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Drug inhibition of glycosyltransferasesMycobacteria, including the species that causes tuberculosis (TB), synthesize a complex cell wall that helps to support and protect the bacterial cells. The major components of the cell wall include complex heteropolysaccharides that are synthesized in the periplasmic space. Zhanget al.determined the cryo–electron microscopy structures of two transmembrane glycosyltransferase enzyme complexes that use a lipid-anchored sugar donor to append arabinose units to the cell wall polysaccharides. They also captured the anti-TB drug ethambutol bound within these complexes and observed that it binds in a site overlapping both donor and acceptor sugars. Mapping of resistance mutants provides a structural understanding of how resistance emerges while preserving function of the enzyme and may help to guide the development of next-generation anti-TB drugs that target these enzymes.Science, this issue p.1211

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. Electronic supplementary material The online version of this article (10.1007/s13238-020-00726-6) contains supplementary material, which is available to authorized users.

Synthetic hexynyl α-D-mannopyranoside and its α-1,6-linked disaccharide counterpart were fluorescently labelled through CuAAC click chemistry with 3-azido-7-hydroxycoumarin. The resulting triazolyl-coumarin adducts, which were amenable to analysis by TLC, HPLC and mass spectrometry, proved to be acceptor substrates for α-1,6-ManT activities in mycobacterial membranes, as well as α- and β-GalT activities in trypanosomal membranes, benchmarking the potential of the fluorescent acceptor approach against earlier radiochemical assays. Following on to explore the glycobiology of the benign protozoan alga Euglena gracilis, α-1,3- and α-1,2-ManT activities were detected in membrane preparations, along with GlcT, Glc-P-T and GlcNAc-P-T activities. These studies serve to demonstrate the potential of readily accessible fluorescent glycans as substrates for exploring carbohydrate active enzymes., Graphical abstract Image 1, Highlights • Assays for the analysis of carbohydrate-active enzymes that rely upon fluorescent acceptor substrates are set out. • New assays are validated by benchmarking against radiochemical work with known glycosyltransferase activities. • The installation of a fluorophore on acceptor substrates was easily achieved through click chemistry. • Fluorescence assays are used to discover GTs activities in Euglena gracilis microsomal membranes.

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-1β/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-1β 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-1β 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.

Graphical abstract, 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 α-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 1 mM concentration, the most active with IC50 400 μM. The library was also tested against Mycobacterium tuberculosis galactosyltransferase GlfT2, and one compound showed effective inhibition at 1 mM concentration.

Mycobacterium tuberculosis H37Rv contains the kshA (Rv3526) and kshB (Rv3571) genes, encoding 3-ketosteroid 9a-hydroxylase (KSH). Consistent with their predicted roles, the Delta kshA and Delta kshB deletion mutants of M. tuberculosis H37Rv were unable to use cholesterol and 4-androstene-3,17-dione as primary carbon and energy sources. Interestingly, Delta kshA and Delta kshB mutants were also unable to metabolize the steroid substrate 5 alpha-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 Delta kshB mutant, but not the Delta kshA mutant. The Delta kshB mutant synthesizes all other types of lipids. The Delta kshB 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 9 alpha-hydroxylase activity is the cause of the highly attenuated phenotype of our M. tuberculosis H37Rv mutants.

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.

Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL-10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M.bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M.marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M.bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M.bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium-host interaction. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd.

Efficient syntheses are reported of the α- l -rhamnosidase inhibitors l -swainsonine [(1 R ,2 S ,8 S ,8a S )-octahydroindolizine-1,2,8-triol], (6 R )- C -methyl- l -swainsonine (1 R ,2 S ,6 R ,8 S ,8a S )-6-methyloctahydro-indolizine-1,2,8-triol, (6 S )- C -methyl- l -swainsonine (1 R ,2 S ,6 S ,8 S ,8a S )-6-methyloctahydro-indolizine-1,2,8-triol and related 6- C -methyl hydroxycastanospermines [(1 R ,2 S ,6 R ,7 R ,8 R ,8a R )-6-methyl-octahydroindolizine-1,2,6,7,8-pentaol and (1 R ,2 S ,6 S ,8 S ,8a S )-6-methyloctahydro-indolizine-1,2,8-triol]. (6 R )- C -Methyl- l -swainsonine [ K i = 0.032 μM] is a significantly more potent naringinase inhibitor than l -swainsonine [ K i = 0.45 μM].

Phenotypic screens for bactericidal compounds against drug-resistant tuberculosis are beginning to yield novel inhibitors. However, reliable target identification remains challenging. Here, we show that tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide (THPP) selectively pulls down EchA6 in a stereospecific manner, instead of the previously assigned target Mycobacterium tuberculosis MmpL3. While homologous to mammalian enoyl-coenzyme A (CoA) hydratases, EchA6 is non-catalytic yet essential and binds long-chain acyl-CoAs. THPP inhibitors compete with CoA-binding, suppress mycolic acid synthesis, and are bactericidal in a mouse model of chronic tuberculosis infection. A point mutation, W133A, abrogated THPP-binding and increased both the in vitro minimum inhibitory concentration and the in vivo effective dose 99 in mice. Surprisingly, EchA6 interacts with selected enzymes of fatty acid synthase II (FAS-II) in bacterial two-hybrid assays, suggesting essentiality may be linked to feeding long-chain fatty acids to FAS-II. Finally, our data show that spontaneous resistance-conferring mutations can potentially obscure the actual target or alternative targets of small molecule inhibitors.

Glycosyltransferases (GTs) play a crucial role in mycobacterial cell wall biosynthesis and are necessary for the survival of mycobacteria. Hence, these enzymes are potential new drug targets for the treatment of tuberculosis (TB), especially multiple drug-resistant TB (MDR-TB). Herein, we report the efficient syntheses of Araf(α 1 → 5)Araf, Galf(β 1 → 5)Galf, and Galf(β 1 → 6)Galf disaccharides possessing a 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl (dansyl) unit that were prepared as fluorescent disaccharide acceptors for arabinosyl- and galactosyl-transferases, respectively. Such analogs may offer advantages relative to radiolabeled acceptors or donors for studying the enzymes and for assay development and compound screening. Additionally, analogs possessing a 5-azidonaphthalene-1-sulfonamidoethyl unit were prepared as photoaffinity probes for their potential utility in studying active site labeling of the GTs (arabinosyl and galactosyl) in Mycobacterium tuberculosis (MTB). Beyond their preparation, initial biological testing and kinetic analysis of these disaccharides as acceptors toward glycosyltransferases are also presented.

The Mycobacterium tuberculosis TetR-type regulator Rv3574 has been implicated in pathogenesis as it is induced in vivo, and genome-wide essentiality studies show it is required for infection. As the gene is highly conserved in the mycobacteria, we deleted the Rv3574 orthologue in Mycobacterium smegmatis (MSMEG_6042) and used real-time quantitative polymerase chain reaction and microarray analyses to show that it represses the transcription both of itself and of a large number of genes involved in lipid metabolism. We identified a conserved motif within its own promoter (TnnAACnnGTTnnA) and showed that it binds as a dimer to 29 bp probes containing the motif. We found 16 and 31 other instances of the motif in intergenic regions of M. tuberculosis and M. smegmatis respectively. Combining the results of the microarray studies with the motif analyses, we predict that Rv3574 directly controls the expression of 83 genes in M. smegmatis, and 74 in M. tuberculosis. Many of these genes are known to be induced by growth on cholesterol in rhodococci, and palmitate in M. tuberculosis. We conclude that this regulator, designated elsewhere as kstR, controls the expression of genes used for utilizing diverse lipids as energy sources, possibly imported through the mce4 system.

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis., Screens for bactericidal compounds have resulted in promising anti-tubercular hits. Here, the authors analyse in detail the target of an indazole sulfonamide (GSK3011724A), and find that it has a different mode of inhibition compared to other Kas inhibitors of fatty acid biosynthesis in bacteria.

The assembly of the arabinan portions of cell wall polysaccharides in mycobacteria involves a family of arabinosyltransferases (AraT's) that promote the polymerization of decaprenolphosphoarabinose. Mycobacterial viability depends upon the ability of the organism to synthesize an intact arabinan and thus compounds that inhibit these AraT's are both useful biochemical tools as well as potential lead compounds for new anti-tuberculosis agents. We describe here the preparation of oligosaccharide fragments of mycobacterial arabinan that contain arabinofuranosyl residues modified at C-3 by the replacement of the hydroxyl group with an amino, azido or methoxy functionality. Subsequent testing of these oligosaccharides as inhibitors of mycobacterial AraT's revealed that all inhibited the enzymes, but to varying degrees. In further studies, each compound was shown to have only low activity as an inhibitor of mycobacterial growth.

Analogs of the α- d -Man p -(1→6)-α- d -Man p -O(CH 2 ) 7 CH 3 disaccharide 4 , a known substrate for a polyprenol monophosphomannose-dependent α-(1→6)-mannosyltransferase involved in mycobacterial LAM biosynthesis, have been synthesized and screened as potential substrates and inhibitors of the enzyme. In the disaccharides synthesized, the hydroxyl groups at C-2 and C-6 on the reducing end residue have been replaced by combinations of amino, fluoro, and methoxy functionalities 9 – 14 . In addition, a disaccharide in which the nonreducing mannopyranose residue was replaced with a 3,6-anhydromannopyranose residue 34 was synthesized from a byproduct formed during one of the reactions leading to 14 . When tested against the enzyme, none were active as substrates, as would be expected as all lack the C-6′ hydroxyl group to which an additional sugar residue would be transferred. Evaluation of these compounds as inhibitors of the enzyme revealed that only three, 11 , 12 , and 13 , all of which contain one or more amino groups, inhibited the enzyme. The most potent inhibitor was the diamino-disaccharide, 11 .

Mannosyltransferases play a crucial role in mycobacterial cell-wall biosynthesis and are potential new drug targets for the treatment of tuberculosis. Herein, we describe the synthesis of α-(1 → 2)- and α-(1 → 6)-linked mannopyranosyl disaccharides possessing a 5-azidonaphthlene-1-sulfonamidoethyl group as photoaffinity probes for active-site labeling studies of mannosyltransferases in Mycobacetrium tuberculosis .

Several octyl 5- O -(α- d -arabinofuranosyl)-α- d -arabinofuranoside disaccharide analogs substituted at the 5-position of the non-reducing end sugar were synthesized and tested in vitro against Mycobacterium tuberculosis ( M.tb. ), Mycobacterium avium complex (MAC) as well as in a cell free assay system for arabinosyltransferase acceptor/inhibitor activity. A few compounds showed interesting inhibitory activity in the cell free assay as well as against the whole microorganism in vitro.

Analogues of the natural antibiotic thiolactomycin, with acetylene-based side chains, have the highest recorded in vitro inhibitory activity against the recombinant Mycobacterium tuberculosis β-ketoacyl-ACP synthase mtFabH condensing enzyme. In particular, 5-[3-(4-acetyl-phenyl)-prop-2-ynyl]-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one exhibited more than an 18-fold increased potency, compared to thiolactomycin, against this key condensing enzyme, involved in M. tuberculosis mycolic acid biosynthesis. Analogues of the antibiotic thiolactomycin, with acetylene-based side chains, have the highest recorded activity against cloned mtFabH condensing enzyme.

Assuming the transition state of glycosyltransferase inhibitors to be similar to those encountered with potent glycosidase inhibitors − i.e. a flattened conformation with a positively charged anomeric centre − we worked out a synthesis of the D-arabino-configured phosphonic acid target molecule 2 derived from an imidazolo-sugar. The key synthetic intermediate is the linear imidazolo L-xylo compound 10 which could be obtained, either from L-threo precursor 6 by a coupling reaction with imidazole derivative 5, or from L-sorbose. A multi-step and site specific iodination of 10 gave the mono-iodo-L-xylo derivative 14 which was cyclised to the D-arabino-configured bicyclic azasugar 15. Phosphorylation of the Grignard derivative of the latter, followed by mono-esterification with citronellol along with some protection-deprotection steps led to target molecule 2. The potential inhibitor 2 is supposed to be protonated at its most basic N atom by a carboxylic acid residue in the arabinosyl-transferase active site. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Dolichol phosphate-mannose (Dol-P-Man) is a mannose donor in various eukaryotic glycosylation processes. So far, two groups of Dol-P-Man synthases have been characterized based on the way they are stabilized in the endoplasmic reticulum membrane. Enzymes belonging to the first group, such as the yeast Dpm1, are typical integral membrane proteins harboring a transmembrane segment (TMS) at their C terminus. In contrast, mammalian Dpm1, enzymes of the second group, lack the typical TMS and require the association with the small hydrophobic proteins Dpm3 to be properly stabilized in the endoplasmic reticulum membrane. In Mycobacterium tuberculosis, the Polyprenol-P-Man synthase MtPpm1 is involved in the biosynthesis of the cell wall-associated glycolipid lipoarabinomannan. MtPpm1 is composed of two domains. The C-terminal catalytic domain is homologous to eukaryotic Dol-P-Man synthases. The N-terminal domain of MtPpm1 contains six TMS that anchor the enzyme in the cytoplasmic membrane. In contrast, in Mycobacterium smegmatis, orthologs of the two domains of MtPpm1 are encoded by two distinct open reading frames, Msppm1 and Msppm2, organized as an operon. No TMS are predicted in MsPpm1, and subcellular fractionation experiments indicate that this enzyme is cytosolic when produced in Escherichia coli. Computer-assisted topology predictions and alkaline phosphatase insertions showed that MsPpm2 is an integral membrane protein. Using a recently developed bacterial two-hybrid system, it was found that MsPpm2 interacts with MsPpm1 to stabilize the synthase MsPpm1 in the bacterial membrane. This interaction is reminiscent of that of mammalian Dpm1 with Dpm3 and mimics the structure of MtPpm1 as demonstrated by the capacity of the two domains of MtPpm1 to spontaneously interact when co-expressed in E. coli.

Phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM) are an important class of bacterial factors termed modulins that are found in tuberculosis and leprosy. Although their structures are well established, little is known with respect to the molecular aspects of the biosynthetic machinery involved in the synthesis of these glycolipids. On the basis of sequence similarity to other glycosyltransferases and our previous studies defining an α-mannosyltransferase from Mycobacterium tuberculosis, named PimB [Schaeffer, Khoo, Besra, Chatterjee, Brennan, Belisle and Inamine (1999) J. Biol. Chem. 274, 31625–31631], which catalysed the formation of triacyl (Ac3)-PIM2 (i.e. the dimannoside), we have identified a related gene from M. tuberculosis CDC1551, now designated pimC. The use of a cell-free assay containing GDP-[14C]mannose, amphomycin and membranes from Myobacterium smegmatis overexpressing PimC led to the synthesis of a new alkali-labile PIM product. Fast-atom-bombardment MS established the identity of the new enzymically synthesized product as Ac3PIM3 (i.e. the trimannoside). The results indicate that pimC encodes an α-mannosyltransferase involved in Ac3PIM3 biosynthesis. However, inactivation of pimC in Myobacterium bovis Bacille Calmette—Guérin (BCG) did not affect the production of higher PIMs, LM and LAM when compared with wild-type M. bovis BCG, suggesting the existence of redundant gene(s) or an alternate pathway that may compensate for this PimC deficiency. Further analyses, which compared the distribution of pimC in a panel of M. tuberculosis strains, revealed that pimC was present in only 22% of the clinical isolates examined.

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.

The appearance multi-drug resistant Mycobacterium tuberculosis (MTB) throughout the world has prompted a search for new, safer and more active agents against tuberculosis. Based on studies of the biosynthesis of mycobacterial cell wall polysaccharides, octyl 5- O -(α- d -arabinofuranosyl)-α- d -arabinofuranoside analogues were synthesized and evaluated as inhibitors for M. tuberculosis and Mycobacterium avium . A cell free assay system has been used for the evaluation of these disaccharides as substrates for mycobacterial arabinosyltransferase activity.

The discovery of the CD1 antigen presentation pathway has expanded the spectrum of T-cell antigens to include lipids, but the range of natural lipid antigens and functions of CD1-restricted T cells in vivo remain poorly understood. Here we show that the T-cell antigen receptor and the CD1c protein mediate recognition of an evolutionarily conserved family of isoprenoid glycolipids whose members include essential components of protein glycosylation and cell-wall synthesis pathways. A CD1c-restricted, mycobacteria-specific T-cell line recognized two previously unknown mycobacterial hexosyl-1-phosphoisoprenoids and structurally related mannosyl-beta1-phosphodolichols. Responses to mannosyl-beta1-phosphodolichols were common among CD1c-restricted T-cell lines and peripheral blood T lymphocytes of human subjects recently infected with M. tuberculosis, but were not seen in naive control subjects. These results define a new class of broadly distributed lipid antigens presented by the CD1 system during infection in vivo and suggest an immune mechanism for recognition of senescent or transformed cells that are known to have altered dolichol lipids.

Two iminosugars have been designed and synthesized as potential inhibitors of UDP-Galf transferase, an enzyme involved in Mycobacterium tuberculosis cell wall biosynthesis. The design is based on a proposed model of the transition state for the transferase reaction. One of the two racemic compounds is the first reported inhibitor of the target enzyme from M. smegmatis.

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.

Tuberculosis (TB) remains a leading cause of mortality worldwide. With the emergence of multidrug resistant TB, extensively drug resistant TB and HIV-associated TB it is imperative that new drug targets be identified. The potential of Mycobacterium tuberculosis inosine monophosphate dehydrogenase (IMPDH) as a novel drug target was explored in the present study. IMPDH exclusively catalyzes the conversion of inosine monophosphate (IMP) to xanthosine monophosphate (XMP) in the presence of the cofactor nicotinamide adenine dinucleotide (NAD(+)). Although the enzyme is a dehydrogenase, the enzyme does not catalyze the reverse reaction i.e. the conversion of XMP to IMP. Unlike other bacteria, M. tuberculosis harbors three IMPDH-like genes, designated as Mt-guaB1, Mt-guaB2 and Mt-guaB3 respectively. Of the three putative IMPDH's, we previously confirmed that Mt-GuaB2 was the only functional ortholog by characterizing the enzyme kinetically. Using an in silico approach based on designed scaffolds, a series of novel classes of inhibitors was identified. The inhibitors possess good activity against M. tuberculosis with MIC values in the range of 0.4 to 11.4 µg mL(-1). Among the identified ligands, two inhibitors have nanomolar K(i)s against the Mt-GuaB2 enzyme.

In contrast with most bacteria, which harbour a single inosine monophosphate dehydrogenase (IMPDH) gene, the genomic sequence ofMycobacterium tuberculosisH37Rv predicts three genes encoding IMPDH:guaB1,guaB2andguaB3. These three genes were cloned and expressed inEscherichia colito evaluate functional IMPDH activity. Purified recombinant Mt-GuaB2, which uses inosine monophosphate as a substrate, was identified as the only active GuaB orthologue inM. tuberculosisand showed optimal activity at pH 8.5 and 37 °C. Mt-GuaB2 was inhibited significantlyin vitroby a panel of diphenyl urea-based derivatives, which were also potent anti-mycobacterial agents againstM. tuberculosisandMycobacterium smegmatis, with MICs in the range of 0.2–0.5 μg ml−1. When Mt-GuaB2 was overexpressed on a plasmidin transinM. 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 plasmidin transinM. smegmatis, they also conferred resistance, suggesting that although these Mt-GuaB orthologues were inactivein vitro, they presumably titrate the effect of the inhibitory properties of the active compoundsin vivo.

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 alpha-(1,6)-mannosyltransferases, the highest activity (IC50 = 0.14-0.22 mM) being registered with the hexamannoside and octamannoside.

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.

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 theMycobacterium tuberculosiscomplex. 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 PIM6, which contains terminal α(1→2)-linked mannosyl residues identical to the mannose cap on ManLAM, but not for di- and tetramannosylated PIM2and PIM4, respectively. To determine the role of PIM6in the binding of whole mycobacteria to DC-SIGN, a mutant strain ofM. bovisbacillus Calmette-Guérin deficient in the production of PIM6(ΔpimE) was created, as well as a double knockout deficient in the production of both PIM6and the mannose caps on LAM (ΔpimEΔcapA). 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, PIM6represents a bona fide DC-SIGN ligand but that other, as-yet-unknown, ligands dominate in the interaction between mycobacteria and DCs.

Previously mutations in a putative protein O-mannosyltransferase (SCO3154, Pmt) and a polyprenol phosphate mannose synthase (SCO1423, Ppm1) were found to cause resistance to phage, phiC31, in the antibiotic producing bacteria Streptomyces coelicolor A3(2). It was proposed that these two enzymes were part of a protein O-glycosylation pathway that was necessary for synthesis of the phage receptor. Here we provide the evidence that Pmt and Ppm1 are indeed both required for protein O-glycosylation. The phosphate binding protein PstS was found to be glycosylated with a trihexose in the S. coelicolor parent strain, J1929, but not in the pmt(-) derivative, DT1025. Ppm1 was necessary for the transfer of mannose to endogenous polyprenol phosphate in membrane preparations of S. coelicolor. A mutation in ppm1 that conferred an E218V substitution in Ppm1 abolished mannose transfer and glycosylation of PstS. Mass spectrometry analysis of extracted lipids showed the presence of a glycosylated polyprenol phosphate (PP) containing nine repeated isoprenyl units (C(45)-PP). S. coelicolor membranes were also able to catalyse the transfer of mannose to peptides derived from PstS, indicating that these could be targets for Pmt in vivo.

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-α 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 ΔmmaA4 mutant stimulated increased IL-12p40, similar to the increase observed from ΔmmaA4 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., Author Summary Currently, one-third of the world's population has tuberculosis (TB). TB, an ancient foe, has reemerged to become a threat to global public health. A central problem in TB research is to investigate why the host immune system cannot sterilize the infection caused by the bacterium Mycobacterium tuberculosis. Interleukin-12 (IL-12), a molecule produced by macrophages in response to pathogens, plays an important role in orchestrating sterilizing immunity. However, M. tuberculosis has evolved mechanisms that block IL-12 production and thereby assist the bacterium in establishing chronic infection. We discovered that mutation of the mycobacterial mmaA4 gene, which controls the chemical modification of complex lipids of M. tuberculosis called mycolic acids, renders the bacterium unable to block IL-12 production. Mycolic acids incorporated into a secreted bacterial molecule called trehalose dimycolate (TDM) from M. tuberculosis had the ability on their own to suppress the production of IL-12 by activated macrophages; we also showed that TDM from the mmaA4 mutant of M. tuberculosis is attenuated for suppression. Our results identify a critical part of the genetic basis and mechanism for an important immune evasion function in M. tuberculosis, and should contribute to the design of future vaccines and immunotherapies for disease caused by this pathogen.

The causative agent of tuberculosis,Mycobacterium tuberculosis, has two chaperonin (Cpn60) proteins and one cochaperonin (Cpn10) protein. We show here thatcpn60.2andcpn10, but notcpn60.1, 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°C). However, infection of mice with the Δcpn60.1mutant revealed a major difference from the wild-type organism. In spite of having equal numbers of bacteria in infected sites, the Δcpn60.1mutant 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 inM. tuberculosisinfection.

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.

Herein we report the synthesis of symmetrical C-linked and pseudo-symmetrical O-linked disaccharides structurally related to Araf motifs present in the cell wall of MTB. Their activity in a competition-based arabinosyltransferase assay using [14C]-DPA as the glycosyl donor is also presented. In addition, in vitro inhibitory activity for the disaccharides was determined in a colorimetric broth microdilution assay system against MTB H37Ra and Mycobacterium avium.

Mycobacterium tuberculosis , the causative agent of tuberculosis, has two distinguishing characteristics: its ability to stain acid-fast and its ability to cause long-term latent infections in humans. Although this distinctive staining characteristic has often been attributed to its lipid-rich cell wall, the specific dye-retaining components were not known. Here we report that targeted deletion of kasB , one of two M. tuberculosis genes encoding distinct β-ketoacyl- acyl carrier protein synthases involved in mycolic acid synthesis, results in loss of acid-fast staining. Biochemical and structural analyses revealed that the Δ kasB mutant strain synthesized mycolates with shorter chain lengths. An additional and unexpected outcome of kasB deletion was the loss of ketomycolic acid trans -cyclopropanation and a drastic reduction in methoxymycolic acid trans -cyclopropanation, activities usually associated with the trans -cyclopropane synthase CmaA2. Although deletion of kasB also markedly altered the colony morphology and abolished classic serpentine growth (cording), the most profound effect of kasB deletion was the ability of the mutant strain to persist in infected immunocompetent mice for up to 600 days without causing disease or mortality. This long-term persistence of Δ kasB represents a model for studying latent M. tuberculosis infections and suggests that this attenuated strain may represent a valuable vaccine candidate against tuberculosis.

The methyl-branched fatty acyl components of sulfolipid-I (SL-I), a major glycolipid of the human pathogen Mycobacterium tuberculosis, are synthesized by the polyketide synthase Pks2. Rv3824c (papA1), located downstream of pks2, encodes a protein that belongs to a subfamily of acyltransferases associated with mycobacterial polyketide synthases [polyketide synthase-associated proteins (PAPs)]. The presence of a conserved acyltransferase motif (HX(3)DX(14)Y) suggested a role for PapA1 in acylation of sulfated trehalose to form SL-I. Targeted deletion of the H37Rv papA1 resulted in loss of SL-I, demonstrating its role in mycobacterial sulfolipid biosynthesis. Furthermore, SL-I synthesis was restored in the mutant strain following complementation with papA1, but not with mutant alleles of papA1 containing alterations of key residues in the acyltransferase motif, confirming that PapA1 was an acyltransferase. While other M. tuberculosis pks clusters are associated with a single PAP-encoding gene, it was demonstrated that another open reading frame, Rv3820c (papA2), located 5.8 kb downstream of papA1 is also an acyltransferase gene involved in SL-I biosynthesis: deletion of papA2 abolished SL-I production. The absence of any partially acylated intermediates in either null mutant indicated that both PapA1 and PapA2 were required for all acylation steps of SL-I assembly.

A panel of α-(1→6)-linked mannose disaccharides ( 5 – 8 ) in which the 2′-OH group has been replaced, independently, by deoxy, fluoro, amino, and methoxy functionalities has been synthesized. Evaluation of these compounds as potential substrates or inhibitors of a polyprenol monophosphomannose-dependent α-(1→6)-mannosyltransferase involved in mycobacterial LAM biosynthesis demonstrated that the enzyme is somewhat tolerant substitution at this site. The enzyme recognizes the disaccharides with groups similar or smaller in size than the native hydroxyl ( 6 – 8 ), but not the disaccharide with the more sterically demanding methoxy group ( 5 ). The 2′-OH appears not form a critical hydrogen bonding interaction with the protein as the 2′-deoxy analog is a substrate for the enzyme.

PPM (polyprenol monophosphomannose) has been shown to act as a glycosyl donor in the biosynthesis of the Man (mannose)-rich mycobacterial lipoglycans LM (lipomannan) and LAM (lipoarabinomannan). The Mycobacterium tuberculosis PPM synthase (Mt-Ppm1) catalyses the transfer of Man from GDP-Man to polyprenyl phosphates. The resulting PPM then serves as a donor of Man residues leading to the formation of an alpha(1--6)LM intermediate through a PPM-dependent alpha(1--6)mannosyltransferase. In the present study, we prepared a series of ten novel prenyl-related photoactivatable probes based on benzophenone with lipophilic spacers replacing several internal isoprene units. These probes were excellent substrates for the recombinant PPM synthase Mt-Ppm1/D2 and, on photoactivation, several inhibited its activity in vitro. The protection of the PPM synthase activity by a 'natural' C(75) polyprenyl acceptor during phototreatment is consistent with probe-mediated photoinhibition occurring via specific covalent modification of the enzyme active site. In addition, the unique mannosylated derivatives of the photoreactive probes were all donors of Man residues, through a PPM-dependent mycobacterial alpha(1--6)mannosyltransferase, to a synthetic Manp(1--6)-Manp-O-C(10:1) disaccharide acceptor (where Manp stands for mannopyranose). Photoactivation of probe 7 led to striking-specific inhibition of the M. smegmatis alpha(1--6)mannosyltransferase. The present study represents the first application of photoreactive probes to the study of mycobacterial glycosyltransferases involved in LM and LAM biosynthesis. These preliminary findings suggest that the probes will prove useful in investigating the polyprenyl-dependent steps of the complex biosynthetic pathways to the mycobacterial lipoglycans, aiding in the identification of novel glycosyltransferases.

Analogues of the natural antibiotic thiolactomycin, with acetylene-based side chains, have the highest recorded in vitro inhibitory activity against the recombinant Mycobacterium tuberculosis beta-ketoacyl-ACP synthase mtFabH condensing enzyme. In particular, 5-[3-(4-acetyl-phenyl)-prop-2-ynyl]-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one exhibited more than an 18-fold increased potency, compared to thiolactomycin, against this key condensing enzyme, involved in M. tuberculosis mycolic acid biosynthesis. Analogues of the antibiotic thiolactomycin, with acetylene-based side chains, have the highest recorded activity against cloned mtFabH condensing enzyme.