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1.
Curr Opin Infect Dis ; 30(3): 309-315, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28134679

RESUMO

PURPOSE OF REVIEW: We wished to overview recent data on a subset of epigenetic changes elicited by intracellular bacteria in human cells. Reprogramming the gene expression pattern of various host cells may facilitate bacterial growth, survival, and spread. RECENT FINDINGS: DNA-(cytosine C5)-methyltransferases of Mycoplasma hyorhinis targeting cytosine-phosphate-guanine (CpG) dinucleotides and a Mycobacterium tuberculosis methyltransferase targeting non-CpG sites methylated the host cell DNA and altered the pattern of gene expression. Gene silencing by CpG methylation and histone deacetylation, mediated by cellular enzymes, also occurred in M. tuberculosis-infected macrophages. M. tuberculosis elicited cell type-specific epigenetic changes: it caused increased DNA methylation in macrophages, but induced demethylation, deposition of euchromatic histone marks and activation of immune-related genes in dendritic cells. A secreted transposase of Acinetobacter baumannii silenced a cellular gene, whereas Mycobacterium leprae altered the epigenotype, phenotype, and fate of infected Schwann cells. The 'keystone pathogen' oral bacterium Porphyromonas gingivalis induced local DNA methylation and increased the level of histone acetylation in host cells. These epigenetic changes at the biofilm-gingiva interface may contribute to the development of periodontitis. SUMMARY: Epigenetic regulators produced by intracellular bacteria alter the epigenotype and gene expression pattern of host cells and play an important role in pathogenesis.


Assuntos
Bactérias/patogenicidade , Epigênese Genética , Regulação Bacteriana da Expressão Gênica/genética , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/patogenicidade , Bactérias/enzimologia , Metilação de DNA , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Mycobacterium leprae/enzimologia , Mycobacterium leprae/patogenicidade , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/patogenicidade , Mycoplasma hyorhinis/enzimologia , Mycoplasma hyorhinis/patogenicidade , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/patogenicidade , Células de Schwann/metabolismo , Células de Schwann/microbiologia
2.
J Bacteriol ; 198(15): 2020-8, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27185825

RESUMO

UNLABELLED: Phthiocerol dimycocerosates (PDIM) are a group of cell surface-associated apolar lipids of Mycobacterium tuberculosis and closely related mycobacteria, such as Mycobacterium bovis and Mycobacterium leprae A characteristic methoxy group of these lipids is generated from the methylation of a hydroxyl group of the direct precursors, the phthiotriols. The precursors arise from the reduction of phthiodiolones, the keto intermediates, by a ketoreductase. The putative phthiodiolone ketoreductase (PKR) is encoded by Rv2951c in M. tuberculosis and BCG_2972c in M. bovis BCG, and these open reading frames (ORFs) encode identical amino acid sequences. We investigated the cofactor requirement of the BCG_2972c protein. A comparative analysis based on the crystallographic structures of similar enzymes identified structural elements for binding of coenzyme F420 and hydrophobic phthiodiolones in PKR. Coenzyme F420 is a deazaflavin coenzyme that serves several key functions in pathogenic and nonpathogenic mycobacteria. We found that an M. bovis BCG mutant lacking F420-dependent glucose-6-phosphate dehydrogenase (Fgd), which generates F420H2 (glucose-6-phosphate + F420 → 6-phosphogluconate + F420H2), was devoid of phthiocerols and accumulated phthiodiolones. When the mutant was provided with F420H2, a broken-cell slurry of the mutant converted accumulated phthiodiolones to phthiocerols; F420H2 was generated in situ from F420 and glucose-6-phosphate by the action of Fgd. Thus, the reaction mixture was competent in reducing phthiodiolones to phthiotriols (phthiodiolones + F420H2 → phthiotriols + F420), which were then methylated to phthiocerols. These results established the mycobacterial phthiodiolone ketoreductase as an F420H2-dependent enzyme (fPKR). A phylogenetic analysis of close homologs of fPKR revealed potential F420-dependent lipid-modifying enzymes in a broad range of mycobacteria. IMPORTANCE: Mycobacterium tuberculosis is the causative agent of tuberculosis, and phthiocerol dimycocerosates (PDIM) protect this pathogen from the early innate immune response of an infected host. Thus, the PDIM synthesis system is a potential target for the development of effective treatments for tuberculosis. The current study shows that a PDIM synthesis enzyme is dependent on the coenzyme F420 F420 is universally present in mycobacteria and absent in humans. This finding expands the number of experimentally validated F420-dependent enzymes in M. tuberculosis to six, each of which helps the pathogen to evade killing by the host immune system, and one of which activates an antituberculosis drug, PA-824. This work also has relevance to leprosy, since similar waxy lipids are found in Mycobacterium leprae.


Assuntos
Proteínas de Bactérias/metabolismo , Desidrogenases de Carboidrato/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Lipídeos/biossíntese , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Desidrogenases de Carboidrato/genética , Mycobacterium bovis/enzimologia , Mycobacterium tuberculosis/enzimologia , Filogenia
3.
FEBS Lett ; 590(4): 453-60, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26823273

RESUMO

Pyridoxal 5'-phosphate (PLP) biosynthesis is essential for the survival and virulence of Mycobacterium tuberculosis (Mtb). PLP functions as a cofactor for 58 putative PLP-binding proteins encoded by the Mtb genome and could also act as a potential antioxidant. De novo biosynthesis of PLP in Mtb takes place through the 'deoxyxylulose 5'-phosphate (DXP)-independent' pathway, whereas PdxH enzymes, possessing pyridoxine/pyridoxamine 5'-phosphate oxidase (PNPOx) activity, are involved in the PLP salvage pathway. In this study, we demonstrate that the annotated PdxH enzymes from various mycobacterial species are bona fide members of the classical PNPOx enzyme family, capable of producing PLP using both pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) substrates.


Assuntos
Proteínas de Bactérias/química , Mycobacterium leprae/enzimologia , Mycobacterium marinum/enzimologia , Mycobacterium tuberculosis/enzimologia , Piridoxaminafosfato Oxidase/química , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/biossíntese , Fosfato de Piridoxal/química , Piridoxamina/análogos & derivados , Piridoxamina/química , Piridoxaminafosfato Oxidase/classificação , Piridoxaminafosfato Oxidase/genética , Especificidade por Substrato
4.
PLoS Negl Trop Dis ; 9(11): e0004198, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26571269

RESUMO

Zoonotic pathogens that cause leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis complex, MTBC) continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect M. leprae as well as MTBC in wild marmosets. All marmosets were negative for M. leprae DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts.


Assuntos
Reservatórios de Doenças/veterinária , Programas de Rastreamento/métodos , Técnicas de Diagnóstico Molecular/métodos , Infecções por Mycobacterium/veterinária , Mycobacterium leprae/isolamento & purificação , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Callithrix , RNA Polimerases Dirigidas por DNA/genética , Reservatórios de Doenças/microbiologia , Infecções por Mycobacterium/microbiologia , Mycobacterium leprae/enzimologia , Mycobacterium leprae/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Sensibilidade e Especificidade
5.
Bioorg Med Chem ; 22(9): 2816-24, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24690527

RESUMO

The flavin-dependent thymidylate synthase X (ThyX), rare in eukaryotes and completely absent in humans, is crucial in the metabolism of thymidine (a DNA precursor) in many microorganisms including several human pathogens. Conserved in mycobacteria, including Mycobacterium leprae, and Mycobacterium tuberculosis, it represents a prospective anti-mycobacterial therapeutic target. In a M. tuberculosis ThyX-enzyme inhibition assay, N-(3-(5-(2'-deoxyuridine-5'-phosphate))prop-2-ynyl)octanamide was reported to be the most potent and selective 5-substituted 2'-deoxyuridine monophosphate analogue. In this study, we masked the two charges at the phosphate moiety of this compound using our ProTide technology in order to increase its lipophilicity and then allow permeation through the complex mycobacterial cell wall. A series of N-(3-(5-(2'-deoxyuridine))prop-2-ynyl)octanamide phosphoroamidates were chemically synthesized and their biological activity as potential anti-tuberculars was evaluated. In addition to mycobacteria, several DNA viruses depend on ThyX for their DNA biosynthesis, thus these prodrugs were also screened for their antiviral properties.


Assuntos
Amidas/química , Antituberculosos/química , Antivirais/química , Desoxiuridina/química , Antituberculosos/síntese química , Antituberculosos/farmacologia , Antivirais/síntese química , Antivirais/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Linhagem Celular , Herpesvirus Humano 3/efeitos dos fármacos , Herpesvirus Humano 3/enzimologia , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/enzimologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Simplexvirus/efeitos dos fármacos , Simplexvirus/enzimologia , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/metabolismo
6.
Biochim Biophys Acta ; 1814(12): 1802-11, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22001565

RESUMO

Mycobacterium leprae is closely related to Mycobacterium tuberculosis, yet causes a very different illness. Detailed genomic comparison between these two species of mycobacteria reveals that the decaying M. leprae genome contains less than half of the M. tuberculosis functional genes. The reduction of genome size and accumulation of pseudogenes in the M. leprae genome is thought to result from multiple recombination events between related repetitive sequences, which provided the impetus to investigate the recombination-like activities of RecA protein. In this study, we have cloned, over-expressed and purified M. leprae RecA and compared its activities with that of M. tuberculosis RecA. Both proteins, despite being 91% identical at the amino acid level, exhibit strikingly different binding profiles for single-stranded DNA with varying GC contents, in the ability to catalyze the formation of D-loops and to promote DNA strand exchange. The kinetics and the extent of single-stranded DNA-dependent ATPase and coprotease activities were nearly equivalent between these two recombinases. However, the degree of inhibition exerted by a range of ATP:ADP ratios was greater on strand exchange promoted by M. leprae RecA compared to its M. tuberculosis counterpart. Taken together, our results provide insights into the mechanistic aspects of homologous recombination and coprotease activity promoted by M. lepare RecA, and further suggests that it differs from the M. tuberculosis counterpart. These results are consistent with an emerging concept of DNA-sequence influenced structural differences in RecA nucleoprotein filaments and how these differences reflect on the multiple activities associated with RecA protein.


Assuntos
Mycobacterium leprae/enzimologia , Mycobacterium tuberculosis/enzimologia , Recombinases Rec A/química , Recombinases Rec A/fisiologia , Homologia Estrutural de Proteína , Sequência de Aminoácidos , Composição de Bases , Sítios de Ligação/genética , Clonagem Molecular , DNA de Cadeia Simples/metabolismo , Dados de Sequência Molecular , Mycobacterium leprae/química , Mycobacterium leprae/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Ligação Proteica , Estrutura Secundária de Proteína , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência , Especificidade da Espécie , Especificidade por Substrato
7.
Artigo em Inglês | MEDLINE | ID: mdl-21821883

RESUMO

The 1.5 Šresolution crystal structure of the Mycobacterium tuberculosis soluble inorganic pyrophosphatase Rv3628 at pH 7.0 is reported. The M. tuberculosis and M. leprae genomes include genes for the only two family I inorganic pyrophosphatases known to contain two histidines in the active site. The role of these two residues in catalysis is not fully understood. Mutational and functional studies of the M. tuberculosis enzyme showed that His21 and His86 are not essential for pyrophosphate hydrolysis, but are responsible for a shift in the optimal pH for the reaction compared with the Escherichia coli enzyme. Comparison with the structure previously reported at pH 5.0 provides further insight into the role of the two histidines. Two potassium-binding sites are found as a result of the high potassium concentration in the mother liquor.


Assuntos
Pirofosfatase Inorgânica/química , Mycobacterium tuberculosis/enzimologia , Sítios de Ligação , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Terciária de Proteína , Solubilidade , Homologia Estrutural de Proteína
8.
Nihon Hansenbyo Gakkai Zasshi ; 80(1): 17-27, 2011 Feb.
Artigo em Japonês | MEDLINE | ID: mdl-21404592

RESUMO

Drugs included in new-quinolone are used for the treatment of leprosy with single lesion. These drugs are also known to be effective drugs for the treatment of multi-drug resistant M. tuberculosis. Recent emergence of new-quinolone resistant M. leprae and M. tuberculosis enforced the urgent elucidation of the mode of emergence of new-quinolone resistant strains. In this review, new-quinolone drugs, their mode of action and mechanism of acquisition of resistance by M. leprae and M. tuberculosis were explained. And rapid differentiation methods for resistant bacilli were also introduced.


Assuntos
Antibacterianos/farmacologia , Técnicas Bacteriológicas/métodos , Farmacorresistência Bacteriana/genética , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/isolamento & purificação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Quinolonas/farmacologia , DNA Girase/genética , DNA Topoisomerase IV/genética , Testes de Sensibilidade Microbiana/métodos , Mutação , Mycobacterium leprae/enzimologia , Mycobacterium tuberculosis/enzimologia , Inibidores da Topoisomerase II
9.
Chem Biol ; 15(1): 51-61, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18158259

RESUMO

Phenolic glycolipids (PGLs) are polyketide-derived virulence factors produced by Mycobacterium tuberculosis, M. leprae, and other mycobacterial pathogens. We have combined bioinformatic, genetic, biochemical, and chemical biology approaches to illuminate the mechanism of chain initiation required for assembly of the p-hydroxyphenyl-polyketide moiety of PGLs. Our studies have led to the identification of a stand-alone, didomain initiation module, FadD22, comprised of a p-hydroxybenzoic acid adenylation domain and an aroyl carrier protein domain. FadD22 forms an acyl-S-enzyme covalent intermediate in the p-hydroxyphenyl-polyketide chain assembly line. We also used this information to develop a small-molecule inhibitor of PGL biosynthesis. Overall, these studies provide insights into the biosynthesis of an important group of small-molecule mycobacterial virulence factors and support the feasibility of targeting PGL biosynthesis to develop new drugs to treat mycobacterial infections.


Assuntos
Coenzima A Ligases , Inibidores Enzimáticos/farmacologia , Glicolipídeos , Macrolídeos/farmacologia , Mycobacterium tuberculosis/enzimologia , Fenóis , Fatores de Virulência , Adenosina/química , Adenosina/metabolismo , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/metabolismo , Glicolipídeos/antagonistas & inibidores , Glicolipídeos/biossíntese , Glicolipídeos/química , Humanos , Macrolídeos/química , Modelos Químicos , Mycobacterium tuberculosis/genética , Parabenos/química , Parabenos/metabolismo , Fenóis/antagonistas & inibidores , Fenóis/química , Fenóis/metabolismo , Fatores de Virulência/antagonistas & inibidores , Fatores de Virulência/biossíntese , Fatores de Virulência/química
10.
J Biol Chem ; 283(3): 1419-1427, 2008 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-18006503

RESUMO

Mycolic acids are major and specific lipid components of the cell envelope of mycobacteria that include the causative agents of tuberculosis and leprosy, Mycobacterium tuberculosis and Mycobacterium leprae, respectively. Subtle structural variations that are known to be crucial for both their virulence and the permeability of their cell envelope occur in mycolic acids. Among these are the introduction of cyclopropyl groups and methyl branches by mycolic acid S-adenosylmethionine-dependent methyltransferases (MA-MTs). While the functions of seven of the M. tuberculosis MA-MTs have been either established or strongly presumed nothing is known of the roles of the remaining umaA gene product and those of M. smegmatis MA-MTs. Mutants of the M. tuberculosis umaA gene and its putative M. smegmatis orthologue, MSMEG0913, were created. The lipid extracts of the resulting mutants were analyzed in detail using a combination of analytical techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proton nuclear magnetic resonance spectroscopy, and chemical degradation methods. The M. smegmatis mutants no longer synthesized subtypes of mycolates containing a methyl branch adjacent to either trans cyclopropyl group or trans double bond at the "proximal" position of both alpha- and epoxy-mycolates. Complementation with MSMEG0913, but not with umaA, fully restored the wild-type phenotype in M. smegmatis. Consistently, no modification was observed in the structures of mycolic acids produced by the M. tuberculosis umaA mutant. These data proved that despite their synteny and high similarity umaA and MSMEG0913 are not functionally orthologous.


Assuntos
Proteínas de Bactérias/metabolismo , Metiltransferases/metabolismo , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/enzimologia , Ativação Enzimática , Compostos de Epóxi/isolamento & purificação , Ésteres/isolamento & purificação , Teste de Complementação Genética , Espectroscopia de Ressonância Magnética , Mutação/genética , Ácidos Micólicos/química , Ácidos Micólicos/classificação , Ácidos Micólicos/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
Biochem J ; 408(3): 417-27, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17784850

RESUMO

The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units x mg(-1) at 37 degrees C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.


Assuntos
Lisofosfolipase/metabolismo , Lipídeos de Membrana/metabolismo , Mycobacterium tuberculosis/enzimologia , Sequência de Bases , Catálise , Primers do DNA , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Lisofosfolipase/antagonistas & inibidores , Lisofosfolipase/química , Lisofosfolipase/genética , Lisofosfolipídeos/metabolismo , Mutagênese Sítio-Dirigida , Mycobacterium tuberculosis/genética , Especificidade por Substrato , Temperatura
12.
Proteins ; 69(2): 358-68, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17600834

RESUMO

Mycolic acids, which render unique qualities to mycobacteria, are known to be important for mycobacterial growth, survival, and pathogenicity. It is of interest to understand the evolutionary origins of the mycolic acid pathway (MAP), as well as the common minimum principles critical for generating the capability of mycolic acid biosynthesis. The recent curation of a comprehensive model of the MAP in Mycobacterium tuberculosis and the availability of a large number of genome sequences make it feasible to carry out detailed sequence and phylogenetic analyses, to address these questions. A comprehensive phylogenetic pathway profile analysis was carried out for 318 fully sequenced bacterial genomes, for each of the proteins present in the MAP. The organisms were clustered on the basis of co-occurrence of the MAP proteins in their proteome, while the proteins were clustered on the basis of their phylogenetic profiles. The MAP proteins were also searched against the nonredundant sequence database, to identify similar proteins from other phyla. The pathway profiles indicate that four proteins and certain protein domains stand out as more characteristic to mycolate producing organisms. Further analysis leads to the identification of the desaturases DesA1 and DesA2 and certain domains of Fas and Pks13 as hallmarks of the pathway. The roles of these proteins in some other organisms, as well as the distribution of these proteins across all known genome sequences are also briefly discussed. The clustering of organisms, carried out to group organisms with similar profiles, provides a means of obtaining finer classification as compared to the standard taxonomic method. The results indicate that the MAP and hence the capacity of mycolic acid production in mycobacteria is an example of an emergent property that has come about by recruiting enzymes from unrelated pathways in plants, presumably through lateral gene transfer. The understanding of the hallmarks of mycolic acid biosynthesis will also find application in evaluating drug targets.


Assuntos
Vias Biossintéticas/genética , Genômica/métodos , Ácidos Micólicos/metabolismo , Genoma Bacteriano , Mycobacterium leprae/enzimologia , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Filogenia , Mapeamento de Interação de Proteínas , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos
13.
J Exp Med ; 204(1): 73-8, 2007 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-17227913

RESUMO

Thioamide drugs, ethionamide (ETH) and prothionamide (PTH), are clinically effective in the treatment of Mycobacterium tuberculosis, M. leprae, and M. avium complex infections. Although generally considered second-line drugs for tuberculosis, their use has increased considerably as the number of multidrug resistant and extensively drug resistant tuberculosis cases continues to rise. Despite the widespread use of thioamide drugs to treat tuberculosis and leprosy, their precise mechanisms of action remain unknown. Using a cell-based activation method, we now have definitive evidence that both thioamides form covalent adducts with nicotinamide adenine dinucleotide (NAD) and that these adducts are tight-binding inhibitors of M. tuberculosis and M. leprae InhA. The crystal structures of the inhibited M. leprae and M. tuberculosis InhA complexes provide the molecular details of target-drug interactions. The purified ETH-NAD and PTH-NAD adducts both showed nanomolar Kis against M. tuberculosis and M. leprae InhA. Knowledge of the precise structures and mechanisms of action of these drugs provides insights into designing new drugs that can overcome drug resistance.


Assuntos
Etionamida/farmacologia , Hanseníase/tratamento farmacológico , Protionamida/farmacologia , Tuberculose/tratamento farmacológico , Antituberculosos/química , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Cristalografia por Raios X , Desenho de Fármacos , Farmacorresistência Bacteriana Múltipla , Etionamida/química , Etionamida/metabolismo , Humanos , Técnicas In Vitro , Hansenostáticos/química , Hansenostáticos/metabolismo , Hansenostáticos/farmacologia , Modelos Moleculares , Complexo Mycobacterium avium/efeitos dos fármacos , Complexo Mycobacterium avium/enzimologia , Infecção por Mycobacterium avium-intracellulare/tratamento farmacológico , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/enzimologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , NAD/química , NAD/metabolismo , Oxirredutases/antagonistas & inibidores , Protionamida/química , Protionamida/metabolismo , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico
14.
Nat Med ; 11(6): 638-44, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15895072

RESUMO

Genes involved in fatty acid catabolism have undergone extensive duplication in the genus Mycobacterium, which includes the etiologic agents of leprosy and tuberculosis. Here, we show that prokaryotic- and eukaryotic-like isoforms of the glyoxylate cycle enzyme isocitrate lyase (ICL) are jointly required for fatty acid catabolism and virulence in Mycobacterium tuberculosis. Although deletion of icl1 or icl2, the genes that encode ICL1 and ICL2, respectively, had little effect on bacterial growth in macrophages and mice, deletion of both genes resulted in complete impairment of intracellular replication and rapid elimination from the lungs. The feasibility of targeting ICL1 and ICL2 for chemical inhibition was shown using a dual-specific ICL inhibitor, which blocked growth of M. tuberculosis on fatty acids and in macrophages. The absence of ICL orthologs in mammals should facilitate the development of glyoxylate cycle inhibitors as new drugs for the treatment of tuberculosis.


Assuntos
Ácidos Graxos/metabolismo , Isocitrato Liase/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/patogenicidade , Animais , Células Cultivadas , Feminino , Humanos , Isocitrato Liase/antagonistas & inibidores , Isocitrato Liase/química , Isoenzimas , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Nitrocompostos , Propionatos/farmacologia , Conformação Proteica , Fatores de Tempo , Virulência
15.
Biochem J ; 387(Pt 2): 541-51, 2005 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15500449

RESUMO

Analysis of the genome sequence of Mycobacterium tuberculosis H37Rv has identified 16 genes that are similar to the mammalian adenylate and guanylate cyclases. Rv1647 was predicted to be an active adenylate cyclase but its position in a phylogenetically distant branch from the other enzymes characterized so far from M. tuberculosis makes it an interestingly divergent nucleotide cyclase to study. In agreement with its divergence at the sequence level from other nucleotide cyclases, the cloning, expression and purification of Rv1647 revealed differences in its biochemical properties from the previously characterized Rv1625c adenylate cyclase. Adenylate cyclase activity of Rv1647 was activated by detergents but was resistant to high concentrations of salt. Mutations of substrate-specifying residues to those present in guanylate cyclases failed to convert the enzyme into a guanylate cyclase, and did not alter its oligomeric status. Orthologues of Rv1647 could be found in M. leprae, M. avium and M. smegmatis. The orthologue from M. leprae (ML1399) was cloned, and the protein was expressed, purified and shown biochemically to be an adenylate cyclase, thus representing the first adenylate cyclase to be described from M. leprae. Importantly, Western-blot analysis of subcellular fractions from M. tuberculosis and M. leprae revealed that the Rv1647 and ML1399 gene products respectively were expressed in these bacteria. Additionally, M. tuberculosis was also found to express the Rv1625c adenylate cyclase, suggesting that multiple adenylate cyclase proteins may be expressed simultaneously in this organism. These results suggest that class III cyclase-like gene products probably have an important role to play in the physiology and perhaps the pathology of these medically important bacteria.


Assuntos
Adenilil Ciclases/metabolismo , Mycobacterium leprae/enzimologia , Mycobacterium tuberculosis/enzimologia , Adenilil Ciclases/química , Adenilil Ciclases/genética , Sequência de Aminoácidos , Domínio Catalítico , Expressão Gênica , Dados de Sequência Molecular , Família Multigênica , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
16.
J Biol Chem ; 278(15): 13094-100, 2003 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-12551895

RESUMO

With the advent of the sequencing programs of prokaryotic genomes, many examples of the presence of serine/threonine protein kinases in these organisms have been identified. Moreover, these kinases could be classified as homologues of those belonging to the well characterized superfamily of the eukaryotic serine/threonine and tyrosine kinases. Eleven such kinases were recognized in the genome of Mycobacterium tuberculosis. Here we report the crystal structure of an active form of PknB, one of the four M. tuberculosis kinases that are conserved in the downsized genome of Mycobacterium leprae and are therefore presumed to play an important role in the processes that regulate the complex life cycle of mycobacteria. Our structure confirms again the extraordinary conservation of the protein kinase fold and constitutes a landmark that extends this conservation across the evolutionary distance between high eukaryotes and eubacteria. The structure of PknB, in complex with a nucleotide triphosphate analog, reveals an enzyme in the active state with an unprecedented arrangement of the Gly-rich loop associated with a new conformation of the nucleotide gamma-phosphoryl group. It presents as well a partially disordered activation loop, suggesting an induced fit mode of binding for the so far unknown substrates of this kinase or for some modulating factor(s).


Assuntos
Mycobacterium tuberculosis/enzimologia , /química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
17.
Microbiol Mol Biol Rev ; 66(4): 702-38, table of contents, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12456788

RESUMO

The bacterial acyltransferases of the SxxK superfamily vary enormously in sequence and function, with conservation of particular amino acid groups and all-alpha and alpha/beta folds. They occur as independent entities (free-standing polypeptides) and as modules linked to other polypeptides (protein fusions). They can be classified into three groups. The group I SxxK D,D-acyltransferases are ubiquitous in the bacterial world. They invariably bear the motifs SxxK, SxN(D), and KT(S)G. Anchored in the plasma membrane with the bulk of the polypeptide chain exposed on the outer face of it, they are implicated in the synthesis of wall peptidoglycans of the most frequently encountered (4-->3) type. They are inactivated by penicillin and other beta-lactam antibiotics acting as suicide carbonyl donors in the form of penicillin-binding proteins (PBPs). They are components of a morphogenetic apparatus which, as a whole, controls multiple parameters such as shape and size and allows the bacterial cells to enlarge and duplicate their particular pattern. Class A PBP fusions comprise a glycosyltransferase module fused to an SxxK acyltransferase of class A. Class B PBP fusions comprise a linker, i.e., protein recognition, module fused to an SxxK acyltransferase of class B. They ensure the remodeling of the (4-->3) peptidoglycans in a cell cycle-dependent manner. The free-standing PBPs hydrolyze D,D peptide bonds. The group II SxxK acyltransferases frequently have a partially modified bar code, but the SxxK motif is invariant. They react with penicillin in various ways and illustrate the great plasticity of the catalytic centers. The secreted free-standing PBPs, the serine beta-lactamases, and the penicillin sensors of several penicillin sensory transducers help the D,D-acyltransferases of group I escape penicillin action. The group III SxxK acyltransferases are indistinguishable from the PBP fusion proteins of group I in motifs and membrane topology, but they resist penicillin. They are referred to as Pen(r) protein fusions. Plausible hypotheses are put forward on the roles that the Pen(r) protein fusions, acting as L,D-acyltransferases, may play in the (3-->3) peptidoglycan-synthesizing molecular machines. Shifting the wall peptidoglycan from the (4-->3) type to the (3-->3) type could help Mycobacterium tuberculosis and Mycobacterium leprae survive by making them penicillin resistant.


Assuntos
Aciltransferases/metabolismo , Proteínas de Bactérias , Proteínas de Transporte/metabolismo , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Mycobacterium tuberculosis/enzimologia , Resistência às Penicilinas/fisiologia , Penicilinas/farmacologia , Peptidil Transferases , Aciltransferases/genética , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Ligação às Penicilinas , Penicilinas/metabolismo , Peptidoglicano/metabolismo
18.
Biochem J ; 363(Pt 3): 437-47, 2002 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-11964144

RESUMO

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 alpha-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 (Ac(3))-PIM(2) (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-[(14)C]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 Ac(3)PIM(3) (i.e. the trimannoside). The results indicate that pimC encodes an alpha-mannosyltransferase involved in Ac(3)PIM(3) 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.


Assuntos
Manosiltransferases/genética , Mycobacterium tuberculosis/enzimologia , Fosfatidilinositóis/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias , DNA Bacteriano/isolamento & purificação , Guanosina Difosfato Manose/metabolismo , Lipopolissacarídeos/metabolismo , Manosiltransferases/isolamento & purificação , Dados de Sequência Molecular , Mycobacterium bovis/genética , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/genética , Alinhamento de Sequência , Espectrometria de Massas de Bombardeamento Rápido de Átomos
19.
Biochem J ; 365(Pt 2): 441-50, 2002 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-11931640

RESUMO

Dolichol monophosphomannose (DPM) is an ever-present donor of mannose (Man) in various eukaryotic glycosylation processes. Intriguingly, the related polyprenol monophosphomannose (PPM) is involved in the biosynthesis of lipomannan and lipoarabinomanan, key bacterial factors termed modulins that are found in mycobacteria. Based on similarities to known DPM synthases, we have identified and characterized the PPM synthase of Mycobacterium tuberculosis, now termed Mt-Ppm1. In the present study, we demonstrate that Mt-Ppm1 possesses an unusual two-domain architecture, by which the second domain is sufficient for PPM synthesis. However, when overexpressed separately in mycobacteria, domain 1 of Mt-Ppm1 appears to increase the synthesis of PPM. Interestingly, other mycobacteria such as M. smegmatis, M. avium and M. leprae produce two distinct proteins, which are similar to the two domains found in Mt-Ppm1. Using an in vitro assay, we also demonstrate that Mt-Ppm1 transfers Man from GDP-Man to a structurally diverse range of lipid monophosphate acceptors. The identification of the PPM synthase as a key enzyme in lipoarabinomannan biosynthesis now provides an attractive candidate for gene disruption to generate mutants for subsequent immunological studies. PPM synthase can also be exploited as a target for specific inhibitors of M. tuberculosis.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , Primers do DNA , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray
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