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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

SB-RA-2001 did not affect the membrane potential of B.subtilis 168 cells. The fluorescence spectra of PBS (■), B. subtilis 168 cells (▲), and B. subtilis 168 cells in the presence of DMSO (○), only DiOC2 (leftward-pointing triangles), 30 μM SB-RA-2001 in DiOC2 (▼), and DiOC2 in the presence of 0.2 μMCCCP (+) are shown. Also shown are spectra of the B. subtilis 168 cells without (●) and with 20 (×) and 30 μM(◇) SB-RA-2001 and 0.2 μM CCCP (□) in the presenceof DiOC2.
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fig9: SB-RA-2001 did not affect the membrane potential of B.subtilis 168 cells. The fluorescence spectra of PBS (■), B. subtilis 168 cells (▲), and B. subtilis 168 cells in the presence of DMSO (○), only DiOC2 (leftward-pointing triangles), 30 μM SB-RA-2001 in DiOC2 (▼), and DiOC2 in the presence of 0.2 μMCCCP (+) are shown. Also shown are spectra of the B. subtilis 168 cells without (●) and with 20 (×) and 30 μM(◇) SB-RA-2001 and 0.2 μM CCCP (□) in the presenceof DiOC2.

Mentions: Vehicle-treated B.subtilis 168 cells displayed weak DiOC2 fluorescence(Figure 9). As expected, a strong increasein DiOC2 fluorescence was observed in the presence of CCCP(a positive control), indicating that CCCP treatment perturbed themembrane potential of B. subtilis 168 cells.38,39 However, 20 and 30 μM SB-RA-2001-treated B. subtilis 168 cells showed emission spectra similar to that of the vehicle-treatedcells, suggesting that SB-RA-2001 did not affect the membrane potentialof B. subtilis 168 cells (Figure 9).


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)

SB-RA-2001 did not affect the membrane potential of B.subtilis 168 cells. The fluorescence spectra of PBS (■), B. subtilis 168 cells (▲), and B. subtilis 168 cells in the presence of DMSO (○), only DiOC2 (leftward-pointing triangles), 30 μM SB-RA-2001 in DiOC2 (▼), and DiOC2 in the presence of 0.2 μMCCCP (+) are shown. Also shown are spectra of the B. subtilis 168 cells without (●) and with 20 (×) and 30 μM(◇) SB-RA-2001 and 0.2 μM CCCP (□) in the presenceof DiOC2.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: SB-RA-2001 did not affect the membrane potential of B.subtilis 168 cells. The fluorescence spectra of PBS (■), B. subtilis 168 cells (▲), and B. subtilis 168 cells in the presence of DMSO (○), only DiOC2 (leftward-pointing triangles), 30 μM SB-RA-2001 in DiOC2 (▼), and DiOC2 in the presence of 0.2 μMCCCP (+) are shown. Also shown are spectra of the B. subtilis 168 cells without (●) and with 20 (×) and 30 μM(◇) SB-RA-2001 and 0.2 μM CCCP (□) in the presenceof DiOC2.
Mentions: Vehicle-treated B.subtilis 168 cells displayed weak DiOC2 fluorescence(Figure 9). As expected, a strong increasein DiOC2 fluorescence was observed in the presence of CCCP(a positive control), indicating that CCCP treatment perturbed themembrane potential of B. subtilis 168 cells.38,39 However, 20 and 30 μM SB-RA-2001-treated B. subtilis 168 cells showed emission spectra similar to that of the vehicle-treatedcells, suggesting that SB-RA-2001 did not affect the membrane potentialof B. subtilis 168 cells (Figure 9).

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