The antibacterial cell division inhibitor PC190723 is an FtsZ polymer-stabilizing agent that induces filament assembly and condensation

Abstract

Cell division protein FtsZ can form single-stranded filaments with a cooperative behavior by self-switching assembly. Subsequent condensation and bending of FtsZ filaments are important for the formation and constriction of the cytokinetic ring. PC190723 is an effective bactericidal cell division inhibitor that targets FtsZ in the pathogen Staphylococcus aureus and Bacillus subtilis and does not affect Escherichia coli cells, which apparently binds to a zone equivalent to the binding site of the antitumor drug taxol in tubulin (Haydon, D. J., Stokes, N. R., Ure, R., Galbraith, G., Bennett, J. M., Brown, D. R., Baker, P. J., Barynin, V. V., Rice, D. W., Sedelnikova, S. E., Heal, J. R., Sheridan, J. M., Aiwale, S. T., Chauhan, P. K., Srivastava, A., Taneja, A., Collins, I., Errington, J., and Czaplewski, L. G. (2008) Science 312, 1673-1675). We have found that the benzamide derivative PC190723 is an FtsZ polymer-stabilizing agent. PC190723 induced nucleated assembly of Bs-FtsZ into single-stranded coiled protofilaments and polymorphic condensates, including bundles, coils, and toroids, whose formation could be modulated with different solution conditions. Under conditions for reversible assembly of Bs-FtsZ, PC190723 binding reduced the GTPase activity and induced the formation of straight bundles and ribbons, which was also observed with Sa-FtsZ but not with nonsusceptible Ec-FtsZ. The fragment 2,6-difluoro-3-methoxybenzamide also induced Bs-FtsZ bundling. We propose that polymer stabilization by PC190723 suppresses in vivo FtsZ polymer dynamics and bacterial division. The biochemical action of PC190723 on FtsZ parallels that of the microtubule-stabilizing agent taxol on the eukaryotic structural homologue tubulin. Both taxol and PC190723 stabilize polymers against disassembly by preferential binding to each assembled protein. It is yet to be investigated whether both ligands target structurally related assembly switches.

FIGURE 1.

Assembly of bundles of Bs-FtsZ (10 μm) induced by PC (20 μm). A, light scattering time courses with and without PC in Hepes50 assembly buffer, 10 mm MgCl₂, pH 6.8, 25 °C. GTP (2 mm) was added at time 0 and 2 mm GDP as indicated by the arrows. B, electron micrograph of Bs-FtsZ with PC. Bar, 200 nm. C, an enlarged bundle. D, its computed diffractogram. The corresponding spacing of the main equatorial spot and the first layer line are indicated in nm. E, Bs-FtsZ polymer pelleting assays at varying pH and ionic strength. F, Bs-FtsZ without PC, magnified as in B.

FIGURE 2.

PC and magnesium-induced Bs-FtsZ assemblies. Electron micrograph of negatively stained polymers formed by Bs-FtsZ (10 μm) in Hepes250 assembly buffer with 0.1 mm GMPCPP (A), 0.1 mm GMPCPP and 20 μm PC (B), 0.1 mm GMPCPP and 10 mm MgCl₂ (C), and 0.1 mm GMPCPP and 10 mm MgCl₂ and 20 μm PC (D). The bar indicates 200 nm.

FIGURE 3.

Bs-FtsZ filament assembly induced by PC. A, solid line, light scattering changes induced on Bs-FtsZ (10 μm) with PC (20 μm) by additions of GMPCPP (25 μm) and MgCl₂ (10 mm) (arrows) in Hepes250, pH 6.8, 25 °C. Dashed line, same without PC. B, example of sedimentation of Bs-FtsZ polymers formed without Mg²⁺ (top) and pellet quantification (bottom). Filled circles, PC and GMPCPP (0.1 mm); filled squares, PC and GTP (2 mm); open symbols, without PC; triangle, PC and GDP; inverted triangle, PC and GMPCP. C, cryoelectron micrograph of Bs-FtsZ filaments assembled with PC and GTP under the same conditions without Mg²⁺. Inset, negatively stained filaments. Bar, 200 nm. D, cryoelectron microscopy filament width distribution, indicating that the filaments are one Bs-FtsZ molecule-wide.

FIGURE 4.

Binding of PC to Bs-FtsZ polymers. A, Bs-FtsZ (10 μm) sedimentation assays in Hepes250, pH 6.8, 25 °C, and quantification of polymer with increasing concentrations of PC. Open circles, with 2 mm GTP; filled circles, with 2 mm GTP and Mg²⁺; void squares, with 0.1 mm GMPCPP. Triangles, corresponding data with GDP, GDP, and Mg²⁺ or GMPCP. B, HPLC analysis of PC (10 μm total) co-sedimentation with Bs-FtsZ polymers. Solid lines and arrow, with GTP and Mg²⁺; dashed gray lines and arrow, control with GDP and Mg²⁺. Taxol is an internal standard here. In this experiment, the pelleted PC and FtsZ were 4.8 and 10 μm, respectively. mAU, milliabsorbance units.

FIGURE 5.

Bs-FtsZ polymers induced by PC fragments in Mes50 assembly buffer with 10 mm MgCl₂ and 2 mm GTP. A, 1 mm fragment of DFMBA. B, 1 mm fragment of analogue CTPM (similar to control without fragment, as in Fig. 1F). Bar, 200 nm. C, chemical structure of PC and low speed FtsZ polymers pelleting (15,000 × g, 20 min) under the same conditions (PC, 20 μm). DFMBA also induced a FtsZ light scattering increase that was not observed with CTPM. The Cr for assembly in Hepes50 was reduced from ∼5 μm to ∼1 μm FtsZ with DFMBA (4 mm).

FIGURE 6.

Effects of PC on the GTPase activity of FtsZ (10 μm), with 1 mm GTP and 10 mm MgCl_2, pH 6.8 at 25 °C. Bs-FtsZ: solid circles, Hepes50; triangles, Hepes250; squares, Mes50. Ec-FtsZ: open circles, Hepes50 (average of three different protein preparations). The lines are drawn solely to show the trend of the data. The increase in GTPase activity of Ec-FtsZ was confirmed by measurements at varying protein concentrations with excess 15 μm PC (not shown).

FIGURE 7.

Polymorphic assemblies of Bs-FtsZ, in addition to bundles and ribbons (see Fig. 1B). A, filaments observed in Hepes50, pH 6.8, with GTP and PC. B, toroids in another field of the same sample. C, helical bundles in Hepes50 at pH 7.5 with GTP, 10 mm MgCl₂, and PC. D, coiled filaments and toroids formed without PC or dimethyl sulfoxide in Mes50 with MgCl₂ and GMPCPP. Similar polymers were observed in Hepes50, pH 6.8, or with GTP. Bar, 200 nm.

FIGURE 8.

Effects of PC on the polymerization of FtsZ (10 μm) from susceptible (B. subtilis and S. aureus) and resistant (E. coli) bacteria. A, FtsZ polymer versus PC concentration. Circles, Bs-FtsZ; squares, Sa-FtsZ; triangles, Ec-FtsZ. The gray symbols are corresponding controls without magnesium. The conditions were Hepes50 buffer with 10 mm MgCl₂ and 1 mm GTP, pH 6.8, 25 °C. A GTP regenerating system (1 unit/ml acetate kinase and 15 mm acetyl phosphate) was added for the measurements in A. This was required to reproducibly pellet the Ec-FtsZ polymers in the experiment time and did not modify the pelleting of Bs-FtsZ and Sa-FtsZ. B–D, electron micrographs of Sa-FtsZ with GTP (B), with GTP and 20 μm PC in two fields from the same sample (C) and with 0.1 mm GMPCPP and 20 μm PC (D, upper panel displayed with reduced contrast). E, Ec-FtsZ filaments with GTP and 20 μm PC. Controls without PC gave similar images. Bar, 200 nm.