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1.
Artigo em Inglês | MEDLINE | ID: mdl-28717039

RESUMO

Although the de novo folate biosynthesis pathway has been well studied in bacteria, little is known about its regulation. In the present study, the sigB gene in Mycobacterium tuberculosis was deleted. Subsequent drug susceptibility tests revealed that the M. tuberculosis ΔsigB strain was more sensitive to para-aminosalicylic acid (PAS) and sulfamethoxazole. Comparative transcriptional analysis was performed, and downregulation of pabB was observed in the ΔsigB strain, which was further verified by a quantitative reverse transcription-PCR and Western blot assay. Then, the production levels of para-aminobenzoic acid (pABA) were compared between the sigB deletion mutant and wild-type strain, and the results showed that sigB deletion resulted in decreased production of pABA. In addition, SigB was able to recognize the promoter of pabB in vitro Furthermore, we found that deleting pabC also caused increased susceptibility to PAS. Taken together, our data revealed that, in M. tuberculosis, sigB affects susceptibility to antifolates through multiple ways, primarily by regulating the expression of pabB To our knowledge, this is the first report showing that SigB modulates pABA biosynthesis and thus affecting susceptibility to antifolates, which broadens our understanding of the regulation of bacterial folate metabolism and mechanisms of susceptibility to antifolates.


Assuntos
Ácido 4-Aminobenzoico/metabolismo , Ácido Aminossalicílico/farmacologia , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Fator sigma/genética , Sulfametoxazol/farmacologia , Ácido Fólico/metabolismo , Deleção de Genes , Liases/genética , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento
2.
Antimicrob Agents Chemother ; 58(3): 1479-87, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24366731

RESUMO

The mechanistic basis for the resistance of Mycobacterium tuberculosis to para-aminosalicylic acid (PAS), an important agent in the treatment of multidrug-resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursor para-aminobenzoic acid, PAS is ultimately bioactivated to hydroxy dihydrofolate, which inhibits dihydrofolate reductase and disrupts the operation of folate-dependent metabolic pathways. As a result, the mutation of dihydrofolate synthase, an enzyme needed for the bioactivation of PAS, causes PAS resistance in M. tuberculosis strain H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates of M. tuberculosis and Mycobacterium bovis. From a panel of 85 multidrug-resistant M. tuberculosis clinical isolates, 5 were found to harbor mutations in the folC gene within the H2Pte binding pocket, resulting in PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished the bioactivation of hydroxy dihydropteroate to hydroxy dihydrofolate. Consistent with this model for abolished bioactivation, the introduction of a wild-type copy of folC fully restored PAS susceptibility in folC mutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics for M. tuberculosis clinical isolates and for further defining the mode of action of this important tuberculosis drug.


Assuntos
Ácido Aminossalicílico/farmacologia , Antibacterianos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeo Sintases/fisiologia , Alelos , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/fisiologia , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/enzimologia , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo
3.
Sci Rep ; 7(1): 6471, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28743871

RESUMO

MarR family proteins are transcriptional regulators that control expression of bacterial proteins involved in metabolism, virulence, stress responses and multi-drug resistance, mainly via ligand-mediated attenuation of DNA binding. Greater understanding of their underlying regulatory mechanism may open up new avenues for the effective treatment of bacterial infections. To gain molecular insight into the mechanism of Rv2887, a MarR family protein in M. tuberculosis, we first showed that it binds salicylate (SA) and para-aminosalicylic acid (PAS), its structural analogue and an antitubercular drug, in a 1:1 stoichiometry with high affinity. Subsequent determination and analysis of Rv2887 crystal structures in apo form, and in complex with SA, PAS and DNA showed that SA and PAS bind to Rv2887 at similar sites, and that Rv2887 interacts with DNA mainly by insertion of helix α4 into the major groove. Ligand binding triggers rotation of the wHTH domain of Rv2887 toward the dimerization domain, causing changes in protein conformation such that it can no longer bind to a 27 bp recognition sequence in the upstream region of gene Rv0560c. The structures provided here lay a foundation for the design of small molecules that target Rv2887, a potential new approach for the development of anti-mycobacterials.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Mycobacterium tuberculosis/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Ácido Aminossalicílico/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Genfibrozila/metabolismo , Modelos Moleculares , Regiões Promotoras Genéticas , Conformação Proteica , Salicilatos/metabolismo , Fatores de Transcrição/genética
4.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 6): 741-5, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26057805

RESUMO

Rv0880 from the pathogen Mycobacterium tuberculosis is classified as a MarR family protein in the Pfam database. It consists of 143 amino acids and has an isoelectric point of 10.9. Crystals of Rv0880 belonged to space group P1, with unit-cell parameters a = 54.97, b = 69.60, c = 70.32 Å, α = 103.71, ß = 111.06, γ = 105.83°. The structure of the MarR family transcription regulator Rv0880 was solved at a resolution of 2.0 Å with an R(cryst) and R(free) of 21.2 and 24.9%, respectively. The dimeric structure resembles that of other MarR proteins, with each subunit comprising a winged helix-turn-helix domain connected to an α-helical dimerization domain.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Mycobacterium tuberculosis/química , Fatores de Transcrição/química , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Cristalização , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium tuberculosis/metabolismo , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia Estrutural de Proteína , Fatores de Transcrição/genética
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