Ppm1-Encoded Polyprenyl Monophosphomannose Synthase Activity Is Essential for Lipoglycan Synthesis and Survival in Mycobacteria

The biosynthesis of mycobacterial mannose-containing lipoglycans, such as lipomannan (LM) and the immunomodulator lipoarabinomanan (LAM), is carried out by the GT-C superfamily of glycosyltransferases that require polyprenylphosphate-based mannose (PPM) as a sugar donor. The essentiality of lipoglycan synthesis for growth makes the glycosyltransferase that synthesizes PPM, a potential drug target in Mycobacterium tuberculosis, the causative agent of tuberculosis. In M. tuberculosis, PPM has been shown to be synthesized by Ppm1 in enzymatic assays. However, genetic evidence for its essentiality and in vivo role in LM/LAM and PPM biosynthesis is lacking. In this study, we demonstrate that MSMEG3859, a Mycobacterium smegmatis gene encoding the homologue of the catalytic domain of M. tuberculosis Ppm1, is essential for survival. Depletion of MSMEG3859 in a conditional mutant of M. smegmatis resulted in the loss of higher order phosphatidyl-myo-inositol mannosides (PIMs) and lipomannan. We were also able to demonstrate that two other M. tuberculosis genes encoding glycosyltransferases that either had been shown to possess PPM synthase activity (Rv3779), or were involved in synthesizing similar polyprenol-linked donors (ppgS), were unable to compensate for the loss of MSMEG3859 in the conditional mutant.     

Identification of a novel alpha(1-->6) mannopyranosyltransferase MptB from Corynebacterium glutamicum by deletion of a conserved gene, NCgl1505, affords a lipomannan- and lipoarabinomannan-deficient mutant

Mycobacterium tuberculosis and Corynebacterium glutamicum share a similar cell wall structure and orthologous enzymes involved in cell wall assembly. Herein, we have studied C. glutamicum NCgl1505, the orthologue of putative glycosyltransferases Rv1459c from M. tuberculosis and MSMEG3120 from Mycobacterium smegmatis. Deletion of NCgl1505 resulted in the absence of lipomannan (Cg-LM-A), lipoarabinomannan (Cg-LAM) and a multi-mannosylated polymer (Cg-LM-B) based on a 1,2-di-O-C(16)/C(18:1)-(alpha-D-glucopyranosyluronic acid)-(1-->3)-glycerol (GlcAGroAc(2)) anchor, while syntheses of triacylated-phosphatidyl-myo-inositol dimannoside (Ac(1)PIM(2)) and Man(1)GlcAGroAc(2) were still abundant in whole cells. Cell-free incubation of C. glutamicum membranes with GDP-[(14)C]Man established that C. glutamicum synthesized a novel alpha(1-->6)-linked linear form of Cg-LM-A and Cg-LM-B from Ac(1)PIM(2) and Man(1)GlcAGroAc(2) respectively. Furthermore, deletion of NCgl1505 also led to the absence of in vitro synthesized linear Cg-LM-A and Cg-LM-B, demonstrating that NCgl1505 was involved in core alpha(1-->6) mannan biosynthesis of Cg-LM-A and Cg-LM-B, extending Ac(1)PI[(14)C]M(2) and [(14)C]Man(1)GlcAGroAc(2) primers respectively. Use of the acceptor alpha-D-Manp-(1-->6)-alpha-D-Manp-O-C(8) in an in vitro cell-free assay confirmed NCgl1505 as an alpha(1-->6) mannopyranosyltransferase, now termed MptB. While Rv1459c and MSMEG3120 demonstrated similar in vitroalpha(1-->6) mannopyranosyltransferase activity, deletion of the Rv1459c homologue in M. smegmatis did not result in loss of mycobacterial LM/LAM, indicating a functional redundancy for this enzyme in mycobacteria.     

Analysis of a new mannosyltransferase required for the synthesis of phosphatidylinositol mannosides and lipoarbinomannan reveals two lipomannan pools in corynebacterineae

The cell walls of the Corynebacterineae, which includes the important human pathogen Mycobacterium tuberculosis, contain two major lipopolysaccharides, lipoarabinomannan (LAM) and lipomannan (LM). LAM is assembled on a subpool of phosphatidylinositol mannosides (PIMs), whereas the identity of the LM lipid anchor is less well characterized. In this study we have identified a new gene (Rv2188c in M. tuberculosis and NCgl2106 in Corynebacterium glutamicum) that encodes a mannosyltransferase involved in the synthesis of the early dimannosylated PIM species, acyl-PIM2, and LAM. Disruption of the C. glutamicum NCgl2106 gene resulted in loss of synthesis of AcPIM2 and accumulation of the monomannosylated precursor, AcPIM1. The synthesis of a structurally unrelated mannolipid, Gl-X, was unaffected. The synthesis of AcPIM2 in C. glutamicum DeltaNCgl2106 was restored by complementation with M. tuberculosis Rv2188c. In vivo labeling of the mutant with [3H]Man and in vitro labeling of membranes with GDP-[3H]Man confirmed that NCgl2106/Rv2188c catalyzed the second mannose addition in PIM biosynthesis, a function previously ascribed to PimB/Rv0557. The C. glutamicum Delta NCgl2106 mutant lacked mature LAM but unexpectedly still synthesized the major pool of LM. Biochemical analyses of the LM core indicated that this lipopolysaccharide was assembled on Gl-X. These data suggest that NCgl2106/Rv2188c and the previously studied PimB/Rv0557 transfer mannose residues to distinct mannoglycolipids that act as precursors for LAM and LM, respectively.     

Biosynthesis of mycobacterial arabinogalactan: identification of a novel alpha(1-->3) arabinofuranosyltransferase

The cell wall mycolyl-arabinogalactan–peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis and is the target of several antitubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. A bioinformatics approach identified putative integral membrane proteins, MSMEG2785 in Mycobacterium smegmatis , Rv2673 in Mycobacterium tuberculosis and NCgl1822 in Corynebacterium glutamicum , with 10 predicted transmembrane domains and a glycosyltransferase motif (DDX), features that are common to the GT-C superfamily of glycosyltransferases. Deletion of M. smegmatis MSMEG2785 resulted in altered growth and glycosyl linkage analysis revealed the absence of AG α(1→3)-linked arabinofuranosyl (Ara f ) residues. Complementation of the M. smegmatis deletion mutant was fully restored to a wild-type phenotype by MSMEG2785 and Rv2673, and as a result, we have now termed this previously uncharacterized open reading frame, a rabino f uranosyl t ransferase C ( aftC ). Enzyme assays using the sugar donor β- d -arabinofuranosyl-1-monophosphoryl-decaprenol (DPA) and a newly synthesized linear α(1→5)-linked Ara₅ neoglycolipid acceptor together with chemical identification of products formed, clearly identified AftC as a branching α(1→3) arabinofuranosyltransferase. This newly discovered glycosyltransferase sheds further light on the complexities of Mycobacterium cell wall biosynthesis, such as in M. tuberculosis and related species and represents a potential new drug target.     

Identification of an alpha(1-->6) mannopyranosyltransferase (MptA), involved in Corynebacterium glutamicum lipomanann biosynthesis, and identification of its orthologue in Mycobacterium tuberculosis

Corynebacterium glutamicum and Mycobacterium tuberculosis share a similar cell wall architecture, and the availability of their genome sequences has enabled the utilization of C. glutamicum as a model for the identification and study of, otherwise essential, mycobacterial genes involved in lipomannan (LM) and lipoarabinomannan (LAM) biosynthesis. We selected the putative glycosyltransferase-Rv2174 from M. tuberculosis and deleted its orthologue NCgl2093 from C. glutamicum. This resulted in the formation of a novel truncated lipomannan (Cg-t-LM) and a complete ablation of LM/LAM biosynthesis. Purification and characterization of Cg-t-LM revealed an overall decrease in molecular mass, a reduction of alpha(1-->6) and alpha(1-->2) glycosidic linkages illustrating a reduced degree of branching compared with wild-type LM. The deletion mutant's biochemical phenotype was fully complemented by either NCgl2093 or Rv2174. Furthermore, the use of a synthetic neoglycolipid acceptor in an in vitro cell-free assay utilizing the sugar donor beta-D-mannopyranosyl-1-monophosphoryl-decaprenol together with the neoglycolipid acceptor alpha-D-Manp-(1-->6)-alpha-D-Manp-O-C8 as a substrate, confirmed NCgl2093 and Rv2174 as an alpha(1-->6) mannopyranosyltransferase (MptA), involved in the latter stages of the biosynthesis of the alpha(1-->6) mannan core of LM. Altogether, these studies have identified a new mannosyltransferase, MptA, and they shed further light on the biosynthesis of LM/LAM in Corynebacterianeae.     

Genetic basis for the biosynthesis of methylglucose lipopolysaccharides in Mycobacterium tuberculosis

Mycobacteria produce two unusual polymethylated polysaccharides, the 6-O-methylglucosyl-containing lipopolysaccharides (MGLP) and the 3-O-methylmannose polysaccharides, which have been shown to regulate fatty acid biosynthesis in vitro. A cluster of genes dedicated to the synthesis of MGLP was identified in Mycobacterium tuberculosis and Mycobacterium smegmatis. Overexpression of the putative glycosyltransferase gene Rv3032 in M. smegmatis greatly stimulated MGLP production, whereas the targeted disruption of Rv3032 in M. tuberculosis and that of the putative methyltransferase gene MSMEG2349 in M. smegmatis resulted in a dramatic reduction in the amounts of MGLP synthesized and in the accumulation of precursors of these molecules. Disruption of Rv3032 also led to a significant decrease in the glycogen content of the tubercle bacillus, indicating that the product of this gene is likely to be involved in the elongation of more than one alpha-(1-->4)-glucan in this bacterium. Results thus suggest that Rv3032 encodes the alpha-(1-->4)-glucosyltransferase responsible for the elongation of MGLP, whereas MSMEG2349 encodes the O-methyltransferase required for the 6-O-methylation of these compounds.     

Identification of the required acyltransferase step in the biosynthesis of the phosphatidylinositol mannosides of mycobacterium species

Fatty acyl functions of the glycosylated phosphatidylinositol (GPI) anchors of the phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM) of mycobacteria play a critical role in both the physical properties and biological activities of these molecules. In a search for the acyltransferases that acylate the GPI anchors of PIM, LM, and LAM, we examined the function of the mycobacterial Rv2611c gene that encodes a putative acyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis. A Rv2611c mutant of Mycobacterium smegmatis was constructed which exhibited severe growth defects and contained an increased amount of phosphatidylinositol mono- and di-mannosides and a decreased amount of acylated phosphatidylinositol di-mannosides compared with the wild-type parental strain. In cell-free assays, extracts from M. smegmatis overexpressing the M. tuberculosis Rv2611c gene incorporated [14C]palmitate into acylated phosphatidylinositol mono- and di-mannosides, and transferred cold endogenous fatty acids onto 14C-labeled phosphatidylinositol mono- and di-mannosides more efficiently than extracts from the wild-type strain. Cell-free extracts from the Rv2611c mutant of M. smegmatis were greatly impaired in these respects. This work provides evidence that Rv2611c is the acyltransferase that catalyzes the acylation of the 6-position of the mannose residue linked to position 2 of myo-inositol in phosphatidylinositol mono- and di-mannosides, with the mono-mannosylated lipid acceptor being the primary substrate of the enzyme. We also provide the first evidence that two distinct pathways lead to the formation of acylated PIM2 from PIM1 in mycobacteria.     

Identification of a novel protein with a role in lipoarabinomannan biosynthesis in mycobacteria

All species of Mycobacteria synthesize distinctive cell walls that are rich in phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). PIM glycolipids, having 2-4 mannose residues, can either be channeled into polar PIM species (with 6 Man residues) or hypermannosylated to form LM and LAM. In this study, we have identified a Mycobacterium smegmatis gene, termed lpqW, that is required for the conversion of PIMs to LAM and is highly conserved in all mycobacteria. A transposon mutant, Myco481, containing an insertion near the 3' end of lpqW exhibited altered colony morphology on complex agar medium. This mutant was unstable and was consistently overgrown by a second mutant, represented by Myco481.1, that had normal growth and colony characteristics. Biochemical analysis and metabolic labeling studies showed that Myco481 synthesized the complete spectrum of apolar and polar PIMs but was unable to make LAM. LAM biosynthesis was restored to near wild type levels in Myco481.1. However, this mutant was unable to synthesize the major polar PIM (AcPIM6) and accumulated a smaller intermediate, AcPIM4. Targeted disruption of the lpqW gene and complementation of the initial Myco481 mutant with the wild type gene confirmed that the phenotype of this mutant was due to loss of LpqW. These studies suggest that LpqW has a role in regulating the flux of early PIM intermediates into polar PIM or LAM biosynthesis. They also suggest that AcPIM4 is the likely branch point intermediate in polar PIM and LAM biosynthesis.     

Structural definition of acylated phosphatidylinositol mannosides from Mycobacterium tuberculosis: definition of a common anchor for lipomannan and lipoarabinomannan

Based on chemical analysis, we have previously concluded thatthe biologically important lipoarabinomannan (LAM) and lipomannan(LM) from Mycobacterium are multiglycosylated forms of the phosphatidylinositolmannosides (PIMs), the characteristic cell envelope mannophosphoinositidesof mycobacteria Using definitive analytical techniques, we havenow re-examined the reported multiacylated nature of PIMs inorder to gain a better insight into their possible roles asbiosynthethic precursors of LM and LAM. High-sensitivity fastatom bombardment-mass spectrometry analyses of the perdeuteroacetyland permethyl derivatives of PIMs from Mycobacterium tuberculosisand Mycobacterium leprae enabled us to define the exact fattyacyl compositions of the multiacylated, heterogeneous PIM families,notably the dimannoside (PIM₂) and the hexamannoside (PIM₆).Specifically, in conjunction with other chemical and gas chromatography-massspectrometry (GCMS) analyses, the additional C16 fatty acylsubstituent on PIM₂ and its lyso form were defined as attachedto the C6 position of mannose. We also present evidence fortriacylated mannophosphoinositide as a common lipid anchor forboth LM and LAM, and further postulate that acylation of PIM₂may constitute a key regulatory step in their biosynthesis. FAB-MS lipoarabinomannan lipomannan Mycobacterium tuberculosis phosphatidylinositol mannosides     

Structural characterization and functional properties of a novel lipomannan variant isolated from a Corynebacterium glutamicum pimB′ mutant

The genus Corynebacterium is part of the phylogenetic group nocardioform actinomycetes, which also includes the genus Mycobacterium. Members of this phylogenetic group have a characteristic cell envelope structure, which is dominated by complex lipids and amongst these, lipoglycans are of particular interest. The disruption of NCgl2106 in C. glutamicum resulted in a mutant devoid of monoacylated phosphatidyl-myo-inositol dimannoside (Ac(1)PIM(2)) resulting in the accumulation of Ac(1)PIM(1) and cessation of phosphatidyl-myo-inositol (PI) based lipomannan (Cg-LM, now also termed 'Cg-LM-A') and lipoarabinomannan (Cg-LAM) biosynthesis. Interestingly, SDS-analysis of the lipoglycan fraction from the mutant revealed the synthesis of a single novel lipoglycan, now termed 'Cg-LM-B'. Further chemical analyses established the lipoglycan possessed an alpha-D: -glucopyranosyluronic acid-(1 --> 3)-glycerol (GlcAGroAc(2)) based anchor which was then further glycosylated by 8-22 mannose residues, with Man(12-20)GlcAGroAC(2) molecular species being the most abundant, to form a novel lipomannan structure (Cg-LM-B). The deletion of NCgl2106 in C. glutamicum has now provided a useful strain, in addition with a deletion mutant of NCgl0452 in C. glutamicum for the purification of Cg-LM-A and Cg-LM-B. Interestingly, both Cg-LM species induced a similar production of TNF-alpha by a human macrophage cell line suggesting that the phospho-myo-inositol residue of the PI-anchor does not play a key role in lipoglycan pro-inflammatory activity.     

Overexpression of Mycobacterium tuberculosis manB, a phosphomannomutase that increases phosphatidylinositol mannoside biosynthesis in Mycobacterium smegmatis and mycobacterial association with human macrophages

Mycobacterium tuberculosis (M. tb) pathogenesis involves the interaction between the mycobacterial cell envelope and host macrophage, a process mediated, in part, by binding of the mannose caps of M. tb lipoarabinomannan (ManLAM) to the macrophage mannose receptor (MR). A presumed critical step in the biosynthesis of ManLAM, and other mannose-containing glycoconjugates, is the conversion of mannose-6-phosphate to mannose-1-phosphate, by a phosphomannomutase (PMM), to produce GDP-mannose, the primary mannose-donor in mycobacteria. We have identified four M. tb H37Rv genes with similarity to known PMMs. Using in vivo complementation of PMM and phosphoglucomutase (PGM) deficient strains of Pseudomonas aeruginosa, and an in vitro enzyme assay, we have identified both PMM and PGM activity from one of these genes, Rv3257c (MtmanB). MtmanB overexpression in M. smegmatis produced increased levels of LAM, lipomannan, and phosphatidylinositol mannosides (PIMs) compared with control strains and led to a 13.3 +/- 3.9-fold greater association of mycobacteria with human macrophages, in a mannan-inhibitable fashion. This increased association was mediated by the overproduction of higher order PIMs that possess mannose cap structures. We conclude that MtmanB encodes a functional PMM involved in the biosynthesis of mannosylated lipoglycans that participate in the association of mycobacteria with macrophage phagocytic receptors.     

The two chorismate mutases from both Mycobacterium tuberculosis and Mycobacterium smegmatis: biochemical analysis and limited regulation of promoter activity by aromatic amino acids

Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate in the biosynthetic pathway that forms phenylalanine and tyrosine in bacteria, fungi, plants, and apicomplexan parasites. Since this enzyme is absent from mammals, it represents a promising target for the development of new antimycobacterial drugs, which are needed to combat Mycobacterium tuberculosis, the causative agent of tuberculosis. Until recently, two putative open reading frames (ORFs), Rv0948c and Rv1885c, showing low sequence similarity to CMs have been described as "conserved hypothetical proteins" in the M. tuberculosis genome. However, we and others demonstrated that these ORFs are in fact monofunctional CMs of the AroQ structural class and that they are differentially localized in the mycobacterial cell. Since homologues to the M. tuberculosis enzymes are also present in Mycobacterium smegmatis, we cloned the coding sequences corresponding to ORFs MSMEG5513 and MSMEG2114 from the latter. The CM activities of both ORFs was determined, as well as their translational start sites. In addition, we analyzed the promoter activities of three M. tuberculosis loci related to phenylalanine and tyrosine biosynthesis under a variety of conditions using M. smegmatis as a surrogate host. Our results indicate that the aroQ (Rv0948c), *aroQ (Rv1885c), and fbpB (Rv1886c) genes from M. tuberculosis are constitutively expressed or subjected to minor regulation by aromatic amino acids levels, especially tryptophan.     

PimF, a mannosyltransferase of mycobacteria, is involved in the biosynthesis of phosphatidylinositol mannosides and lipoarabinomannan

Phosphatidylinositol mannosides (PIMs) and their related molecules lipomannan (LM) and lipoarabinomannan (LAM) are important components of the mycobacterial cell wall. These molecules mediate host-pathogen interactions and exhibit immunomodulatory activities. The biosynthesis of these lipoglycans is not fully understood. In this study, we have identified a mycobacterial gene (Rv1500) that is involved in the synthesis of PIMs. We have named this gene pimF. Transposon mutagenesis of pimF of Mycobacterium marinum resulted in multiple phenotypes, including altered colony morphology, disappearance of tetracyl-PIM(7), and accumulation of tetraacyl-PIM(5). The syntheses of LAM and LM were also affected. In addition, the pimF mutant exhibited a defect during infection of cultured macrophage cells. Although the mutant was able to replicate and persist within macrophages, the initial cell entry step was inefficient. Transformation of the M. marinum mutant with the pimF homolog of Mycobacterium tuberculosis complemented all of the above mentioned phenotypes. These results provide evidence that PimF is a mannosyltransferase. However, sequence analysis indicates that PimF is distinct from mannosyltransferases involved in the early steps of PIM synthesis. PimF catalyzes the formation of high molecular weight PIMs, which are precursors for the synthesis of LAM and LM. As such, this work marks the first analysis of a mannosyltransferase involved in the later stages of PIM synthesis.     

Lipoglycans are putative ligands for the human pulmonary surfactant protein A attachment to mycobacteria. Critical role of the lipids for lectin-carbohydrate recognition

The human pulmonary surfactant protein A (hSP-A) has been implicated in the early capture and phagocytosis of the pathogenic Mycobacterium tuberculosis by alveolar macrophages. In this report, we examined the interaction of alveolar proteinosis patient hSP-A with Mycobacterium bovis BCG, the vaccinating strain, as a model of pathogenic mycobacteria, and Mycobacterium smegmatis, a nonpathogenic strain. We found that hSP-A binds to the surface of M. bovis BCG, but also to a slightly lesser extent, to M. smegmatis, indicating that hSP-A does not discriminate between virulent and nonpathogenic strains. Among the various glycoconjugates isolated from the mycobacterial envelope, we found that the best ligands are the two major lipoglycans: the mannosylated lipoarabinomannan (ManLAM) and the lipomannan. In contrast, the mannose-capped arabinomannan, structurally close to the ManLAM, as well as the LAMs from the non pathogenic M. smegmatis are poorly recognized by hSP-A. These results clearly show that the presence of both the terminal mannose residues and the phophatidyl-myo-inositol anchor are necessary to achieve the highest binding affinity. Selective removal of either the terminal mannose or the acyl residues esterifying the glycerol moiety of the ManLAM abrogates the interaction with hSP-A, further supporting the notion that the hSP-A recognition of the carbohydrate epitopes of the lipoglycans is dependent of the presence of the fatty acids.     

Evidence for the presence of a phosphatidylinositol anchor on the lipoarabinomannan and lipomannan of Mycobacterium tuberculosis

The recent availability (Hunter, S.W., Gaylord, H., and Brennan, P.J. (1986) J. Biol. Chem. 261, 12345-12351) of the well known arabinomannan of Mycobacterium leprae and Mycobacterium tuberculosis as the pure native lipoarabinomannan has resulted in its implication in key aspects of the immunopathogenesis of leprosy and tuberculosis. We had indicated that the lipid moiety of lipoarabinomannan is probably based on a diacylglycerol unit in that glycerol and the two fatty acids, hexadecanoate and 10-methyloctadecanoate, were identified. In addition, lipoarabinomannan was also shown to contain myo-inositol 1-phosphate. Evidence is now presented, based on selective radiolabeling and analysis of various cleavage fragments, that the inositol phosphate exists as both an alkalilable phosphodiester and as part of a phosphatidylinositol "membrane anchor." The mannan of M. tuberculosis was also isolated as the native lipomannan. It also apparently contains a phosphatidylinositol unit but is devoid of the alkali-labile inositol phosphate residues. These lipopolysaccharides are apparently multiglycosylated versions of the well known myocobacterial mannosyl phosphatidylinositols and are prokaryotic versions of the growing list of phosphatidylinositol-anchored macromolecules. Immunogold labeling demonstrates that lipoarabinomannan is a true antigenic capsular or extracellular product of M. tuberculosis. The presence of a phosphatidylinositol residue on lipoarabinomannan may explain its interaction with macrophage membranes and role in mycobacterial pathogenesis.     

Lipoarabinomannan and related glycoconjugates: structure, biogenesis and role in Mycobacterium tuberculosis physiology and host-pathogen interaction

Approximately one third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. This bacterium has an unusual lipid-rich cell wall containing a vast repertoire of antigens, providing a hydrophobic impermeable barrier against chemical drugs, thus representing an attractive target for vaccine and drug development. Apart from the mycolyl-arabinogalactan-peptidoglycan complex, mycobacteria possess several immunomodulatory constituents, notably lipomannan and lipoarabinomannan. The availability of whole-genome sequences of M. tuberculosis and related bacilli over the past decade has led to the identification and functional characterization of various enzymes and the potential drug targets involved in the biosynthesis of these glycoconjugates. Both lipomannan and lipoarabinomannan possess highly variable chemical structures, which interact with different receptors of the immune system during host-pathogen interactions, such as Toll-like receptors-2 and C-type lectins. Recently, the availability of mutants defective in the synthesis of these glycoconjugates in mycobacteria and the closely related bacterium, Corynebacterium glutamicum, has paved the way for host-pathogen interaction studies, as well as, providing attenuated strains of mycobacteria for the development of new vaccine candidates. This review provides a comprehensive account of the structure, biosynthesis and immunomodulatory properties of these important glycoconjugates.     

Phosphatidylinositol is an essential phospholipid of mycobacteria

Phosphatidylinositol (PI) and metabolically derived products such as the phosphatidylinositol mannosides and linear and mature branched lipomannan and lipoarabinomannan are prominent phospholipids/lipoglycans of Mycobacterium sp. believed to play important roles in the structure and physiology of the bacterium as well as during host infection. To determine if PI is an essential phospholipid of mycobacteria, we identified the pgsA gene of Mycobacterium tuberculosis encoding the phosphatidylinositol synthase enzyme and constructed a pgsA conditional mutant of Mycobacterium smegmatis. The ability of this mutant to synthesize phosphatidylinositol synthase and subsequently PI was dependent on the presence of a functional copy of the pgsA gene carried on a thermosensitive plasmid. The mutant grew like the control strain under permissive conditions (30 degrees C), but ceased growing when placed at 42 degrees C, a temperature at which the rescue plasmid is lost. Loss of cell viability at 42 degrees C was observed when PI and phosphatidylinositol dimannoside contents dropped to approximately 30 and 50% of the wild-type levels, respectively. This work provides the first evidence of the essentiality of PI to the survival of mycobacteria. PI synthase is thus an essential enzyme of Mycobacterium that shows promise as a drug target for anti-tuberculosis therapy.     

N-Acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis

Mycothiol is a novel thiol produced only by actinomycetes and is the major low-molecular-weight thiol in mycobacteria. Mycothiol was previously shown to be synthesized from 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside by ligation with cysteine followed by acetylation. A novel mycothiol-dependent detoxification enzyme, mycothiol conjugate amidase, was recently identified in Mycobacterium smegmatis and shown to have a homolog, Rv1082, in Mycobacterium tuberculosis. In the present study we found that a protein encoded by the M. tuberculosis open reading frame Rv1170, a homolog of Rv1082, possesses weak mycothiol conjugate amidase activity but shows substantial deacetylation activity with 1-D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins), a hypothetical mycothiol biosynthetic precursor. The availability of this protein enabled us to develop an assay for GlcNAc-Ins, which was used to demonstrate that GlcNAc-Ins is present in M. smegmatis at a level about twice that of mycothiol. It was shown that GlcNAc-Ins is absent in mycothiol-deficient mutant strain 49 of M. smegmatis and that this strain can concentrate GlcNAc-Ins from the medium and convert it to mycothiol. This demonstrates that GlcNAc-Ins is a key intermediate in the pathway of mycothiol biosynthesis. Assignment of Rv1170 as the gene coding the deacetylase in the M. tuberculosis genome represents the first identification of a gene of the mycothiol biosynthesis pathway. The presence of a large cellular pool of substrate for this enzyme suggests that it may be important in regulating mycothiol biosynthesis.     

Identification of the missing trans-acting enoyl reductase required for phthiocerol dimycocerosate and phenolglycolipid biosynthesis in Mycobacterium tuberculosis

Phthiocerol dimycocerosates (DIM) and phenolglycolipids (PGL) are functionally important surface-exposed lipids of Mycobacterium tuberculosis. Their biosynthesis involves the products of several genes clustered in a 70-kb region of the M. tuberculosis chromosome. Among these products is PpsD, one of the modular type I polyketide synthases responsible for the synthesis of the lipid core common to DIM and PGL. Bioinformatic analyses have suggested that this protein lacks a functional enoyl reductase activity domain required for the synthesis of these lipids. We have identified a gene, Rv2953, that putatively encodes an enoyl reductase. Mutation in Rv2953 prevents conventional DIM formation and leads to the accumulation of a novel DIM-like product. This product is unsaturated between C-4 and C-5 of phthiocerol. Consistently, complementation of the mutant with a functional pks15/1 gene from Mycobacterium bovis BCG resulted in the accumulation of an unsaturated PGL-like substance. When an intact Rv2953 gene was reintroduced into the mutant strain, the phenotype reverted to the wild type. These findings indicate that Rv2953 encodes a trans-acting enoyl reductase that acts with PpsD in phthiocerol and phenolphthiocerol biosynthesis.     

The Mycobacterium tuberculosis serine/threonine kinase PknL phosphorylates Rv2175c: mass spectrometric profiling of the activation loop phosphorylation sites and their role in the recruitment of Rv2175c

Although Mycobacterium tuberculosis (M. tb) comprises 11 serine/threonine protein kinases, the mechanisms of regulation of these kinases and the nature of their endogenous substrates remain largely unknown. Herein, we characterized the M. tb kinase PknL by demonstrating that it expresses autophosphorylation activity and phosphorylates Rv2175c. On-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, five phosphorylated threonine residues were identified in PknL. Among them, we showed that the activation loop phosphorylated residues Thr173 and Thr175 were essential for the autophosphorylation activity of PknL. Phosphorylation of the activation loop Thr173 residue is also required for optimal PknL-mediated phosphorylation of Rv2175c. Together, our results indicate that phosphorylation of the PknL activation loop Thr residues not only controls PknL kinase activity but is also required for recruitment and phosphorylation of its substrate. Rv2175c was found to be phosphorylated when overexpressed and purified from Mycobacterium smegmatis as 2-DE indicated the presence of different phosphorylated isoforms. Given the presence of the dcw gene cluster in the close vicinity of the pknL/Rv2175c locus, and its conservation in all mycobacterial species, we propose that PknL/Rv2175c may represent a functional pair in the regulation of mycobacterial cell division and cell envelope biosynthesis.