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SB-RA-2001 inhibits bacterial proliferation by targeting FtsZ assembly.

Singh D, Bhattacharya A, Rai A, Dhaked HP, Awasthi D, Ojima I, Panda D - Biochemistry (2014)

Bottom Line: 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 B. subtilis 168 cells.The agent also did not appear to perturb the membrane potential of B. subtilis 168 cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay , Mumbai 400076, India.

ABSTRACT
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 Bacillus subtilis 168 and Mycobacterium smegmatis 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 B. subtilis 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 B. subtilis 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 B. subtilis 168 cells. The agent also did not appear to perturb the membrane potential of B. subtilis 168 cells. In vitro, 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.

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Related in: MedlinePlus

Binding interactionof SB-RA-2001 and FtsZ monitored by fluorescencespectroscopy. (A) FtsZ increased the fluorescence intensity of SB-RA-2001.The fluorescence spectra of 1 μM SB-RA-2001 in the absence ofFtsZ (+) and in the presence of 1 (◀), 2 (◇), 3 (●),4 (△), and 5 (■) μM FtsZ are shown. (B). FtsZ(2 μM) was incubated with different concentrations of SB-RA-2001for 15 min at 25 °C. The change in the fluorescence intensityof the FtsZ–SB-RA-2001 complex is plotted vs SB-RA-2001 concentration.
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fig4: Binding interactionof SB-RA-2001 and FtsZ monitored by fluorescencespectroscopy. (A) FtsZ increased the fluorescence intensity of SB-RA-2001.The fluorescence spectra of 1 μM SB-RA-2001 in the absence ofFtsZ (+) and in the presence of 1 (◀), 2 (◇), 3 (●),4 (△), and 5 (■) μM FtsZ are shown. (B). FtsZ(2 μM) was incubated with different concentrations of SB-RA-2001for 15 min at 25 °C. The change in the fluorescence intensityof the FtsZ–SB-RA-2001 complex is plotted vs SB-RA-2001 concentration.

Mentions: A fixed concentration of SB-RA-2001, upon incubation withdifferentconcentrations of FtsZ, showed a concentration-dependent increasein the fluorescence intensity of SB-RA-2001 (Figure 4A). The fluorescence emission maximum of SB-RA-2001 was foundto be 440 and 400 nm in the absence and presence of 5 μM FtsZ,respectively. SB-RA-2001 showed a large blue shift in its emissionmaximum upon interacting with FtsZ, and the fluorescence intensitywas also enhanced, suggesting that SB-RA-2001 binds to FtsZ.


SB-RA-2001 inhibits bacterial proliferation by targeting FtsZ assembly.

Singh D, Bhattacharya A, Rai A, Dhaked HP, Awasthi D, Ojima I, Panda D - Biochemistry (2014)

Binding interactionof SB-RA-2001 and FtsZ monitored by fluorescencespectroscopy. (A) FtsZ increased the fluorescence intensity of SB-RA-2001.The fluorescence spectra of 1 μM SB-RA-2001 in the absence ofFtsZ (+) and in the presence of 1 (◀), 2 (◇), 3 (●),4 (△), and 5 (■) μM FtsZ are shown. (B). FtsZ(2 μM) was incubated with different concentrations of SB-RA-2001for 15 min at 25 °C. The change in the fluorescence intensityof the FtsZ–SB-RA-2001 complex is plotted vs SB-RA-2001 concentration.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4020581&req=5

fig4: Binding interactionof SB-RA-2001 and FtsZ monitored by fluorescencespectroscopy. (A) FtsZ increased the fluorescence intensity of SB-RA-2001.The fluorescence spectra of 1 μM SB-RA-2001 in the absence ofFtsZ (+) and in the presence of 1 (◀), 2 (◇), 3 (●),4 (△), and 5 (■) μM FtsZ are shown. (B). FtsZ(2 μM) was incubated with different concentrations of SB-RA-2001for 15 min at 25 °C. The change in the fluorescence intensityof the FtsZ–SB-RA-2001 complex is plotted vs SB-RA-2001 concentration.
Mentions: A fixed concentration of SB-RA-2001, upon incubation withdifferentconcentrations of FtsZ, showed a concentration-dependent increasein the fluorescence intensity of SB-RA-2001 (Figure 4A). The fluorescence emission maximum of SB-RA-2001 was foundto be 440 and 400 nm in the absence and presence of 5 μM FtsZ,respectively. SB-RA-2001 showed a large blue shift in its emissionmaximum upon interacting with FtsZ, and the fluorescence intensitywas also enhanced, suggesting that SB-RA-2001 binds to FtsZ.

Bottom Line: 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 B. subtilis 168 cells.The agent also did not appear to perturb the membrane potential of B. subtilis 168 cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay , Mumbai 400076, India.

ABSTRACT
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 Bacillus subtilis 168 and Mycobacterium smegmatis 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 B. subtilis 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 B. subtilis 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 B. subtilis 168 cells. The agent also did not appear to perturb the membrane potential of B. subtilis 168 cells. In vitro, 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.

Show MeSH
Related in: MedlinePlus