homology to E. coli IspD. Rv3582c was cloned and expressed and the purified protein is capable of catalyzing formation of CDP‐ME from MEP and CTP. The enzyme is active between pH 6.0 – 9.0 with optimal activity at pH 8.0 and requires divalent cations. Rv3582c has Km values of 54 μM and 49 μM for MEP and CTP, respectively. The Kcat and Kcat/Km values were calculated to be 3.29 × 104min−1 and 6.13 × 105M−1min−1 and 4.28 × 104 min−1 and 8.66 × 105M−1min−1for MEP and CTP, respectively. Allelic disruption of Rv3582c in M. tuberculosis provided direct evidence that the protein encoded by this gene is essential for M. tuberculosis survival. Thus, Rv3582c encodes a functional IspD in M. tuberculosis, and provides a new target for the development of novel anti‐tuberculosis drugs.

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 Man p residues, and the absence of 2,6-linked and terminal Man p . 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.