RESUMO
The antigen 85 (Ag85) complex of Mycobacterium tuberculosis represents a promising candidate as a novel drug target and pathogenesis factor. Ag85 comprises three proteins Ag85A, B and C, (encoded by the genes fbpA, B, and C), which participate in cell wall biosynthesis, and interact with the host macrophage as fibronectin-binding proteins (fbps). Ag85 is also involved in the response to isoniazid (INH) treatment. The objective of this study was to identify potential fbp gene activators involved in the over-expression of fbp genes in response to INH. The biotinylated upstream promoter regions of fbpA and fbpC were used together with streptavidin-coated magnetic beads in DNA-binding assays, to isolate proteins with high-binding affinities from cytosolic extracts of INH-treated M. tuberculosis. Resolution of the DNA-binding proteins by 1D SDS-PAGE revealed 6 proteins with high-affinity for the fbpA promoter, and 7 with specificity the fbpC promoter. Mass spectrometric analyses [LC-ES(MS/MS)] identified proteins associated with drug resistance and stress/treatment responses, intermediary metabolism and cellular division, hypothetical proteins including a member of the MarR family of bacterial transcriptional regulators. The DNA-binding MarR protein shows potential as an authentic activator of fbp genes and functional validation of this factor is warranted.
Assuntos
Aciltransferases/genética , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Parede Celular/genética , Proteínas de Ligação a DNA/genética , Amplificação de Genes/genética , Proteínas de Membrana Transportadoras/genética , Mycobacterium tuberculosis/genética , Fatores de Transcrição/genética , Tuberculose/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Mycobacterium tuberculosis/isolamento & purificação , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Tuberculose/metabolismo , Regulação para CimaRESUMO
We have demonstrated that fluoroquinolones, a class of antibacterial agents that act through inhibition of type II DNA topoisomerases, exert selective action against intracellular amastigotes of Leishmania (Viannia) panamensis at concentrations that are achievable in vivo. Drug cytotoxicity assays employing the luciferase reporter gene revealed that intracellular amastigotes were 6.6- to 25.9-fold more sensitive than human macrophages (P < 0.05) to second-generation fluoroquinolones in vitro. The most selective agents (enoxacin and ciprofloxacin) exhibited 2 orders of magnitude greater potency against parasites (50% effective dose [ED50] = 54.9-83.4 microM) than host cells (ED50 = 1,425-1,740 microM). Linear regression analysis of ED50 data confirmed a complete lack of correlation (r = 0.001) between the relative drug sensitivities of parasites and host cells. A potential relationship between the structures of fluoroquinolones and their relative leishmanicidal activities was observed. The key substituents of the basic pyridone beta-carboxylic acid nucleus accounting for enhanced antiparasite potency and selectivity appear to be a nitrogen at position 8 of the bicyclic nucleus (enoxacin), a cyclopropyl substituent at the R1 site (ciprofloxacin), and linkage of the R1 and X8 groups by a CH3CHO bridge to form a tricyclic compound (ofloxacin). These findings support the potential of fluoroquinolones and derivatives as novel antileishmanials and encourage their clinical evaluation.
