RESUMO
OBJECTIVES: The search for new TB drugs has become one of the great challenges for modern medicinal chemistry. An improvement in the outcomes of TB chemotherapy can be achieved by the development of new, shorter, cheap, safe and effective anti-TB regimens. METHODS: Chalcones (1a-1o) were synthesized and evaluated for their antimycobacterial activity against Mycobacterium bovis BCG using growth inhibition assays. Compound 1a was selected as a 'hit' compound. The mode of action of compound 1a, was identified by mycolic acid methyl esters (MAMEs) and fatty acid methyl esters (FAMEs) analysis using thin layer chromatography. Dose dependent experiments were conducted by over-expressing components of FAS-II in M. bovis BCG to confirm the target. Ligand binding using intrinsic tryptophan assay and molecular docking were used to further validate the target. RESULTS: MAMEs and FAMEs analysis showed dose-dependent reduction of MAMEs with the overall abundance of FAMEs suggesting that compound 1a targets mycolic acid biosynthesis. Direct binding of 1a to InhA was observed using an intrinsic tryptophan fluorescence binding assay, and a 2-fold IC50 shift was observed with an InhA overexpressing strain confirming InhA as the cellular target. CONCLUSION: The chalcone 1a exhibits potent antimycobacterial activity, displays a good safety profile and is a direct inhibitor of InhA, a key component in mycolic acid synthesis, validating this series for further anti-TB drug development.
RESUMO
We have reported previously that treatment of non-obese diabetic (NOD) mice with the invariant natural killer T (iNK T) cell agonist α-galactosylceramide C26:0 (α-GalCer) or its T helper type 2 (Th2)-biasing derivative α-GalCer C20:2 (C20:2) protects against type 1 diabetes (T1D), with C20:2 yielding greater protection. After an initial response to α-GalCer, iNK T cells become anergic upon restimulation. While such anergic iNK T cells can induce tolerogenic dendritic cells (DCs) that mediate protection from T1D, chronic administration of α-GalCer also results in long-lasting anergy accompanied by significantly reduced iNK T cell frequencies, which raises concerns about its long-term therapeutic use. In this study, our objective was to understand more clearly the roles of anergy and induction of tolerogenic DCs in iNK T cell-mediated protection from T1D and to circumvent potential complications associated with α-GalCer. We demonstrate that NOD iNK T cells activated during multi-dose (MD) treatment in vivo with C20:2 enter into and exit from anergy more rapidly than after activation by α-GalCer. Importantly, this shorter duration of iNK T cells in the anergic state promotes the more rapid induction of tolerogenic DCs and reduced iNK T cell death, and enables C20:2 stimulated iNK T cells to elicit enhanced protection from T1D. Our findings further that suggest C20:2 is a more effective therapeutic drug than α-GalCer for protection from T1D. Moreover, the characteristics of C20:2 provide a basis of selection of next-generation iNK T cell agonists for the prevention of T1D.
Assuntos
Anergia Clonal/efeitos dos fármacos , Diabetes Mellitus Tipo 1/prevenção & controle , Galactosilceramidas/farmacologia , Células T Matadoras Naturais/efeitos dos fármacos , Animais , Antígeno B7-1/imunologia , Antígeno B7-1/metabolismo , Antígeno B7-H1 , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Células Cultivadas , Anergia Clonal/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Galactosilceramidas/química , Galactosilceramidas/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Masculino , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Peptídeos/imunologia , Peptídeos/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Fatores de TempoRESUMO
Protection from type 1 diabetes (T1D), a T helper type 1 (Th1)-mediated disease, is achievable in non-obese diabetic (NOD) mice by treatment with alpha-galactosylceramide (alpha-GalCer) glycolipids that stimulate CD1d-restricted invariant natural killer T (iNK T) cells. While we have reported previously that the C20:2 N-acyl variant of alpha-GalCer elicits a Th2-biased cytokine response and protects NOD mice from T1D more effectively than a form of alpha-GalCer that induces mixed Th1 and Th2 responses, it remained to determine whether this protection is accompanied by heightened anti-inflammatory responses. We show that treatment of NOD mice with C20:2 diminished the activation of 'inflammatory' interleukin (IL)-12 producing CD11c(high)CD8+ myeloid dendritic cells (mDCs) and augmented the function of 'tolerogenic' DCs more effectively than treatment with the prototypical iNKT cell activator KRN7000 (alpha-GalCer C26:0) that induces Th1- and Th2-type responses. These findings correlate with a reduced capacity of C20:2 to sustain the early transactivation of T, B and NK cells. They may also explain our observation that C20:2 activated iNK T cells depend less than KRN7000 activated iNK T cells upon regulation by regulatory T cells for cytokine secretion and protection from T1D. The enhanced anti-inflammatory properties of C20:2 relative to KRN7000 suggest that C20:2 should be evaluated further as a drug to induce iNK T cell-mediated protection from T1D in humans.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Células Dendríticas/efeitos dos fármacos , Diabetes Mellitus Tipo 1/prevenção & controle , Galactosilceramidas/uso terapêutico , Hipoglicemiantes/uso terapêutico , Fatores Imunológicos/uso terapêutico , Interleucina-12/metabolismo , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Apresentação de Antígeno/efeitos dos fármacos , Efeito Espectador/efeitos dos fármacos , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Galactosilceramidas/química , Galactosilceramidas/farmacologia , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Organismos Livres de Patógenos Específicos , Baço/efeitos dos fármacos , Baço/imunologia , Relação Estrutura-Atividade , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/metabolismoRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Periplásmicas/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Streptomyces coelicolor/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Glicosilação , Manose/metabolismo , Dados de Sequência Molecular , Proteínas Periplásmicas/genética , Proteínas de Ligação a Fosfato/genética , Análise de Sequência de Proteína , Streptomyces coelicolor/genéticaRESUMO
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.
Assuntos
Cápsulas Bacterianas/fisiologia , Lipopolissacarídeos/metabolismo , Manose/metabolismo , Mycobacterium/fisiologia , Animais , Cápsulas Bacterianas/metabolismo , Elementos de DNA Transponíveis/genética , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Eletroforese em Gel de Poliacrilamida , Feminino , Teste de Complementação Genética , Interações Hospedeiro-Patógeno , Humanos , Immunoblotting , Interleucina-10/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Manose/química , Manose/fisiologia , Manosiltransferases/genética , Manosiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Modelos Moleculares , Mutagênese Insercional , Mutação , Mycobacterium/metabolismo , Infecções por Mycobacterium/metabolismo , Infecções por Mycobacterium/microbiologia , Peixe-ZebraRESUMO
In spite of effective antibiotics to treat TB (tuberculosis) since the early 1960s, we enter the new millennium with TB, currently the leading cause of death from a single infectious agent, killing more than three million people worldwide each year. Thus an understanding of drug-resistance mechanisms, the immunobiology of cell wall components to elucidate host-pathogen interactions and the discovery of new drug targets are now required for the treatment of TB. Above the plasma membrane is a classical chemotype IV PG (peptidoglycan) to which is attached the macromolecular structure, mycolyl-arabinogalactan, via a unique diglycosylphosphoryl bridge. This review will discuss the assembly of the mAGP (mycolyl-arabinogalactan-peptidoglycan), its associated glycolipids and the site of action of EMB (ethambutol), bringing forward a new era in TB research and focus on new drugs to combat multidrug resistant TB.
Assuntos
Antituberculosos/química , Parede Celular/metabolismo , Galactanos/biossíntese , Lipopolissacarídeos/biossíntese , Mycobacterium tuberculosis/metabolismo , Galactanos/metabolismo , Lipopolissacarídeos/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacosRESUMO
AIMS: Phthiocerol dimycocerosate (PDIM) waxes and other lipids are necessary for successful Mycobacterium tuberculosis infection, although the exact role of PDIM in host-pathogen interactions remains unclear. In this study, we investigated the contribution of tesA, drrB, pks6 and pks11 genes in complex lipid biosynthesis in M. tuberculosis. METHODS AND RESULTS: Four mutants were selected from M. tuberculosis H37Rv transposon mutant library. The transposon insertion sites were confirmed to be within the M. tuberculosis open reading frames for tesA (a probable thioesterase), drrB (predicted ABC transporter), pks11 (putative chalcone synthase) and pks6 (polyketide synthase). The first three of these transposon mutants were unable to generate PDIM and the fourth lacked novel polar lipids. CONCLUSIONS: Mycobacterium tuberculosis can be cultivated in vitro without the involvement of certain lipid synthesis genes, which may be necessary for in vivo pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of transposon mutants is a new functional genomic approach for the eventual definition of the mycobacterial 'lipidome'.
Assuntos
Lipídeos/análise , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Policetídeo Sintases/genética , Transportadores de Cassetes de Ligação de ATP/genética , Aciltransferases/genética , Elementos de DNA Transponíveis/genética , Inativação Gênica , Genes Bacterianos/genética , Lipídeos/biossíntese , Família Multigênica/genética , Mutagênese Insercional/genética , Mutação/genética , Mycobacterium tuberculosis/patogenicidade , Palmitoil-CoA Hidrolase/genéticaRESUMO
Corynebacterium diphtheriae is a Gram-positive, non-spore forming, non-motile, pleomorphic rod belonging to the genus Corynebacterium and the actinomycete group of organisms. The organism produces a potent bacteriophage-encoded protein exotoxin, diphtheria toxin (DT), which causes the symptoms of diphtheria. This potentially fatal infectious disease is controlled in many developed countries by an effective immunisation programme. However, the disease has made a dramatic return in recent years, in particular within the Eastern European region. The largest, and still on-going, outbreak since the advent of mass immunisation started within Russia and the newly independent states of the former Soviet Union in the 1990s. We have sequenced the genome of a UK clinical isolate (biotype gravis strain NCTC13129), representative of the clone responsible for this outbreak. The genome consists of a single circular chromosome of 2 488 635 bp, with no plasmids. It provides evidence that recent acquisition of pathogenicity factors goes beyond the toxin itself, and includes iron-uptake systems, adhesins and fimbrial proteins. This is in contrast to Corynebacterium's nearest sequenced pathogenic relative, Mycobacterium tuberculosis, where there is little evidence of recent horizontal DNA acquisition. The genome itself shows an unusually extreme large-scale compositional bias, being noticeably higher in G+C near the origin than at the terminus.
Assuntos
Corynebacterium diphtheriae/genética , Genoma Bacteriano , Idoso , Composição de Bases , Cromossomos Bacterianos/genética , Corynebacterium diphtheriae/metabolismo , Corynebacterium diphtheriae/patogenicidade , DNA Bacteriano/química , DNA Bacteriano/genética , Toxina Diftérica/metabolismo , Feminino , Fímbrias Bacterianas/genética , Humanos , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Virulência/genéticaRESUMO
AIMS: The antigen 85 complex (Ag85) from Mycobacterium tuberculosis consists of three abundantly secreted proteins (FbpA, FbpB and FbpC2) which play a key role in the pathogenesis of tuberculosis and also exhibit cell wall mycolyltransferase activity. A related protein with similarity to the Ag85 complex was recently annotated in the M. tuberculosis genome as FbpC1. An investigation was carried out to determine whether FbpC1 may also possess mycolyltransferase activity, a characteristic feature of the Ag85 complex. METHODS AND RESULTS: Heterologous expression of FbpA, FbpC1 and FbpC2 was performed in Escherichia coli. Recombinant proteins were purified under non-denaturating conditions and used in an in vitro mycolyltransferase assay. CONCLUSIONS: In contrast to FbpA and FbpC2, recombinant FbpC1 did not possess in vitro mycolyltransferase activity and was not recognized by two monoclonal antibodies to the native Ag85. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycolyltransferase activity is restricted to FbpA, FbpbB and FbpC2 only; the actual function of FbpC1 remains to be established.
Assuntos
Aciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
Members of the CD1 family of antigen-presenting molecules bind and present a variety of mammalian and microbial glycolipids for specific recognition by T cells. CD1 proteins accomplish their antigen-presenting function by binding the alkyl chains of the antigens within a deep, hydrophobic groove on the membrane distal surface of CD1, making the hydrophilic elements of the antigen available for contact with the variable regions of antigen-specific T-cell receptors. Most models of CD1-restricted T cells function in infectious, neoplastic, or autoimmune diseases and are based on the premise that CD1-restricted T-cell responses are initiated by alterations in cellular glycolipid content. Although a growing number of self, altered self and foreign glycolipid antigens have been identified, the cellular mechanisms that could lead to the generation of antigenic glycolipids within cells, or control the presentation of particular classes of altered self or microbial glycolipids in disease states have only recently come under investigation. Here we review the structures of known glycolipid antigens for T cells and discuss how the chemical nature of these antigens, which is quite different from that of peptides, influences their recognition by T cells.
Assuntos
Antígenos CD1/imunologia , Glicolipídeos/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Antígenos de Bactérias/imunologia , Galactosilceramidas/imunologia , Humanos , Receptores de Antígenos de Linfócitos T/imunologia , Tolerância a Antígenos Próprios/imunologiaRESUMO
The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (MTB) and the continuing pandemic of tuberculosis emphasizes the urgent need for the development of new anti-tubercular agents with novel drug targets. The recent structural elucidation of the mycobacterial cell wall highlights a large variety of structurally unique components that may be a basis for new drug development. This publication describes the synthesis, characterization, and screening of several octyl Galf(beta,1-->5)Galf and octyl Galf(beta,1-->6)Galf derivatives. A cell-free assay system has been utilized for galactosyltransferase activity using UDP[14C]Galf as the glycosyl donor, and in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). Certain derivatives showed moderate activities against MTB and MAC. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to totally deprotected disaccharides that more closely resemble the natural substrates in cell wall biosynthesis.
Assuntos
Dissacarídeos/química , Dissacarídeos/metabolismo , Galactosiltransferases/metabolismo , Mycobacterium/enzimologia , Antituberculosos/química , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Bioquímica/métodos , Configuração de Carboidratos , Dissacarídeos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Concentração Inibidora 50 , Mycobacterium/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
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-(alpha-D-arabinofuranosyl)-alpha-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.
Assuntos
Antituberculosos/química , Antituberculosos/metabolismo , Dissacarídeos/química , Dissacarídeos/metabolismo , Mycobacterium tuberculosis/enzimologia , Pentosiltransferases/metabolismo , Antituberculosos/farmacologia , Configuração de Carboidratos , Dissacarídeos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Concentração Inibidora 50 , Mycobacterium avium/efeitos dos fármacos , Mycobacterium avium/enzimologia , Mycobacterium tuberculosis/efeitos dos fármacos , Pentosiltransferases/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
A series of synthetic mannosides was screened in a cell-free system for their ability to act as acceptor substrates for mycobacterial mannosyltransferases. Evaluation of these compounds demonstrated the incorporation of [14C]Man from GDP-[14C]Man into a radiolabeled organic-soluble fraction and analysis by thin layer chromatography and autoradiography revealed the formation of two radiolabeled products. Each synthetic acceptor was capable of accepting one or two mannose residues, resulting in a major and a minor mannosylated product. Both products from each acceptor were isolated and their mass was confirmed by fast-atom bombardment-mass spectrometry (FABMS). Characterization of each mannosylated product by exo-glycosidase digestion. acetolysis and linkage analysis by gas chromatography mass spectrometry of partially per-O-methylated alditols, revealed only alpha1-6-linked products. In addition. the antibiotic amphomycin selectively inhibited the formation of mannosylated products suggesting polyprenolmonophosphate-mannose (C15 50-P-Man) was the immediate mannose donor in all mannosylation reactions observed. The ability of synthetic disaccharides to act as acceptor substrates in this system, is most likely due to the action of a mycobacterial polyprenol-P-Man:mannan alpha1-6 mannosyltransferase involved in the biosynthesis of linear alpha1-6-linked lipomannan.
Assuntos
Manosídeos/química , Manosiltransferases/metabolismo , Mycobacterium smegmatis/enzimologia , Sequência de Carboidratos , Cromatografia Gasosa-Espectrometria de Massas , Manosídeos/síntese química , Dados de Sequência Molecular , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in the biosynthesis of fatty acids in all bacteria, including Mycobacterium tuberculosis. MCAT catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP, to generate malonyl-ACP, which is an elongation substrate in fatty acid biosynthesis. To clarify the roles of the mycobacterial acyl carrier protein (AcpM) and MCAT in fatty acid and mycolic acid biosynthesis, we have cloned, expressed, and purified acpM and mtfabD (malonyl-CoA:AcpM transacylase) from M. tuberculosis. According to the culture conditions used, AcpM was produced in Escherichia coli in two or three different forms: apo-AcpM, holo-AcpM, and palmitoylated-AcpM, as revealed by electrospray mass spectrometry. The mtfabD gene encoding a putative MCAT was used to complement a thermosensitive E. coli fabD mutant. Expression and purification of mtFabD resulted in an active enzyme displaying strong MCAT activity in vitro. Enzymatic studies using different ACP substrates established that holo-AcpM constitutes the preferred substrate for mtFabD. In order to provide further insight into the structure-function relationship of mtFabD, different mutant proteins were generated. All mutations (Q9A, R116A, H194A, Q243A, S91T, and S91A) completely abrogated MCAT activity in vitro, thus underlining the importance of these residues in transacylation. The generation and characterization of the AcpM forms and mtFabD opens the way for further studies relating to fatty acid and mycolic acid biosynthesis to be explored in M. tuberculosis. Since a specific type of FabD is found in mycobacterial species, it represents an attractive new drug target waiting to be exploited.
Assuntos
Aciltransferases/química , Proteínas de Bactérias , Proteínas de Transporte/química , Ácido Graxo Sintases/química , Mycobacterium tuberculosis/enzimologia , Proteína de Transporte de Acila S-Maloniltransferase , Sequência de Aminoácidos , Clonagem Molecular , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Ácido Graxo Sintase Tipo II , Teste de Complementação Genética , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , TemperaturaRESUMO
The cell wall of Mycobacterium tuberculosis and related genera is unique among prokaryotes, consisting of a covalently bound complex of mycolic acids, D-arabinan and D-galactan, which is linked to peptidoglycan via a special linkage unit consisting of Rhap-(1-->3)-GlcNAc-P. Information concerning the biosynthesis of this entire polymer is now emerging with the promise of new drug targets against tuberculosis. Accordingly, we have developed a galactosyltransferase assay that utilizes the disaccharide neoglycolipid acceptors beta-d-Galf-(1-->5)-beta-D-Galf-O-C(10:1) and beta-D-Galf-(1-->6)-beta-D-Galf-O-C(10:1), with UDP-Gal in conjunction with isolated membranes. Chemical analysis of the subsequent reaction products established that the enzymatically synthesized products contained both beta-D-Galf linkages ((1-->5) and (1-->6)) found within the mycobacterial cell, as well as in an alternating (1-->5) and (1-->6) fashion consistent with the established structure of the cell wall. Furthermore, through a detailed examination of the M. tuberculosis genome, we have shown that the gene product of Rv3808c, now termed glfT, is a novel UDP-galactofuranosyltransferase. This enzyme possesses dual functionality in performing both (1-->5) and (1-->6) galactofuranosyltransferase reactions with the above neoglycolipid acceptors, using membranes isolated from the heterologous host Escherichia coli expressing Rv3808c. Thus, at a biochemical and genetic level, the polymerization of the galactan region of the mycolyl-arabinogalactan complex has been defined, allowing the possibility of further studies toward substrate recognition and catalysis and assay development. Ultimately, this may also lead to a more rational approach to drug design to be explored in the context of mycobacterial infections.
Assuntos
Galactanos/biossíntese , Galactosiltransferases/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/metabolismo , Especificidade por SubstratoRESUMO
T cells recognize microbial glycolipids presented by CD1 proteins, but there is no information regarding the generation of natural glycolipid antigens within infected tissues. Therefore, we determined the molecular basis of CD1b-restricted T cell recognition of mycobacterial glycosylated mycolates, including those produced during tissue infection in vivo. Transfection of the T cell receptor (TCR) alpha and beta chains from a glucose monomycolate (GMM)-specific T cell line reconstituted GMM recognition in TCR-deficient T lymphoblastoma cells. This TCR-mediated response was highly specific for natural mycobacterial glucose-6-O-(2R, 3R) monomycolate, including the precise structure of the glucose moiety, the stereochemistry of the mycolate lipid, and the linkage between the carbohydrate and the lipid. Mycobacterial production of antigenic GMM absolutely required a nonmycobacterial source of glucose that could be supplied by adding glucose to media at concentrations found in mammalian tissues or by infecting tissue in vivo. These results indicate that mycobacteria synthesized antigenic GMM by coupling mycobacterial mycolates to host-derived glucose. Specific T cell recognition of an epitope formed by interaction of host and pathogen biosynthetic pathways provides a mechanism for immune response to those pathogenic mycobacteria that have productively infected tissues, as distinguished from ubiquitous, but innocuous, environmental mycobacteria.
Assuntos
Antígenos de Bactérias/imunologia , Antígenos CD1/imunologia , Carboidratos/imunologia , Glicolipídeos/imunologia , Mycobacterium/imunologia , Linfócitos T/imunologia , Animais , Tatus , Configuração de Carboidratos , Carboidratos/química , Humanos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Estereoisomerismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Mycobacterium smegmatis has been shown to contain two forms of polyprenyl phosphate (Pol-P), while Mycobacterium tuberculosis contains only one. Utilizing subcellular fractions from M. smegmatis and M. tuberculosis, we show that Pol-P synthesis is different in these species. The specific activities of the prenyl diphosphate synthases in M. tuberculosis are 10- to 100-fold lower than those in M. smegmatis. In M. smegmatis decaprenyl diphosphate and heptaprenyl diphosphate were the main products synthesized in vitro, whereas in M. tuberculosis only decaprenyl diphosphate was synthesized. The data from both organisms suggest that geranyl diphosphate is the allylic substrate for two distinct prenyl diphosphate synthases, one located in the cell membrane that synthesizes omega,E,Z-farnesyl diphosphate and the other present in the cytosol that synthesizes omega,E,E,E-geranylgeranyl diphosphate. In M. smegmatis, the omega,E, Z-farnesyl diphosphate is utilized by a membrane-associated prenyl diphosphate synthase activity to generate decaprenyl diphosphate, and the omega,E,E,E-geranylgeranyl diphosphate is utilized by a membrane-associated activity for the synthesis of the heptaprenyl diphosphate. In M. tuberculosis, however, omega,E,E,E-geranylgeranyl diphosphate is not utilized for the synthesis of heptaprenyl diphosphate. Thus, the difference in the compositions of the Pol-P of M. smegmatis and M. tuberculosis can be attributed to distinct enzymatic differences between these two organisms.
Assuntos
Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Fosfatos de Poli-Isoprenil/biossíntese , Membrana Celular/metabolismo , Cromatografia em Camada Fina , Citosol/metabolismo , Estrutura Molecular , Fosfatos de Poli-Isoprenil/química , Fosfatos de Poli-Isoprenil/isolamento & purificação , Especificidade da EspécieRESUMO
The structural core of the cell walls of Mycobacterium spp. consists of peptidoglycan bound by a linker unit (-alpha-L-Rhap-(1-->3)-D-GlcNAc-P-) to a galactofuran, which in turn is attached to arabinofuran and mycolic acids. The sequence of reactions leading to the biogenesis of this complex starts with the formation of the linker unit on a polyprenyl-P to produce polyprenyl-P-P-GlcNAc-Rha (Mikusová, K., Mikus, M., Besra, G. S., Hancock, I., and Brennan, P. J. (1996) J. Biol. Chem. 271, 7820-7828). We now establish that formation of the galactofuran takes place on this intermediate with UDP-Galf as the Galf donor presented in the form of UDP-Galp and UDP-Galp mutase (the glf gene product) and is catalyzed by galactofuranosyl transferases, one of which, the Mycobacterium tuberculosis H37Rv3808c gene product, has been identified. Evidence is also presented for the growth of the arabinofuran on this polyprenyl-P-P-linker unit-galactan intermediate catalyzed by unidentified arabinosyl transferases, with decaprenyl-P-Araf or 5-P-ribosyl-PP as the Araf donor. The product of these steps, the lipid-linked-LU-galactan-arabinan has been partially characterized in terms of its heterogeneity, size, and composition. Biosynthesis of the major components of mycobacterial cell walls is proving to be extremely complex. However, partial definition of arabinogalactan synthesis, the site of action of several major anti-tuberculosis drugs, facilitates the present day thrust for new drugs to counteract multiple drug-resistant tuberculosis.
Assuntos
Galactanos/biossíntese , Transferases Intramoleculares/metabolismo , Mycobacterium/metabolismo , Parede Celular/química , Clonagem Molecular , Furanos/metabolismo , Galactosiltransferases/genética , Glicopeptídeos/metabolismoRESUMO
The long-chain alpha-alkyl-beta-hydroxy fatty acids, termed mycolic acids, which are characteristic components of the mycobacterial cell wall are produced by successive rounds of elongation catalyzed by a multifunctional (type I) fatty acid synthase complex followed by a dissociated (type II) fatty acid synthase. In bacterial type II systems, the first initiation step in elongation is the condensation of acetyl-CoA with malonyl-acyl carrier protein (ACP) catalyzed by beta-ketoacyl-ACP III (FabH). An open reading frame in the Mycobacterium tuberculosis genome (Rv0533c), now termed mtfabH, was 37.3% identical to Escherichia coli ecFabH and contained the Cys-His-Asn catalytic triad signature. However, the purified recombinant mtFabH clearly preferred long-chain acyl-CoA substrates rather than acyl-ACP primers and did not utilize acetyl-CoA as a primer in comparison to ecFabH. In addition, purified mtFabH was sensitive to thiolactomycin and resistant to cerulenin in an in vitro assay. However, mtFabH overexpression in Mycobacterium bovis BCG did not confer thiolactomycin resistance, suggesting that mtFabH may not be the primary target of thiolactomycin inhibition in vivo and led to several changes in the lipid composition of the bacilli. The data presented is consistent with a role for mtFabH as the pivotal link between the type I and type II fatty acid elongation systems in M. tuberculosis. This study opens up new avenues for the development of selective and novel anti-mycobacterial agents targeted against mtFabH.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , Acil Coenzima A/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Domínio Catalítico , Clonagem Molecular , Escherichia coli/enzimologia , Escherichia coli/genética , Genoma Bacteriano , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The anti-tuberculosis drug ethionamide (ETH), which is a structural analog of isoniazid (INH), is known to strongly inhibit mycolic acid synthesis in Mycobacterium tuberculosis. Although several targets have been identified for INH, only speculative information is available concerning ETH. Mutations within the promoter and the coding region of enoyl-acyl carrier protein reductase (InhA) were found to confer resistance to both drugs, thus leading to the impression that INH and ETH may share a common mode of action. However, a notable distinction between the two drugs lies in the lack of cross-resistance in clinical isolates. This may be attributed in part to the fact that the pro-drug INH must be activated via KatG, and no activation step for ETH has yet been described. Here we report the identification of an activator for ETH. The ETH activator (Rv3854c), which we have termed EthA, was found to be homologous to various monooxygenases and induced ETH sensitivity when overexpressed in mycobacteria. Interestingly, the neighboring open reading frame (Rv3855), which was found homologous to transcriptional repressors of the tetR family, led to ETH resistance when overexpressed. In addition, chromosomal inactivation of this gene by transposition led to ETH hypersensitivity. These data strongly suggest that Rv3855, which we have termed EthR, regulates the production of EthA, which subsequently activates the pro-drug ETH. This study opens up new avenues of research relating to ETH activation in mycobacteria, possibly leading to an improved efficacy of ETH and to the generation of new anti-mycobacterial agents.