RESUMO
The assembly and disassembly of FtsZ play an essential role in bacterial cell division. Using single-cell imaging, we report that short exposure to BT-benzo-29 inhibits Z-ring formation in live Bacillus subtilis cells. Fluorescence recovery after photobleaching of the Z-ring in live bacteria demonstrated that BT-benzo-29 strongly suppressed the assembly dynamics of FtsZ in the Z-ring. Furthermore, B. subtilis cells expressing V275A-FtsZ resisted the antibacterial activity of BT-benzo-29 providing evidence that BT-benzo-29 inhibits bacterial proliferation by targeting FtsZ. In addition, a brief (8 min) exposure of BT-benzo-29 destroyed the Z-ring without perturbing the localization of a late cell division protein, DivIVA, the nucleoid segregation, and membrane permeability. BT-benzo-29, when used in combination with vancomycin and polymyxin B (PMB), produced a much stronger inhibitory effect on Bacillus subtilis and Escherichia coli cells, respectively. The combination index of BT-benzo-29 with vancomycin and PMB was determined to be <1, suggesting that BT-benzo-29 exhibits synergistic inhibitory effects on bacterial proliferation when used along with these antibiotics.
Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Benzimidazóis/farmacologia , Divisão Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/antagonistas & inibidores , Farmacorresistência Bacteriana/genética , Tiofenos/farmacologia , Substituição de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Combinação de Medicamentos , Sinergismo Farmacológico , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Expressão Gênica , Testes de Sensibilidade Microbiana , Mutação , Polimixina B/farmacologia , Análise de Célula Única , Vancomicina/farmacologiaRESUMO
Inhibition of FtsZ assembly has been found to stall bacterial cell division. Here, we report the identification of a potent carbocyclic curcumin analogue (2d) that inhibits Bacillus subtilis 168 cell proliferation by targeting the assembly of FtsZ. 2d also showed potent inhibitory activity (minimum inhibitory concentrations of 2-4 mg/L) against several clinically important species of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. In addition, 2d displayed a significantly reduced inhibitory effect on human cervical cancer cells in comparison to its effect on bacterial cells. Using live cell imaging of GFP-FtsZ by confocal microscopy, 2d was found to rapidly perturb the cytokinetic FtsZ rings in Bacillus subtilis cells. The immunofluorescence imaging of FtsZ also showed that 2d destroyed the Z-ring in bacteria within 5 min. Prolonged treatment with 2d produced filamentous bacteria, but 2d had no detectable effect either on the nucleoids or on the membrane potential of bacteria. 2d inhibited FtsZ assembly in vitro, whereas it had minimal effects on tubulin assembly. Interestingly, 2d strongly enhanced the GTPase activity of FtsZ and reduced the GTPase activity of tubulin. Furthermore, 2d bound to purified FtsZ with a dissociation constant of 4.0 ± 1.1 µM, and the binding of 2d altered the secondary structures of FtsZ. The results together suggested that the non-natural curcumin analogue 2d possesses powerful antibacterial activity against important pathogenic bacteria, and the evidence indicates that 2d inhibits bacterial proliferation by targeting FtsZ.
Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Curcumina/farmacologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Animais , Antibacterianos/síntese química , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sítios de Ligação , Clonagem Molecular , Curcumina/análogos & derivados , Curcumina/síntese química , Ciclização , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Cabras , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Staphylococcus aureus Resistente à Meticilina/ultraestrutura , Testes de Sensibilidade Microbiana , Imagem Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Especificidade da Espécie , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
We have identified a potent antibacterial agent N-(4-sec-butylphenyl)-2-(thiophen-2-yl)-1H-benzo[d]imidazole-4-carboxamide (BT-benzo-29) from a library of benzimidazole derivatives that stalled bacterial division by inhibiting FtsZ assembly. A short (5 min) exposure of BT-benzo-29 disassembled the cytokinetic Z-ring in Bacillus subtilis cells without affecting the cell length and nucleoids. BT-benzo-29 also perturbed the localization of early and late division proteins such as FtsA, ZapA and SepF at the mid-cell. Further, BT-benzo-29 bound to FtsZ with a dissociation constant of 24 ± 3 µm and inhibited the assembly and GTPase activity of purified FtsZ. A docking analysis suggested that BT-benzo-29 may bind to FtsZ at the C-terminal domain near the T7 loop. BT-benzo-29 displayed significantly weaker inhibitory effects on the assembly and GTPase activity of two mutants (L272A and V275A) of FtsZ supporting the prediction of the docking analysis. Further, BT-benzo-29 did not appear to inhibit DNA duplication and nucleoid segregation and it did not perturb the membrane potential of B. subtilis cells. The results suggested that BT-benzo-29 exerts its potent antibacterial activity by inhibiting FtsZ assembly. Interestingly, BT-benzo-29 did not affect the membrane integrity of mammalian red blood cells. BT-benzo-29 bound to tubulin with a much weaker affinity than FtsZ and exerted significantly weaker effects on mammalian cells than on the bacterial cells indicating that the compound may have a strong antibacterial potential.