Inositol monophosphate phosphatase genes of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background  

Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome.     

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.     

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.     

Identification by surface plasmon resonance of the mycobacterial lipomannan and lipoarabinomannan domains involved in binding to CD14 and LPS-binding protein

The mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), regulate host defence mechanisms through their interaction with pattern recognition receptors such as Toll-like receptors (TLRs). We have developed a surface plasmon resonance assay to analyse the molecular basis for the recognition of Mycobacterium kansasii LM or LAM, by immobilized CD14 and LPS-binding protein (LBP) both being capable to promote presentation of bacterial glycolipids to TLRs. The affinity of either LM/LAM was higher to CD14 than to LBP. Kinetic and Scatchard analyses were consistent with a model involving a single class of binding sites. These interactions required the lipidic anchor, but not the carbohydrate domains, of LM or LAM. We also provide evidence that addition of recombinant LBP enhanced the stimulatory effect of LM or LAM on matrix metalloproteinase-9 expression and secretion in macrophages, through a TLR1/TLR2-dependent mechanism.     

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.     

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.     

Polysaccharide structural variability in mycobacteria: identification and characterization of phosphorylated mannan and arabinomannan

Arabinomannan (AMannan) and mannan (Mannan) are major polysaccharides antigens of the mycobacterial capsule. They are highly related to the lipoarabinomannan (LAM) and lipomannan (LM) lipoglycans of the cell wall, known to participate to the immunopathogenesis of mycobacterial infections. Here we present the identification of two related polysaccharides from Mycobacterium kansasii that co-purified with AMannan and Mannan. Structural analysis using GC, MALDI-MS and NMR clearly established these molecules as non-acylated phosphorylated AMannan and Mannan designated P-AMannan and P-Mannan, respectively. These glycoconjugates represent a new source of polysaccharide structural variability in mycobacteria and constitute unique tools for structure-activity relationship studies in order to investigate the role of fatty acids in the biological functions of LAM and LM. The potential participation of these polysaccharides in influencing the outcome of the infection is also discussed.     

Mannan core branching of lipo(arabino)mannan is required for mycobacterial virulence in the context of innate immunity

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, remains an important worldwide health threat. Although TB is one of the oldest infectious diseases of man, a detailed understanding of the mycobacterial mechanisms underlying pathogenesis remains elusive. Here, we studied the role of the α(1→2) mannosyltransferase MptC in mycobacterial virulence, using the Mycobacterium marinum zebrafish infection model. Like its M. tuberculosis orthologue, disruption of M. marinum mptC (mmar_3225) results in defective elongation of mannose caps of lipoarabinomannan (LAM) and absence of α(1→2)mannose branches on the lipomannan (LM) and LAM mannan core, as determined by biochemical analysis (NMR and GC‐MS) and immunoblotting. We found that the M. marinum mptC mutant is strongly attenuated in embryonic zebrafish, which rely solely on innate immunity, whereas minor virulence defects were observed in adult zebrafish. Strikingly, complementation with the Mycobacterium smegmatis mptC orthologue, which restored mannan core branching but not cap elongation, was sufficient to fully complement the virulence defect of the mptC mutant in embryos. Altogether our data demonstrate that not LAM capping, but mannan core branching of LM/LAM plays an important role in mycobacterial pathogenesis in the context of innate immunity.     

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.     

Structural definition of the non-reducing termini of mannose-capped LAM from Mycobacterium tuberculosis through selective enzymatic degradation and fast atom bombardment-mass spectrometry

The application of extracellular arabinases from aCellulomonassp. and fast atom bombardment-mass spectrometry (FAB-MS) providednew insight into the structure of lipoarabinomannan (LAM) ofMycobacterium tuberculosis, a key molecule in the pathogenesisand physiology of the tubercle bacillus. Previously, the non-reducingarabinan ends of LAM from the virulent (Erdman) strain of M.tuberculosiswere shown to be ‘capped’ by short (a1     

Differential Arabinan Capping of Lipoarabinomannan Modulates Innate Immune Responses and Impacts T Helper Cell Differentiation

Toll-like receptors (TLRs) recognize pathogens by interacting with pathogen-associated molecular patterns, such as the phosphatidylinositol-based lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM). Such structures are present in several pathogens, including Mycobacterium tuberculosis, being important for the initiation of immune responses. It is well established that the interaction of LM and LAM with TLR2 is a process dependent on the structure of the ligands. However, the implications of structural variations on TLR2 ligands for the development of T helper (Th) cell responses or in the context of in vivo responses are less studied. Herein, we used Corynebacterium glutamicum as a source of lipoglycan intermediates for host interaction studies. In this study, we have deleted a putative glycosyltransferase, NCgl2096, from C. glutamicum and found that it encodes for a novel α(1→2)arabinofuranosyltransferase, AftE. Biochemical analysis of the lipoglycans obtained in the presence (wild type) or absence of NCgl2096 showed that AftE is involved in the biosynthesis of singular arabinans of LAM. In its absence, the resulting molecule is a hypermannosylated (hLM) form of LAM. Both LAM and hLM were recognized by dendritic cells, mainly via TLR2, and triggered the production of several cytokines. hLM was a stronger stimulus for in vitro cytokine production and, as a result, a more potent inducer of Th17 responses. In vivo data confirmed hLM as a stronger inducer of cytokine responses and suggested the involvement of pattern recognition receptors other than TLR2 as sensors for lipoglycans.     

New Insights into the Early Steps of Phosphatidylinositol Mannoside Biosynthesis in Mycobacteria: PimB�� IS AN ESSENTIAL ENZYME OF MYCOBACTERIUM SMEGMATIS*

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.PIMs³ are unique mannolipids found in abundant quantities in the inner and outer membranes of the cell envelope of Mycobacterium spp. and a few other actinomycetes.⁴ They are based on a phosphatidyl-myo-inositol (PI) lipid anchor carrying one to six Manp residues and up to four acyl chains (for review see Refs. 1, 2). Based on a conserved mannosyl-PI anchor, they are also thought to be the precursors of the two major mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM) (1, 2). PIMs, LM, and LAM are considered not only essential structural components of the mycobacterial cell envelope (3–6), but also important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy (1).Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still fragmentary. According to the currently accepted model, the biosynthetic pathway is initiated by the transfer of two Manp residues and a fatty acyl chain to PI in the cytoplasmic leaflet of the plasma membrane. Based on genetic and biochemical evidence, Korduláková et al. (5) identified PimA (MSMEG_2935 in Mycobacterium smegmatis mc²155) as the enzyme that catalyzes the first mannosylation step of the pathway transferring a Manp residue most likely to the 2-position of the myo-inositol (myo-Ins) ring of PI. In contrast, the identity of PimB′, the enzyme responsible for the transfer of the second Manp to the 6-position of the myo-Ins ring of PIM₁, still remains controversial. The Rv0557 protein of Mycobacterium tuberculosis H37Rv (PimB; MSMEG_1113 in M. smegmatis mc²155) was originally characterized as PimB′ (7). However, the lack of an Rv0557 ortholog in the genome of Mycobacterium leprae and the fact that the disruption of this gene in M. tuberculosis Erdman did not significantly affect the biosynthesis of PIMs suggest that compensatory activities exist in the bacterium or that Rv0557 serves another primary function (8, 9). Somewhat supporting the latter hypothesis, the ortholog of Rv0557 in Corynebacterium glutamicum (NCgl0452, renamed mgtA) was implicated in the mannosylation of a novel glycolipid (1,2-di-O-C₁₆/C_18:1-(α-d-mannosyl)-(1→4)-(α-d-glucopyranosyluronic acid)-(1→3)-glycerol), and Rv0557 from M. tuberculosis was reported to functionally complement for this enzyme in a C. glutamicum knock-out mutant (10). However, to our knowledge this mannosylated glycolipid has never been reported in mycobacteria, and it remains unclear whether PimB serves a similar physiological function in Mycobacterium spp.More recently, Lea-Smith et al. (11) have shown that the biosynthesis of Ac₁PIM₂ from Ac₁PIM₁ in C. glutamicum is catalyzed by NCgl2106 (Cg-PimB′). Disruption of the NCgl2106 gene totally abolished Ac₁PIM₂ production in the mutant, arguing against the existence of a compensatory activity associated with the corynebacterial PimB enzyme. Although Ac₁PIM₂ production in Cg-pimB′ and Cg-pimB′/Cg-pimB knock-out mutants was restored upon complementation with the M. tuberculosis Rv2188c gene (11, 12), direct evidence that Rv2188c carried out the same physiological function in mycobacteria has been lacking. Moreover, in light of the recent work by Torrelles et al. (9) showing an involvement of pimB (Rv0557) in the synthesis of LM and LAM in M. tuberculosis Erdman and of the demonstrated relaxed substrate specificity of the M. tuberculosis PimB (Rv0557) and PimB′ (Rv2188c) enzymes expressed in C. glutamicum (12), whether or not pimB and pimB′ could compensate for one another in mycobacteria remained open to speculation.Both PIM₁ and PIM₂ can be acylated with palmitate at position 6 of the Manp residue transferred by PimA by the acyltransferase MSMEG_2934 (orthologous to Rv2611c from M. tb) to form Ac₁PIM₁ and Ac₁PIM₂, respectively (13). Ac₁PIM₂ can further be acylated at position 3 of the myo-Ins ring by an as yet unidentified acyltransferase to yield Ac₂PIM₂. Importantly, Ac₁PIM₂ and Ac₂PIM₂ are among the most abundant forms of PIMs found in mycobacteria and are considered both metabolic end products and intermediates in the biosynthesis of more polar forms of PIMs (PIM₅ and PIM₆), LM, and LAM.In this work, clear evidence is provided that PimB′ (MSMEG_4253 in M. smegmatis mc²155) is the α-ManT responsible for the biosynthesis of PIM₂ from PIM₁ in mycobacteria and that no other ManT can compensate for a deficiency in this enzyme in M. smegmatis. Like PimA (5), PimB′ is essential to the growth of M. smegmatis. Cell-free assays using purified PimA and PimB′ and M. smegmatis membrane preparations provide new insights into the sequential events leading to the synthesis of the early forms of PIMs in mycobacteria.     

Biosynthesis of hexamannophosphoinositides inMycobacterium smegmatis ATCC 607

The biosynthesis of hexamannophosphoinositides inMycobacterium smegmatis ATCC 607 was examined using labelled tri- and tetraacylated dimannophosphoinositides (PIM₂-3F and PIM₂-4F) as precursors, byin vivo andin vitro incorporation. Tetraacylated dimannoside was metabolically more active as compared to triacylated dimannoside and seems to be the precursor for the synthesis of hexamannophosphoinositides.Abbreviations M. 607 Mycobacterium smegmatis ATCC 607 - PIMs Mannophosphoinositides - PIM₂-2F diacylated dimannoside - PIM₂-3F triacylated dimannoside - PIM₂-4F tetraacylated dimannoside - PIM₃-3F triacylated trimannoside - PIM₆-3F triacylated hexamannoside - PIM₆-4F tetraacylated hexamannoside - GDP-mannose Guanosine diphosphomannose     

Mycobacterial lipoarabinomannan and related lipoglycans: from biogenesis to modulation of the immune response

The cell wall component lipoarabinomannan (ManLAM) from Mycobacterium tuberculosis is involved in the inhibition of phagosome maturation, apoptosis and interferon (IFN)-gamma signalling in macrophages and interleukin (IL)-12 cytokine secretion of dendritic cells (DC). All these processes are important for the host to mount an efficient immune response. Conversely, LAM isolated from non-pathogenic mycobacteria (PILAM) have the opposite effect, by inducing a potent proinflammatory response in macrophages and DCs. LAMs from diverse mycobacterial species differ in the modification of their terminal arabinose residues. The strong proinflammatory response induced by PILAM correlates with the presence of phospho-myo-inositol on the terminal arabinose. Interestingly, recent work indicates that the biosynthetic precursor of LAM, lipomannan (LM), which is also present in the cell wall, displays strong proinflammatory effects, independently of which mycobacterial species it is isolated from. Results from in vitro assays and knock-out mice suggest that LM, like PILAM, mediates its biological activity via Toll-like receptor 2. We hypothesize that the LAM/LM ratio might be a crucial factor in determining the virulence of a mycobacterial species and the outcome of the infection. Recent progress in the identification of genes involved in the biosynthesis of LAM is discussed, in particular with respect to the fact that enzymes controlling the LAM/LM balance might represent targets for new antitubercular drugs. In addition, inactivation of these genes may lead to attenuated strains of M. tuberculosis for the development of new vaccine candidates.     

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.     

Lipoarabinomannan biosynthesis in Corynebacterineae: the interplay of two α(1→2)-mannopyranosyltransferases MptC and MptD in mannan branching

Lipomannan (LM) and lipoarabinomannan (LAM) are key Corynebacterineae glycoconjugates that are integral components of the mycobacterial cell wall, and are potent immunomodulators during infection. LAM is a complex heteropolysaccharide synthesized by an array of essential glycosyltransferase family C (GT-C) members, which represent potential drug targets. Herein, we have identified and characterized two open reading frames from Corynebacterium glutamicum that encode for putative GT-Cs. Deletion of NCgl2100 and NCgl2097 in C. glutamicum demonstrated their role in the biosynthesis of the branching α(1→2)-Manp residues found in LM and LAM. In addition, utilizing a chemically defined nonasaccharide acceptor, azidoethyl 6-O-benzyl-α-D-mannopyranosyl-(1→6)-[α-D-mannopyranosyl-(1→6)](7) -D-mannopyranoside, and the glycosyl donor C(50) -polyprenol-phosphate-[(14) C]-mannose with membranes prepared from different C. glutamicum mutant strains, we have shown that both NCgl2100 and NCgl2097 encode for novel α(1→2)-mannopyranosyltransferases, which we have termed MptC and MptD respectively. Complementation studies and in vitro assays also identified Rv2181 as a homologue of Cg-MptC in Mycobacterium tuberculosis. Finally, we investigated the ability of LM and LAM from C. glutamicum, and C. glutamicumΔmptC and C. glutamicumΔmptD mutants, to activate Toll-like receptor 2. Overall, our study enhances our understanding of complex lipoglycan biosynthesis in Corynebacterineae and sheds further light on the structural and functional relationship of these classes of polysaccharides.     

Lipoarabinomannan. Multiglycosylated form of the mycobacterial mannosylphosphatidylinositols.

The lipopolysaccharides of mycobacteria, lipoarabinomannan (LAM) and lipomannan (LM), of key importance in host-pathogen interaction, were recently shown to contain a phosphatidylinositol "anchoring domain." We now have established that LAM and LM are based on the phosphatidylinositol mannosides, the characteristic glycophospholipids of mycobacteria. Digestion of the arabinose-free LM with an endo-alpha 1----6-mannosidase yielded evidence for the presence of the 1-(sn-glycerol-3-phospho)-D-myo-inositol-2,6-bis-alpha-D-mannopyranoside unit, indistinguishable from that derived from phosphatidylinositol dimannoside. This same inositol substitution pattern was shown to be present in LAM by methylation analysis before and after dephosphorylation. Positions C-2 and C-6 of the inositol unit of LAM are occupied by mannosyl residues and C-1 by a phosphoryl group. Partial acid hydrolysis of per-O-methylated LAM and comparison by gas chromatography-mass spectrometry of the resulting derivatized oligosaccharides with like products from phosphatidylinositol hexamannoside demonstrated that the C-6 of inositol is the point of attachment of the mannan core of LAM, which consists of an alpha 1----6-linked backbone with considerable alpha-1----2 side chains. Thus, a structural and presumably biosynthetic relationship is established between some of the membranous mannosylphosphatidylinositols described some 25 years ago and the newly emerging, biologically active lipopolysaccharides of mycobacteria.     

Tsukamurella paurometabola lipoglycan, a new lipoarabinomannan variant with pro-inflammatory activity

The genus Tsukamurella is a member of the phylogenetic group nocardioform actinomycetes and is closely related to the genus Mycobacterium. The mycobacterial cell envelope contains lipoglycans, and of particular interest is lipoarabinomannan, one of the most potent mycobacterial immunomodulatory molecules. We have investigated the presence of lipoglycans in Tsukamurella paurometabola and report here the isolation and structural characterization of a new lipoarabinomannan variant, designated TpaLAM. Matrix-assisted laser desorption ionization-mass spectrometric analysis revealed that TpaLAM had an average molecular mass of 12.5 kDa and consequently was slightly smaller than Mycobacterium tuberculosis lipoarabinomannan. Using a range of chemical degradations, NMR experiments, capillary electrophoresis, and mass spectrometry analyses, TpaLAM revealed an original carbohydrate structure. Indeed, TpaLAM contained a mannosylphosphatidyl-myo-inositol (MPI) anchor glycosylated by a linear (alpha1-->6)-Manp mannan domain, which is further substituted by an (alpha1-->5)-Araf chain. Half of the Araf units are further substituted at the O-2 position by a Manp-(alpha1-->2)-Manp-(alpha1--> dimannoside motif. Altogether, TpaLAM appears to be the most elaborated non-mycobacterial LAM molecule identified to date. TpaLAM was found to induce the pro-inflammatory cytokine tumor necrosis factor (TNF)-alpha when tested with either human or murine monocyte/macrophage cell lines. This induction was completely abrogated in the presence of an anti-toll-like receptor-2 (TLR-2) antibody, suggesting that TLR-2 participates in the mediation of TNF-alpha production in response to TpaLAM. Moreover, we established that the lipomannan core of TpaLAM is the primary moiety responsible for the observed TNF-alpha-inducing activity. This conclusively demonstrates that a linear (alpha1-->6)-Manp chain, linked to the MPI anchor, is sufficient in providing pro-inflammatory activity.