RESUMO
The arabinogalactan (AG) of slow growing pathogenic Mycobacterium spp. is characterized by the presence of galactosamine (GalN) modifying some of the interior branched arabinosyl residues. The biosynthetic origin of this substituent and its role(s) in the physiology and/or pathogenicity of mycobacteria are not known. We report on the discovery of a polyprenyl-phospho-N-acetylgalactosaminyl synthase (PpgS) and the glycosyltransferase Rv3779 from Mycobacterium tuberculosis required, respectively, for providing and transferring the GalN substrate for the modification of AG. Disruption of either ppgS (Rv3631) or Rv3779 totally abolished the synthesis of the GalN substituent of AG in M. tuberculosis H37Rv. Conversely, expression of ppgS in Mycobacterium smegmatis conferred upon this species otherwise devoid of ppgS ortholog and any detectable polyprenyl-phospho-N-acetylgalactosaminyl synthase activity the ability to synthesize polyprenyl-phospho-N-acetylgalactosamine (polyprenyl-P-GalNAc) from polyprenyl-P and UDP-GalNAc. Interestingly, this catalytic activity was increased 40-50-fold by co-expressing Rv3632, the encoding gene of a small membrane protein apparently co-transcribed with ppgS in M. tuberculosis H37Rv. The discovery of this novel lipid-linked sugar donor and the involvement of a the glycosyltransferase C-type glycosyltransferase in its transfer onto its final acceptor suggest that pathogenic mycobacteria modify AG on the periplasmic side of the plasma membrane. The availability of a ppgS knock-out mutant of M. tuberculosis provides unique opportunities to investigate the physiological function of the GalN substituent and the potential impact it may have on host-pathogen interactions.
Assuntos
Galactanos/química , Galactosamina/química , Mycobacterium tuberculosis/metabolismo , Alelos , Membrana Celular/metabolismo , Glicosilação , Lipídeos/química , Modelos Biológicos , Mutação , Mycobacterium smegmatis/metabolismo , Fenótipo , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The synthesis of conjugates consisting of two or three mannose units interconnected by a 1,2,3-triazole linker installed by the "click" reaction is reported. These conjugates were evaluated in mycobacterial mannosyltransferase (ManT) assay. Detailed analysis of the reaction products showed that these compounds with triazole linker between sugar moieties were tolerated by the enzyme, which elongated them by one or two sugar units with α-(1â6) linkage. The effectiveness of this transfer was reduced in comparison to that observed for the acceptor analogues containing a glycosidic linkage, but still, this is the first report on such unnatural compounds serving as substrates for mycobacterial ManT. The ability of the studied compounds to function as acceptors for the ManT suggests that the relative distance and spatial orientation of acceptor octyl hydrophobic aglycone (optimal length for the ManT) and free primary C-6 hydroxy group of the nonreducing terminal mannose unit (to which glycosyl residue is transferred by the mycobacterial ManT) are important for ManT activity, but at the same time, their variations are tolerated by the enzyme in a relatively wide range.
Assuntos
Glicoconjugados/síntese química , Manosiltransferases/metabolismo , Mycobacterium smegmatis/enzimologia , Oligossacarídeos/síntese química , Triazóis/síntese química , Glicoconjugados/química , Manosiltransferases/química , Estrutura Molecular , Oligossacarídeos/química , Triazóis/químicaRESUMO
Arabinogalactan (AG) and lipoarabinomannan (LAM) are the two major cell wall (lipo)polysaccharides of mycobacteria. They share arabinan chains made of linear segments of alpha-1,5-linked D-Araf residues with some alpha-1,3-branching, the biosynthesis of which offers opportunities for new chemotherapeutics. In search of the missing arabinofuranosyltransferases (AraTs) responsible for the formation of the arabinan domains of AG and LAM in Mycobacterium tuberculosis, we identified Rv0236c (AftD) as a putative membrane-associated polyprenyl-dependent glycosyltransferase. AftD is 1400 amino acid-long, making it the largest predicted glycosyltransferase of its class in the M. tuberculosis genome. Assays using cell-free extracts from recombinant Mycobacterium smegmatis and Corynebacterium glutamicum strains expressing different levels of aftD indicated that this gene encodes a functional AraT with alpha-1,3-branching activity on linear alpha-1,5-linked neoglycolipid acceptors in vitro. The disruption of aftD in M. smegmatis resulted in cell death and a decrease in its activity caused defects in cell division, reduced growth, alteration of colonial morphology, and accumulation of trehalose dimycolates in the cell envelope. Overexpression of aftD in M. smegmatis, in contrast, induced the accumulation of two arabinosylated compounds with carbohydrate backbones reminiscent of that of LAM and a degree of arabinosylation dependent on aftD expression levels. Altogether, our results thus indicate that AftD is an essential AraT involved in the synthesis of the arabinan domain of major mycobacterial cell envelope (lipo)polysaccharides.
Assuntos
Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Mycobacterium smegmatis/enzimologia , Configuração de Carboidratos , Sequência de Carboidratos , Galactanos/química , Galactanos/metabolismo , Glicosiltransferases/química , Glicosiltransferases/isolamento & purificação , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Dados de Sequência Molecular , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismoRESUMO
Two galactosyl transferases can apparently account for the full biosynthesis of the cell wall galactan of mycobacteria. Evidence is presented based on enzymatic incubations with purified natural and synthetic galactofuranose (Galf) acceptors that the recombinant galactofuranosyl transferase, GlfT1, from Mycobacterium smegmatis, the Mycobacterium tuberculosis Rv3782 ortholog known to be involved in the initial steps of galactan formation, harbors dual beta-(1-->4) and beta-(1-->5) Galf transferase activities and that the product of the enzyme, decaprenyl-P-P-GlcNAc-Rha-Galf-Galf, serves as a direct substrate for full polymerization catalyzed by another bifunctional Galf transferase, GlfT2, the Rv3808c enzyme.
Assuntos
Parede Celular/metabolismo , Galactanos/metabolismo , Galactosiltransferases/metabolismo , Mycobacterium smegmatis/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Galactanos/química , Galactosiltransferases/genética , Humanos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The synthesis of a series of alkyl (having from C6 to C20 aglycones), cyclohexyl, and cyclohexylalkyl alpha-d-mannopyranosides, 6-deoxygenated analogs, thioglycosides, and sulfones derived thereof, is reported. Here, under the in vitro assay conditions used, none of the 15 tested compounds acted as an inhibitor of the mannose transfer catalyzed by the enzymes present in mycobacterial membrane and cell wall fractions. Mannopyranosides comprising shorter aliphatic, up to 8 carbon atoms long linear, or cyclic aglycone served as the acceptor substrates in the mycobacterial mannosyltransferase reaction. The thioglycosides exhibited similar behavior, in contrast to the sulfones, which were essentially not recognized by the mycobacterial enzymes. 6-Deoxygenated glycosides were not processed by the enzymes, suggesting that the mannose transfer occurs at position 6 of the acceptors.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glicosídeos/síntese química , Glicosídeos/farmacologia , Manosiltransferases/antagonistas & inibidores , Mycobacterium smegmatis/enzimologia , Alquilação , Biocatálise/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/enzimologia , Parede Celular/metabolismo , Inibidores Enzimáticos/química , Glicosídeos/química , Manosiltransferases/metabolismo , Mycobacterium smegmatis/citologiaRESUMO
UDP-galactofuranose (UDP-Galf) is a substrate for two types of enzymes, UDP-galactopyranose mutase and galactofuranosyltransferases, which are present in many pathogenic organisms but absent from mammals. In particular, these enzymes are involved in the biosynthesis of cell wall galactan, a polymer essential for the survival of the causative agent of tuberculosis, Mycobacterium tuberculosis. We describe here the synthesis of derivatives of UDP-Galf modified at C-5 and C-6 using a chemoenzymatic route. In cell-free assays, these compounds prevented the formation of mycobacterial galactan, via the production of short "dead-end" intermediates resulting from their incorporation into the growing oligosaccharide chain. Modified UDP-furanoses thus constitute novel probes for the study of the two classes of enzymes involved in mycobacterial galactan assembly, and studies with these compounds may ultimately facilitate the future development of new therapeutic agents against tuberculosis.
Assuntos
Antituberculosos/química , Inibidores Enzimáticos/química , Galactanos/biossíntese , Galactose/análogos & derivados , Galactosiltransferases/antagonistas & inibidores , Difosfato de Uridina/análogos & derivados , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Galactanos/antagonistas & inibidores , Galactose/biossíntese , Galactose/química , Galactose/farmacologia , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Transferases Intramoleculares/antagonistas & inibidores , Transferases Intramoleculares/metabolismo , Klebsiella pneumoniae/enzimologia , Mycobacterium smegmatis/enzimologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Difosfato de Uridina/biossíntese , Difosfato de Uridina/química , Difosfato de Uridina/farmacologiaRESUMO
New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.
Assuntos
Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Mycobacterium tuberculosis/efeitos dos fármacos , Polissacarídeos/biossíntese , Racemases e Epimerases/antagonistas & inibidores , Compostos de Espiro/farmacologia , Compostos de Espiro/uso terapêutico , Tiazinas/farmacologia , Tiazinas/uso terapêutico , Tuberculose/tratamento farmacológico , Sequência de Aminoácidos , Animais , Antituberculosos/síntese química , Antituberculosos/química , Arabinose/metabolismo , Parede Celular/metabolismo , Farmacorresistência Bacteriana , Inibidores Enzimáticos/líquido cefalorraquidiano , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Etambutol/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Estrutura Molecular , Mycobacterium/efeitos dos fármacos , Mycobacterium/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Racemases e Epimerases/metabolismo , Compostos de Espiro/síntese química , Compostos de Espiro/química , Tiazinas/síntese química , Tiazinas/química , Tuberculose/microbiologiaRESUMO
The possibility of the Rv3782 protein of Mycobacterium tuberculosis being a putative galactosyl transferase (GalTr) implicated in galactan synthesis arose from its similarity to the known GalTr Rv3808c, its classification as a nucleotide sugar-requiring inverting glycosyltransferase (GT-2 family), and its location within the "possible arabinogalactan biosynthetic gene cluster" of M. tuberculosis. In order to study the function of the enzyme, active membrane and cell wall fractions from Mycobacterium smegmatis containing the overexpressed Rv3782 protein were incubated with endogenous decaprenyldiphosphoryl-N-acetylglucosaminyl-rhamnose (C(50)-P-P-GlcNAc-Rha) as the primary substrate for galactan synthesis and UDP-[(14)C]galactopyranose as the immediate precursor of UDP-[(14)C]galactofuranose, the ultimate source of all of the galactofuranose (Galf) units of galactan. Obvious increased and selective synthesis of C(50)-P-P-GlcNAc-Rha-Galf-Galf, the earliest product in the pathway leading to the fully polymerized galactan, was observed, suggesting that Rv3782 encodes a GalTr involved in the first stages of galactan synthesis. Time course experiments pointed to a possible bifunctional enzyme responsible for the initial synthesis of C(50)-P-P-GlcNAc-Rha-Galf, followed by immediate conversion to C(50)-P-P-GlcNAc-Rha-Galf-Galf. Thus, Rv3782 appears to be the initiator of galactan synthesis, while Rv3808c continues with the subsequent polymerization events.