Structure of the priming arabinosyltransferase AftA required for AG biosynthesis of Mycobacterium tuberculosis.

Arabinogalactan (AG) is an essential cell wall component in mycobacterial species, including the deadly human pathogen Mycobacterium tuberculosis. It plays a pivotal role in forming the rigid mycolyl-AG-peptidoglycan core for in vitro growth. AftA is a membrane-bound arabinosyltransferase and a key enzyme involved in AG biosynthesis which bridges the assembly of the arabinan chain to the galactan chain. It is known that AftA catalyzes the transfer of the first arabinofuranosyl residue from the donor decaprenyl-monophosphoryl-arabinose to the mature galactan chain (i.e., priming); however, the priming mechanism remains elusive. Herein, we report the cryo-EM structure of Mtb AftA. The detergent-embedded AftA assembles as a dimer with an interface maintained by both the transmembrane domain (TMD) and the soluble C-terminal domain (CTD) in the periplasm. The structure shows a conserved glycosyltransferase-C fold and two cavities converging at the active site. A metal ion participates in the interaction of TMD and CTD of each AftA molecule. Structural analyses combined with functional mutagenesis suggests a priming mechanism catalyzed by AftA in Mtb AG biosynthesis. Our data further provide a unique perspective into anti-TB drug discovery.

Cord factor and peptidoglycan recapitulate the Th17-promoting adjuvant activity of mycobacteria through mincle/CARD9 signaling and the inflammasome.

Although adjuvants are critical vaccine components, their modes of action are poorly understood. In this study, we investigated the mechanisms by which the heat-killed mycobacteria in CFA promote Th17 CD4(+) T cell responses. We found that IL-17 secretion by CD4(+) T cells following CFA immunization requires MyD88 and IL-1ß/IL-1R signaling. Through measurement of Ag-specific responses after adoptive transfer of OTII cells, we confirmed that MyD88-dependent signaling controls Th17 differentiation rather than simply production of IL-17. Additional experiments showed that CFA-induced Th17 differentiation involves IL-1ß processing by the inflammasome, as mice lacking caspase-1, ASC, or NLRP3 exhibit partially defective responses after immunization. Biochemical fractionation studies further revealed that peptidoglycan is the major component of heat-killed mycobacteria responsible for inflammasome activation. By assaying Il1b transcripts in the injection site skin of CFA-immunized mice, we found that signaling through the adaptor molecule caspase activation and recruitment domain 9 (CARD9) plays a major role in triggering pro-IL-1ß expression. Moreover, we demonstrated that recognition of the mycobacterial glycolipid trehalose dimycolate (cord factor) by the C-type lectin receptor mincle partially explains this CARD9 requirement. Importantly, purified peptidoglycan and cord factor administered in mineral oil synergized to recapitulate the Th17-promoting activity of CFA, and, as expected, this response was diminished in caspase-1- and CARD9-deficient mice. Taken together, these findings suggest a general strategy for the rational design of Th17-skewing adjuvants by combining agonists of the CARD9 pathway with inflammasome activators.

Selectfluor and NFSI exo-glycal fluorination strategies applied to the enhancement of the binding affinity of galactofuranosyltransferase GlfT2 inhibitors.

Two complementary methods for the synthesis of fluorinated exo-glycals have been developed, for which previously no general reaction had been available. First, a Selectfluor-mediated fluorination was optimized after detailed analysis of all the reaction parameters. A dramatic effect of molecular sieves on the course of the reaction was observed. The reaction was generalized with a set of biologically relevant furanosides and pyranosides. A second direct approach involving carbanionic chemistry and the use of N-fluorobenzenesulfonimide (NFSI) was performed and this method gave better diastereoselectivities. Assignment of the Z/E configuration of all the fluorinated exo-glycals was achieved based on the results of HOESY experiments. Furthermore, fluorinated exo-glycal analogues of UDP-galactofuranose were prepared and assayed against GlfT2, which is a key enzyme involved in the cell-wall biosynthesis of major pathogens. The fluorinated exo-glycals proved to be potent inhibitors as compared with a series of C-glycosidic analogues of UDP-Galf, thus demonstrating the double beneficial effect of the exocyclic enol ether functionality and the fluorine atom.

Structural analysis of phosphoribosyltransferase-mediated cell wall precursor synthesis in Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, Rv3806c is a membrane-bound phosphoribosyltransferase (PRTase) involved in cell wall precursor production. It catalyses pentosyl phosphate transfer from phosphoribosyl pyrophosphate to decaprenyl phosphate, to generate 5-phospho-ß-ribosyl-1-phosphoryldecaprenol. Despite Rv3806c being an attractive drug target, structural and molecular mechanistic insight into this PRTase is lacking. Here we report cryogenic electron microscopy structures for Rv3806c in the donor- and acceptor-bound states. In a lipidic environment, Rv3806c is trimeric, creating a UbiA-like fold. Each protomer forms two helical bundles, which, alongside the bound lipids, are required for PRTase activity in vitro. Mutational and functional analyses reveal that decaprenyl phosphate and phosphoribosyl pyrophosphate bind the intramembrane and extramembrane cavities of Rv3806c, respectively, in a distinct manner to that of UbiA superfamily enzymes. Our data suggest a model for Rv3806c-catalysed phosphoribose transfer through an inverting mechanism. These findings provide a structural basis for cell wall precursor biosynthesis that could have potential for anti-tuberculosis drug development.

The mycobacterial glycoside hydrolase LamH enables capsular arabinomannan release and stimulates growth.

Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are trafficked from the bacterium to the host via unknown mechanisms. Arabinomannan is thought to be a capsular derivative of these molecules, lacking a lipid anchor. However, the mechanism by which this material is generated has yet to be elucidated. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH (Rv0365c in Mycobacterium tuberculosis) which specifically cleaves α-1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures, potentially implicating arabinomannan as a signal for growth phase transition. Finally, we demonstrate that LamH is important for M. tuberculosis survival in macrophages.

DprE2 is a molecular target of the anti-tubercular nitroimidazole compounds pretomanid and delamanid.

Mycobacterium tuberculosis is one of the global leading causes of death due to a single infectious agent. Pretomanid and delamanid are new antitubercular agents that have progressed through the drug discovery pipeline. These compounds are bicyclic nitroimidazoles that act as pro-drugs, requiring activation by a mycobacterial enzyme; however, the precise mechanisms of action of the active metabolite(s) are unclear. Here, we identify a molecular target of activated pretomanid and delamanid: the DprE2 subunit of decaprenylphosphoribose-2'-epimerase, an enzyme required for the synthesis of cell wall arabinogalactan. We also provide evidence for an NAD-adduct as the active metabolite of pretomanid. Our results highlight DprE2 as a potential antimycobacterial target and provide a foundation for future exploration into the active metabolites and clinical development of pretomanid and delamanid.

The mycobacterial glycoside hydrolase LamH enables capsular arabinomannan release and stimulates growth.

Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important amongst these are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are both trafficked out of the bacterium to the host via unknown mechanisms. An important class of exported LM/LAM is the capsular derivative of these molecules which is devoid of its lipid anchor. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH which specifically cleaves α-1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures where arabinomannan acts as a signal for growth phase transition. Finally, we demonstrate that LamH is important for Mycobacterium tuberculosis survival in macrophages. These data provide a new framework for understanding the biological role of LAM in mycobacteria.

3-Ketosteroid 9alpha-hydroxylase is an essential factor in the pathogenesis of Mycobacterium tuberculosis.

Mycobacterium tuberculosis H37Rv contains the kshA (Rv3526) and kshB (Rv3571) genes, encoding 3-ketosteroid 9alpha-hydroxylase (KSH). Consistent with their predicted roles, the DeltakshA and DeltakshB deletion mutants of M. tuberculosis H37Rv were unable to use cholesterol and 4-androstene-3,17-dione as primary carbon and energy sources. Interestingly, DeltakshA and DeltakshB mutants were also unable to metabolize the steroid substrate 5alpha-androstane-3,17-dione, whereas wild-type M. tuberculosis H37Rv could. The deletion of either of these genes lead to rapid death of the microorganism in murine infection models and in macrophages, showing that kshA and kshB are essential factors for M. tuberculosis pathogenesis. Penta-acylated trehalose (PAT) biosynthesis was altered in the DeltakshB mutant, but not the DeltakshA mutant. The DeltakshB mutant synthesizes all other types of lipids. The DeltakshB mutant had a thickened outer layer in its cell wall. KshB thus appears to be involved in multiple processes, probably as a reductase of different oxygenases. We conclude that an impaired 3-ketosteroid 9alpha-hydroxylase activity is the cause of the highly attenuated phenotype of our M. tuberculosis H37Rv mutants.

Identification of novel diphenyl urea inhibitors of Mt-GuaB2 active against Mycobacterium tuberculosis.

In contrast with most bacteria, which harbour a single inosine monophosphate dehydrogenase (IMPDH) gene, the genomic sequence of Mycobacterium tuberculosis H37Rv predicts three genes encoding IMPDH: guaB1, guaB2 and guaB3. These three genes were cloned and expressed in Escherichia coli to evaluate functional IMPDH activity. Purified recombinant Mt-GuaB2, which uses inosine monophosphate as a substrate, was identified as the only active GuaB orthologue in M. tuberculosis and showed optimal activity at pH 8.5 and 37 °C. Mt-GuaB2 was inhibited significantly in vitro by a panel of diphenyl urea-based derivatives, which were also potent anti-mycobacterial agents against M. tuberculosis and Mycobacterium smegmatis, with MICs in the range of 0.2-0.5 µg ml(-1). When Mt-GuaB2 was overexpressed on a plasmid in trans in M. smegmatis, a diphenyl urea analogue showed a 16-fold increase in MIC. Interestingly, when Mt-GuaB orthologues (Mt-GuaB1 and 3) were also overexpressed on a plasmid in trans in M. smegmatis, they also conferred resistance, suggesting that although these Mt-GuaB orthologues were inactive in vitro, they presumably titrate the effect of the inhibitory properties of the active compounds in vivo.

Mycolic acid modification by the mmaA4 gene of M. tuberculosis modulates IL-12 production.

Mycobacterium tuberculosis has evolved many strategies to evade elimination by the host immune system, including the selective repression of macrophage IL-12p40 production. To identify the M. tuberculosis genes responsible for this aspect of immune evasion, we used a macrophage cell line expressing a reporter for IL-12p40 transcription to screen a transposon library of M. tuberculosis for mutants that lacked this function. This approach led to the identification of the mmaA4 gene, which encodes a methyl transferase required for introducing the distal oxygen-containing modifications of mycolic acids, as a key locus involved in the repression of IL-12p40. Mutants in which mmaA4 (hma) was inactivated stimulated macrophages to produce significantly more IL-12p40 and TNF-alpha than wild-type M. tuberculosis and were attenuated for virulence. This attenuation was not seen in IL-12p40-deficient mice, consistent with a direct linkage between enhanced stimulation of IL-12p40 by the mutant and its reduced virulence. Treatment of macrophages with trehalose dimycolate (TDM) purified from the DeltammaA4 mutant stimulated increased IL-12p40, similar to the increase observed from DeltammaA4 mutant-infected macrophages. In contrast, purified TDM isolated from wild-type M. tuberculosis inhibited production of IL-12p40 by macrophages. These findings strongly suggest that M. tuberculosis has evolved mmaA4-derived mycolic acids, including those incorporated into TDM to manipulate IL-12-mediated immunity and virulence.

Modular approach to triazole-linked 1,6-α-D-oligomannosides to the discovery of inhibitors of Mycobacterium tuberculosis cell wall synthetase.

Aiming at developing inhibitors of mannosyltransferases, the enzymes that participate in the biosynthesis of the cell envelope of Mycobacterium tuberculosis, the synthesis of a range of designed triazole-linked 1,6-oligomannosides up to a hexadecamer has been accomplished by a modular approach centered on the Cu(I)-catalyzed azide-alkyne cycloaddition as key process. The efficiency and fidelity of the cycloaddition are substantiated by high yields (76-96%) and exclusive formation of the expected 1,4-disubstituted triazole ring in all oligomer assembling reactions. Key features of oligomers thus prepared are the anomeric carbon-carbon bond of all mannoside residues and the 6-deoxymannoside capping residue. Suitable bioassays with dimer, tetramer, hexamer, octamer, decamer, and hexadecamer showed variable inhibitor activity against mycobacterial α-(1,6)-mannosyltransferases, the highest activity (IC(50) = 0.14-0.22 mM) being registered with the hexamannoside and octamannoside.

Inhibition of Escherichia coli glycosyltransferase MurG and Mycobacterium tuberculosis Gal transferase by uridine-linked transition state mimics.

Glycosyltransferase MurG catalyses the transfer of N-acetyl-d-glucosamine to lipid intermediate I on the bacterial peptidoglycan biosynthesis pathway, and is a target for development of new antibacterial agents. A transition state mimic was designed for MurG, containing a functionalised proline, linked through the alpha-carboxylic acid, via a spacer, to a uridine nucleoside. A set of 15 functionalised prolines were synthesised, using a convergent dipolar cycloaddition reaction, which were coupled via either a glycine, proline, sarcosine, or diester linkage to the 5'-position of uridine. The library of 18 final compounds were tested as inhibitors of Escherichia coli glycosyltransferase MurG. Ten compounds showed inhibition of MurG at 1mM concentration, the most active with IC(50) 400microM. The library was also tested against Mycobacterium tuberculosis galactosyltransferase GlfT2, and one compound showed effective inhibition at 1mM concentration.

Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB2-AcpM2.

Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.

Structures of cell wall arabinosyltransferases with the anti-tuberculosis drug ethambutol.

The arabinosyltransferases EmbA, EmbB, and EmbC are involved in Mycobacterium tuberculosis cell wall synthesis and are recognized as targets for the anti-tuberculosis drug ethambutol. In this study, we determined cryo-electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits arabinosyltransferases by binding to the same site as both substrates in EmbB and EmbC. Most drug-resistant mutations are located near the ethambutol binding site. Collectively, our work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and the development of new anti-tuberculosis agents.

Role of phosphatidylinositol mannosides in the interaction between mycobacteria and DC-SIGN.

The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is the major receptor on DCs for mycobacteria of the Mycobacterium tuberculosis complex. Recently, we have shown that although the mannose caps of the mycobacterial surface glycolipid lipoarabinomannan (ManLAM) are essential for the binding to DC-SIGN, genetic removal of these caps did not diminish the interaction of whole mycobacteria with DC-SIGN and DCs. Here we investigated the role of the structurally related glycolipids phosphatidylinositol mannosides (PIMs) as possible ligands for DC-SIGN. In a binding assay with both synthetic and natural PIMs, DC-SIGN exhibited a high affinity for hexamannosylated PIM(6), which contains terminal alpha(1-->2)-linked mannosyl residues identical to the mannose cap on ManLAM, but not for di- and tetramannosylated PIM(2) and PIM(4), respectively. To determine the role of PIM(6) in the binding of whole mycobacteria to DC-SIGN, a mutant strain of M. bovis bacillus Calmette-Guérin deficient in the production of PIM(6) (Delta pimE) was created, as well as a double knockout deficient in the production of both PIM(6) and the mannose caps on LAM (Delta pimE Delta capA). Compared to the wild-type strain, both mutant strains bound similarly well to DC-SIGN and DCs. Furthermore, the wild-type and mutant strains induced comparable levels of interleukin-10 and interleukin-12p40 when used to stimulate DCs. Hence, we conclude that, like ManLAM, PIM(6) represents a bona fide DC-SIGN ligand but that other, as-yet-unknown, ligands dominate in the interaction between mycobacteria and DCs.

Inactivation of Mycobacterium tuberculosis mannosyltransferase pimB reduces the cell wall lipoarabinomannan and lipomannan content and increases the rate of bacterial-induced human macrophage cell death.

The Mycobacterium tuberculosis (M.tb) cell wall contains an important group of structurally related mannosylated lipoglycans called phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM), and mannose-capped lipoarabinomannan (ManLAM), where the terminal alpha-[1-->2] mannosyl structures on higher order PIMs and ManLAM have been shown to engage C-type lectins such as the macrophage mannose receptor directing M.tb phagosome maturation arrest. An important gene described in the biosynthesis of these molecules is the mannosyltransferase pimB (Rv0557). Here, we disrupted pimB in a virulent strain of M.tb. We demonstrate that the inactivation of pimB in M.tb does not abolish the production of any of its cell wall mannosylated lipoglycans; however, it results in a quantitative decrease in the ManLAM and LM content without affecting higher order PIMs. This finding indicates gene redundancy or the possibility of an alternative biosynthetic pathway that may compensate for the PimB deficiency. Furthermore, infection of human macrophages by the pimB mutant leads to an alteration in macrophage phenotype concomitant with a significant increase in the rate of macrophage death.

Synthesis of deoxygenated alpha(1-->5)-linked arabinofuranose disaccharides as substrates and inhibitors of arabinosyltransferases of Mycobacterium tuberculosis.

Arabinosyltransferases (AraTs) play a critical role in mycobacterial cell wall biosynthesis and are potential drug targets for the treatment of tuberculosis, especially multi-drug resistant forms of M. tuberculosis (MTB). Herein, we report the synthesis and acceptor/inhibitory activity of Araf alpha(1-->5) Araf disaccharides possessing deoxygenation at the reducing sugar of the disaccharide. Deoxygenation at either the C-2 or C-3 position of Araf was achieved via a free radical procedure using xanthate derivatives of the hydroxyl group. The alpha(1-->5)-linked disaccharides were produced by coupling n-octyl alpha-Araf 2-/3-deoxy, 2-fluoro glycosyl acceptors with an Araf thioglycosyl donor. The target disaccharides were tested in a cell free mycobacterial AraTs assay as well as an in vitro assay against MTB H(37)Ra and M. avium complex strains.

A Mycobacterium tuberculosis mutant lacking the groEL homologue cpn60.1 is viable but fails to induce an inflammatory response in animal models of infection.

The causative agent of tuberculosis, Mycobacterium tuberculosis, has two chaperonin (Cpn60) proteins and one cochaperonin (Cpn10) protein. We show here that cpn60.2 and cpn10, but not cpn60.1, are essential for cell survival. A mutant lacking Cpn60.1 was indistinguishable from the wild-type organism in plate and broth culture and within murine macrophages, although it showed increased sensitivity to high temperature (55 degrees C). However, infection of mice with the Deltacpn60.1 mutant revealed a major difference from the wild-type organism. In spite of having equal numbers of bacteria in infected sites, the Deltacpn60.1 mutant failed to produce granulomatous inflammation in either mice or guinea pigs. This was associated with reduced cytokine expression in infected animals and macrophages. Cell wall lipid acid composition was not altered in the mutant strain. Thus, it appears that Cpn60.1 is an important agent in the regulation of the cytokine-dependent granulomatous response in M. tuberculosis infection.

Expression, purification and characterisation of soluble GlfT and the identification of a novel galactofuranosyltransferase Rv3782 involved in priming GlfT-mediated galactan polymerisation in Mycobacterium tuberculosis.

The arabinogalactan (AG) component of the mycobacterial cell wall is an essential branched polysaccharide which tethers mycolic acids (m) to peptidoglycan (P), forming the mAGP complex. Much interest has been focused on the biosynthetic machinery involved in the production of this highly impermeable shield, which is the target for numerous anti-tuberculosis agents. The galactan domain of AG is synthesised via a bifunctional galactofuranosyltransferase (GlfT), which utilises UDP-Galf as its high-energy substrate. However, it has proven difficult to study the protein in its recombinant form due to difficulties in recovering pure soluble protein using standard expression systems. Herein, we describe the effects of glfT co-induction with a range of chaperone proteins, which resulted in an appreciable yield of soluble protein at 5 mg/L after a one-step purification procedure. We have shown that this purified enzyme transfers [14C]Galf to a range of both beta(1-->5) and beta(1-->6) linked digalactofuranosyl neoglycolipid acceptors with a distinct preference for the latter. Ligand binding studies using intrinsic tryptophan fluorescence have provided supporting evidence for the apparent preference of this enzyme to bind the beta(1-->6) disaccharide acceptor. However, we could not detect binding or galactofuranosyltransferase activity with an n-octyl beta-d-Gal-(1-->4)-alpha-l-Rha acceptor, which mimics the reducing terminus of galactan in the mycobacterial cell wall. Conversely, after an extensive bioinformatics analysis of the H37Rv genome, further cloning, expression and functional analysis of the Rv3792 open reading frame indicates that this protein affords galactofuranosyltransferase activity against such an acceptor and paves the way for a better understanding of galactan biosynthesis in Mycobacterium tuberculosis.

The singular Corynebacterium glutamicum Emb arabinofuranosyltransferase polymerises the α(1 → 5) arabinan backbone in the early stages of cell wall arabinan biosynthesis.

The arabinan-containing polysaccharides, arabinogalactan (AG) and lipoarabinomannan (LAM), are key cell wall components of the Corynebacterineae, which include Corynebacteria, Norcadia and Mycobacteria. Both AG and LAM contain elaborate arabinan domains composed of distinct structural motifs. Mycobacterial EmbA, EmbB and EmbC, collectively known as the Emb proteins, have been identified as arabinosyltransferases (ArafTs), which are targeted by the front-line anti-tubercular drug ethambutol. Previous studies have established that EmbA and EmbB play a role in the synthesis of the characteristic terminal hexa-arabinosuranosyl motif, whilst EmbC is involved exclusively in the biosynthesis of LAM. Herein, we have investigated the role of the singular Emb protein from Corynebacterium glutamicum through the detailed biochemical and chemical analysis of a double ΔaftAΔemb mutant, where the priming Cg-AftA protein, which generates the substrate for Cg-Emb has been deleted. Analysis of its cell wall revealed a complete absence of arabinose resulting in a truncated cell wall containing only a galactan backbone accompanied with complete loss of cell wall bound mycolates. In vitro cell-free assays using C. glutamicumΔaftA, C. glutamicumΔemb, C. glutamicumΔaftAΔemb and C. glutamicumΔaftBΔaftD and two synthetic acceptors, which mimick the arabinofuranose (Araf) "primed" galactan chain, demonstrated that Cg-Emb is able to transfer an Araf residue to the C5 of the Araf positioned on the synthetic acceptor(s). These results indicate that Cg-Emb acts as an α(1 → 5) ArafT and elongates the arabinan core during the early stages of arabinan biosynthesis in C. glutamicum.