8 research outputs found
ZipA induced bundling of FtsZ and co-polymerized with FtsZ.
<p>FtsZ (2 µM) was polymerized in 25 mM PIPES buffer, pH 6.8 containing 1 mM MgCl<sub>2</sub> and 1 mM GTP at 37°C in the absence and presence of ZipA. FtsZ polymers were observed using a fluorescence microscope and a differential interference contrast microscope. FtsZ was polymerized in the absence (i), and presence of 0.5 (ii), 1.0 (iii) and 2.0 (iv) µM FITC-ZipA (Panel A). 0.5 (i), 1.0 (ii) and 2.0 (iii) µM of FITC-ZipA in the absence of FtsZ are shown (Panel B). FITC-FtsZ was polymerized in the absence (i), and presence of 0.5 (ii), 1.0 (iii) and 2.0 (iv) µM ZipA (Panel C), respectively. Scale bar is 10 µm.</p
Effects of ZipA on the assembly and bundling of FtsZ at different pHs.
<p>FtsZ (6 µM) was polymerized in 50 mM PIPES buffer containing 1 mM MgCl<sub>2</sub> and 1 mM GTP at 37°C in the absence (i, ii, iii) and presence (iv, v, vi) of ZipA (2 µM) at pHs of 6.0 (Panel A), 6.8 (Panel B) and 8.0 (Panel C). ZipA formed aggregates at different pHs used in this study (vii, viii, ix). Scale bar is 1000 nm.</p
ZipA prevented dilution-induced disassembly of FtsZ polymers.
<p>FtsZ (25 µM) was polymerized as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028262#s2" target="_blank">materials and methods</a> section. The preformed FtsZ polymers were diluted 20 times in warm 25 mM PIPES buffer, pH 6.8 without and with different concentrations of ZipA and incubated for an additional 5 min at 37°C. The polymers were collected through centrifugation and the amount of FtsZ in the pellet was estimated using Coomassie blue stained SDS-PAGE. Lanes 1–5 denote FtsZ polymers pelleted in the absence and presence of 1, 2, 3 and 4 µM ZipA, respectively (Panel A). The relative amount of FtsZ in the pellets with respect to control was plotted against ZipA concentration (Panel B).</p
Characterization of equilibrium binding of FITC-ZipA to FtsZ.
<p>The fluorescence emission spectra of FITC-ZipA (0.5 µM) at pH 6.8 in the absence (▪) and presence of 10 (▴), 25 (▵), 50 (•), 100 (○), 200 (♦), 300 (◊), 500 (x), 600 (*), 1000 (+), 1500 (−) nM FtsZ (Panel A). Excitation wavelength used was 495 nm. The changes in the fluorescence intensities of FITC-ZipA in the presence of different concentrations of FtsZ at pHs of 6.0 (i), 6.8 (ii) and 8.0 (iii) (Panel B) and in the presence of 500 mM NaCl at pH 6.8 (Panel C) are shown.</p
Effects of ZipA on the assembly kinetics of FtsZ.
<p>FtsZ (6 µM) was polymerized in the absence (○) and presence of 2 µM (•) and 4 µM (▪) of ZipA (Panel A). FtsZ (6 µM) was polymerized in the absence of ZipA for 5 min. Then, 4 µM ZipA was added to the reaction milieu (indicated by an arrow) and the assembly kinetics of FtsZ was monitored for an additional 10 min (Panel B).</p
Bis-ANS inhibited the interaction of FtsZ and ZipA.
<p>Bis-ANS inhibited the binding of FITC-ZipA to FtsZ (Panel A). FITC-ZipA (0.5 µM) was incubated without or with different concentrations of bis-ANS for 5 min at 25°C. Then, 0.5 µM FtsZ was added to the reaction mixtures and incubated for an additional 15 min at 25°C and the fluorescence spectra were recorded. Bis-ANS inhibited the effects of ZipA on the assembly of FtsZ (Panel B). FtsZ (6 µM) was polymerized in the presence of 4 µM ZipA without or with different concentrations (5 and 10 µM) of bis-ANS. The polymeric FtsZ was collected by sedimentation and the amount of FtsZ in the pellets was estimated by coomassie-blue staining of the SDS-PAGE. The experiment was performed five times. Panel C shows electron micrographs of ZipA-induced FtsZ polymers in the absence (i) and presence of 5 µM (ii) and 10 µM (iii) bis-ANS, respectively. Scale bar is 1000 nm.</p
SB-RA-2001 Inhibits Bacterial Proliferation by Targeting FtsZ Assembly
FtsZ
has been recognized as a promising antimicrobial drug target
because of its vital role in bacterial cell division. In this work,
we found that a taxane SB-RA-2001 inhibited the proliferation of <i>Bacillus subtilis</i> 168 and <i>Mycobacterium smegmatis</i> cells with minimal inhibitory concentrations of 38 and 60 μM,
respectively. Cell lengths of these microorganisms increased remarkably
in the presence of SB-RA-2001, indicating that it inhibits bacterial
cytokinesis. SB-RA-2001 perturbed the formation of the FtsZ ring in <i>B. subtilis</i> 168 cells and also affected the localization
of the late cell division protein, DivIVA, at the midcell position.
Flow cytometric analysis of the SB-RA-2001-treated cells indicated
that the compound did not affect the duplication of DNA in <i>B. subtilis</i> 168 cells. Further, SB-RA-2001 treatment did
not affect the localization of the chromosomal partitioning protein,
Spo0J, along the two ends of the nucleoids and also had no discernible
effect on the nucleoid segregation in <i>B. subtilis</i> 168 cells. The agent also did not appear to perturb the membrane
potential of <i>B. subtilis</i> 168 cells. <i>In vitro</i>, SB-RA-2001 bound to FtsZ with modest affinity, promoted the assembly
and bundling of FtsZ protofilaments, and reduced the GTPase activity
of FtsZ. GTP did not inhibit the binding of SB-RA-2001 to FtsZ, suggesting
that it does not bind to the GTP binding site on FtsZ. A computational
analysis indicated that SB-RA-2001 binds to FtsZ in the cleft region
between the C-terminal domain and helix H7, and the binding site of
SB-RA-2001 on FtsZ resembled that of PC190723, a well-characterized
inhibitor of FtsZ. The findings collectively suggested that SB-RA-2001
inhibits bacterial proliferation by targeting the assembly dynamics
of FtsZ, and this can be exploited further to develop potent FtsZ-targeted
antimicrobials
A Carbocyclic Curcumin Inhibits Proliferation of Gram-Positive Bacteria by Targeting FtsZ
Inhibition
of FtsZ assembly has been found to stall bacterial cell
division. Here, we report the identification of a potent carbocyclic
curcumin analogue (<b>2d</b>) that inhibits <i>Bacillus
subtilis</i> 168 cell proliferation by targeting the assembly
of FtsZ. <b>2d</b> 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 <i>Staphylococcus aureus</i>. In addition, <b>2d</b> 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, <b>2d</b> was found
to rapidly perturb the cytokinetic FtsZ rings in <i>Bacillus
subtilis</i> cells. The immunofluorescence imaging of FtsZ also
showed that <b>2d</b> destroyed the Z-ring in bacteria within
5 min. Prolonged treatment with <b>2d</b> produced filamentous
bacteria, but <b>2d</b> had no detectable effect either on the
nucleoids or on the membrane potential of bacteria. <b>2d</b> inhibited FtsZ assembly <i>in vitro</i>, whereas it had
minimal effects on tubulin assembly. Interestingly, <b>2d</b> strongly enhanced the GTPase activity of FtsZ and reduced the GTPase
activity of tubulin. Furthermore, <b>2d</b> bound to purified
FtsZ with a dissociation constant of 4.0 ± 1.1 μM, and
the binding of <b>2d</b> altered the secondary structures of
FtsZ. The results together suggested that the non-natural curcumin
analogue <b>2d</b> possesses powerful antibacterial activity
against important pathogenic bacteria, and the evidence indicates
that <b>2d</b> inhibits bacterial proliferation by targeting
FtsZ