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1.
Microbiol Spectr ; 10(4): e0128822, 2022 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-35862962

RESUMO

Mycolic acids, a hallmark of the genus Mycobacterium, are unique branched long-chain fatty acids produced by a complex biosynthetic pathway. Due to their essentiality and involvement in various aspects of mycobacterial pathogenesis, the synthesis of mycolic acids-and the identification of the enzymes involved-is a valuable target for drug development. Although most of the core pathway is comparable between species, subtle structure differences lead to different structures delineating the mycolic acid repertoire of tuberculous and some nontuberculous mycobacteria. We here report the characterization of an α'-mycolic acid-deficient Mycobacterium smegmatis mutant obtained by chemical mutagenesis. Whole-genome sequencing and bioinformatic analysis identified a premature stop codon in MSMEG_4301, encoding an acyl-CoA synthetase. Orthologs of MSMEG_4301 are present in all mycobacterial species containing α'-mycolic acids. Deletion of the Mycobacterium abscessus ortholog MAB_1915 abrogated synthesis of α'-mycolic acids; likewise, deletion of MSMEG_4301 in an otherwise wild-type M. smegmatis background also caused loss of these short mycolates. IMPORTANCE Mycobacterium abscessus is a nontuberculous mycobacterium responsible for an increasing number of hard-to-treat infections due to the impervious nature of its cell envelope, a natural barrier to several antibiotics. Mycolic acids are key components of that envelope; thus, their synthesis is a valuable target for drug development. Our results identify the first enzyme involved in α'-mycolic acids, a short-chain member of mycolic acids, loss of which greatly affects growth of this opportunistic pathogen.


Assuntos
Mycobacterium abscessus , Mycobacterium , Vias Biossintéticas/genética , Ácidos Graxos/metabolismo , Mycobacterium/metabolismo , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Ácidos Micólicos/metabolismo , Micobactérias não Tuberculosas
2.
Microbiology (Reading) ; 164(12): 1567-1582, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30311878

RESUMO

Mycobacterium smegmatis is intrinsically resistant to thiacetazone, an anti-tubercular thiourea; however we report here that it causes a mild inhibition in growth in liquid medium. Since mycolic acid biosynthesis was affected, we cloned and expressed Mycobacterium smegmatis mycolic acid methyltransferases, postulated as targets for thiacetazone in other mycobacterial species. During this analysis we identified MSMEG_1350 as the methyltransferase involved in epoxy mycolic acid synthesis since its deletion led to their total loss. Phenotypic characterization of the mutant strain showed colony morphology alterations at all temperatures, reduced growth and a slightly increased susceptibility to SDS, lipophilic and large hydrophilic drugs at 20 °C with little effect at 37 °C. No changes were detected between parental and mutant strains in biofilm formation, sliding motility or sedimentation rate. Intriguingly, we found that several mycobacteriophages severely decreased their ability to form plaques in the mutant strain. Taken together our results prove that, in spite of being a minor component of the mycolic acid pool, epoxy-mycolates are required for a proper assembly and functioning of the cell envelope. Further studies are warranted to decipher the role of epoxy-mycolates in the M. smegmatis cell envelope.


Assuntos
Proteínas de Bactérias/genética , Metiltransferases/genética , Micobacteriófagos/fisiologia , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/virologia , Ácidos Micólicos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Temperatura Baixa , Metiltransferases/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Mycobacterium smegmatis/fisiologia , Deleção de Sequência
3.
Tuberculosis (Edinb) ; 112: 69-78, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30205971

RESUMO

Clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis are differentially susceptible to 2-Thiophen Hydrazide (TCH); however its mechanism of action or the reasons for that difference are unknown. We report herein that under our experimental conditions, TCH inhibits M. tuberculosis in solid but not in liquid medium, and that in spite of resembling Isoniazid and Ethionamide, it does not affect mycolic acid synthesis. To understand the mechanisms of action of TCH we isolated M. tuberculosis TCH resistant mutants which fell into two groups; one resistant to TCH and Isoniazid but not to Ethionamide or Triclosan, and the other resistant only to TCH with no, or marginal, cross resistance to Isoniazid. A S315T katG mutation conferred resistance to TCH while katG expression from a plasmid reduced M. tuberculosis MIC to this drug, suggesting a possible involvement of KatG in TCH activation. Whole genome sequencing of mutants from this second group revealed a single mutation in the alkylhydroperoxide reductase ahpC promoter locus in half of the mutants, while the remaining contained mutations in dispensable genes. This is the first report of the genetics underlying the action of TCH and of the involvement of ahpC as the sole basis for resistance to an anti-tubercular compound.


Assuntos
Antituberculosos/farmacologia , Ácidos Carboxílicos/farmacologia , Catalase/genética , Farmacorresistência Bacteriana/genética , Etionamida/farmacologia , Isoniazida/farmacologia , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Peroxirredoxinas/genética , Regiões Promotoras Genéticas , Proteínas de Bactérias , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo
4.
Expert Rev Anti Infect Ther ; 11(4): 429-40, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23566152

RESUMO

One of the first approaches undertaken in the quest for antitubercular compounds was that of understanding the mechanism of action of old drugs and proposing chemical modifications or other strategies to improve their activity, generally lost to the mechanisms of resistance developed by Mycobacterium tuberculosis. A leading case was the work carried out on a set of compounds with proven activity on the essential pathway of the synthesis of mycolic acids. As a result, different solutions were presented, improving the activity of those inhibitors or producing novel compounds acting on the same molecular target(s), but avoiding the most common resistance strategies developed by the tubercle bacilli. This review focuses on the activity of those compounds, developed following the completion of the studies on several of the classic antitubercular drugs.


Assuntos
Antituberculosos/síntese química , Mycobacterium tuberculosis/efeitos dos fármacos , Ácidos Micólicos/antagonistas & inibidores , Antituberculosos/farmacologia , Desenho de Fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Etionamida/análogos & derivados , Etionamida/síntese química , Etionamida/farmacologia , Humanos , Isoniazida/análogos & derivados , Isoniazida/síntese química , Isoniazida/farmacologia , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Feniltioureia/análogos & derivados , Feniltioureia/síntese química , Feniltioureia/farmacologia , Relação Estrutura-Atividade , Tioacetazona/análogos & derivados , Tioacetazona/síntese química , Tioacetazona/farmacologia , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia
5.
Mol Microbiol ; 86(3): 568-79, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22994892

RESUMO

It has recently been shown that the anti-mycobacterial pro-drug thiacetazone (TAC) inhibits the conversion of double bonds of mycolic acid precursors into cyclopropyl rings in Mycobacterium bovis var BCG, M. marimum and M. chelonae by affecting the cyclopropyl mycolic acid synthases (CMASs) as judged by the build-up of unsaturated mycolate precursors. In our hands, TAC inhibits mycolic acid biosynthesis in Mycobacterium tuberculosis and M. kansasii with almost negligible accumulation of those precursors. Our observations that 'de novo' biosynthesis of all the mycolic acid families decreased upon TAC treatment prompted us to analyse the role of each one of the Type II Fatty Acid Synthase (FASII) enzymes. Overexpression of the hadABC operon, encoding the essential FASII dehydratase complex, but not of any of the remaining FASII genes acting on the elongation of fatty acyl chains leading to the synthesis of meromycolic acids, resulted in high level of resistance to TAC in M. tuberculosis. Spontaneous M. tuberculosis and M. kansasii TAC-resistant mutants isolated during this work revealed mutations in the hadABC genes strongly supporting our proposal that these enzymes are new players in the resistance to this anti-mycobacterial compound.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Enoil-CoA Hidratase/genética , Mycobacterium kansasii/enzimologia , Mycobacterium tuberculosis/enzimologia , Tioacetazona/farmacologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Enoil-CoA Hidratase/química , Enoil-CoA Hidratase/metabolismo , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Dados de Sequência Molecular , Mutação , Mycobacterium kansasii/química , Mycobacterium kansasii/efeitos dos fármacos , Mycobacterium kansasii/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Ácidos Micólicos/metabolismo , Óperon , Alinhamento de Sequência
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