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

Low Ply expression promotes pneumococcal trafficking across BBB.

<p>Balb/c mice were infected via i.v. route with 10⁶ CFU of WT, WT:Ply-High or WT:Ply-Low SPN strain and at 14 h p.i. animals were sacrificed. PBS injected mice served as control. <b>A.</b> Confocal micrographs depicting heterogeneous Ply expression (red) within WT SPN population (blue) in the blood of infected mice. Blood smears were stained with Anti-Ply antibody and DAPI. Scale bar, 5 μm. <b>B.</b> Ratio of transcript levels of Ply in brain to blood of mice infected with WT SPN. Transcript levels were normalized to 16S rRNA and expressed as fold change compared to Ply transcript levels of WT SPN grown <i>in vitro</i> in THY medium. <b>C.</b> Kaplan-Meier survival curve of mice (n = 5 per group) following i.v. injection with different SPN strains. Experiments were repeated twice and one representative graph is shown. Statistical analysis was performed using Log-rank test. <b>D.</b> Net growth of SPN strains in the blood of infected mice (n = 5 per group) at indicated time points post infection. Experiments were repeated twice and one representative graph is shown. Statistical analysis was performed using two-way ANOVA (Bonferroni test); ns, non-significant. <b>E.</b> Quantification of bacterial counts (CFU) in various tissue homogenates and blood of mice infected with different Ply expressing SPN strains. Each dot represents one mouse; black bars show average values. n = 12 per group (2 experiments with n = 6 per group). Statistical analysis for each tissue was individually performed using non-parametric test (Mann-Whitney test). <i>p</i> values are mentioned in the graph. <b>F—H.</b> Ratio of bacterial CFU in brain to blood <b>(F)</b>, lungs to blood <b>(G)</b> and spleen to blood <b>(H)</b> of individual infected mice. Each dot represents one mouse; black bars show average values. n = 12 per group (2 experiments with n = 6 per group). Statistical analysis was performed using non-parametric test (Mann-Whitney test). <i>p</i> values are mentioned in the graph. <b>I.</b> Transcript abundance of pro-inflammatory cytokines in total RNA isolated from brains of mice that were injected with PBS or infected with different SPN strains. Transcript levels were normalized to 18S rRNA and expressed as fold change compared to control mice. Statistical analysis was performed using two-way ANOVA (Bonferroni test); ns, nonsignificant. <b>J.</b> Histopathology of H & E stained representative brain tissue samples of control (PBS injected) or mice infected with different strains of SPN. Scale bar, 100 μm.</p

Heterogeneous Ply expression is a serotype independent phenomenon.

<p><b>A.</b> Expression of Ply on pneumococcal cell surface was assessed by flow cytometry following staining with Anti-Ply Ab. Different SPN strains used for the study included; D39 (serotype 2), TIGR4 (serotype 4), Tupelo (serotype 14) and A60 (serotype 19F). Pink curve depicts unstained SPN cells while blue curve represents antibody stained SPN cells. Experiments were repeated thrice and representative histograms are shown. <b>B.</b> Percentage of Ply positive cells in different SPN serotype strains as analyzed by flow cytometry. Data is represented as mean ± SD of triplicate experiments. Statistical analysis was performed using one-way ANOVA (Tukey’s Multiple comparison test). ns, non-significant; **<i>p<</i>0.005.</p

Damage to endosomal membrane by Ply triggers autophagy mediated clearance of SPN in brain endothelium.

<p><b>A.</b> Intracellular survival efficiency of WT SPN, Δ<i>ply</i> mutant and Δ<i>ply</i>:pPly strains of R6 (serotype 2) in hBMECs were calculated as percent survival at indicated time points relative to 0 h. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using one-way ANOVA (Tukey’s multiple comparison test). *<i>p</i><0.05; ***<i>p</i><0.001. <b>B.</b> Confocal micrographs showing association of Gal8 with GFP expressing WT and Δ<i>ply</i> SPN strains at 3 h p.i. in hBMECs. Arrowhead designates bacteria shown in insets. Event localized at z-stack no. 6 and 5 out of 10 and 11 for WT and Δ<i>ply</i>, respectively. Scale bar, 5 μm. <b>C.</b> Percent co-localization of Gal8 with WT and Δ<i>ply</i> SPN strains at indicated time points post-infection. n ≥ 100 bacteria per coverslip. Data are presented as mean ± SD of triplicate hBMEC cultures. <b>D.</b> Confocal micrograph showing association of WT SPN (blue) with Gal8 (red), NDP52 (pink) and LC3 (green) at 3 h p.i.. hBMECs stably expressing GFP-LC3 were infected with SPN and stained with anti-Gal8 Ab, anti-NDP52 Ab and DAPI. Arrowhead designates bacteria shown in insets. Event localized at z-stack no. 4 out of 9. Scale bar, 5 μm. <b>E.</b> Fluorescent line scan across the yellow line in the merged inset in “<b>D</b>”, depicting co-localization of SPN with Gal8, NDP52 and LC3. <b>F.</b> Quantification of co-localization of LC3 with Gal8 positive SPN in hBMECs at 3 h p.i. Data are presented as mean ± SD of triplicate experiments. n ≥ 50 bacteria per coverslip. <b>G.</b> Western blot demonstrating knock-down in expression of Gal8 in hBMEC cultures following transfection with a targeted siRNA against <i>LGALS8</i> (siGal8). GAPDH served as loading control. Relative ratio of Gal8 to GAPDH band intensities (normalized to siControl) is mentioned on top of the blot. <b>H.</b> Quantification of LC3 co-localization with WT SPN under Gal8 knockdown conditions using siGal8. Cells transfected with siScrambled served as control (siControl). n ≥ 100 bacteria per coverslip. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using Students t-test. ***<i>p<</i>0.001. <b>I.</b> Intracellular survival of WT SPN in hBMECs following transfection with siGal8 or siControl at indicated time points relative to 0 h. Data are presented as mean ± SD of triplicate hBMEC cultures. Statistical analysis was performed using two-way ANOVA (Bonferroni test). ns, non-significant, *<i>p</i><0.05, **<i>p</i><0.005. <b>J–L.</b> Transmission electron micrograph depicting WT SPN <b>(J)</b> in vacuolar compartments inside hBMECs. Scale bar, 1 μm. Zoomed in view of the boxed area in “<b>J</b>” and “<b>K</b>” are shown in “<b>K</b>” (scale bar, 0.2 μm) and “<b>L</b>”, respectively. Arrow in “J” points towards SPN sequestered in a vacuole while arrows in “L” show disruptions in the PCV membrane.</p

Degradation of cytosolic SPN via autophagy–dependent or independent pathway is reliant on Ply induced ubiquitination.

<p><b>A, C.</b> Representative time-lapse montage for degradation of cytosolic SPN following escape from autophagosomes in LC3-dependent <b>(A)</b> or independent manner <b>(C)</b>. hBMECs stably expressing LC3-GFP (green) and Ubq-mStrawberry (red) were infected with DRAQ5 stained WT SPN (white). Live imaging of infected cells was performed for extended hours beginning at 6 h p.i. under a confocal microscope and time-lapse series with intervals of 60 min <b>(A)</b> or 50 min <b>(C)</b> are represented. The stills in “<b>A</b>” and “<b>C</b>” correspond to movies shown as <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007168#ppat.1007168.s010" target="_blank">S3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007168#ppat.1007168.s011" target="_blank">S4</a> Movies, respectively. Scale bar, 2 μm. <b>B, D.</b> Temporal quantification of ubiquitin, LC3 and SPN fluorescence intensities either relative to fluorescence in the cytosol (Cyt) (for LC3 and Ubq) or fluorescence signal of hBMEC nuclei (Nuc) (for SPN). Relative mean intensity for LC3 (I_PCV/I_Cyt) in case of ubiquitinated SPN displayed values > 1.0, depicting LC3-dependent killing of SPN <b>(B)</b>. Ubiquitinated SPN that is degraded in LC3 independent manner, displayed relative mean intensity value ~ 1.0 for LC3 <b>(D)</b>. In both cases relative mean intensity values for bacteria (I_SPN/I_Nuc) remains > 1.0 for substantially long period of time before finally disappearing. <b>E.</b> Percentage of ubiquitinated SPN undergone LC3-dependent or independent killing as examined by time-lapse imaging of hBMECs stably expressing GFP-LC3 and Ubq-mStrawberry following infection with DRAQ5 stained SPN. n = 41. <b>F.</b> Intracellular survival percentages of WT SPN following treatment with MG132 (10 μM) at indicated time points relative to 0 h. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using two-way ANOVA (Bonferroni test). ns, non-significant, ***<i>p</i><0.001.</p

Phenotypic heterogeneity of SPN inside brain endothelium.

<p>Confocal images of different population of WT SPN inside hBMECs at 6 h p.i. <b>(A)</b> Gal8⁺ Ubq^<b>–</b>LC3^<b>–</b>, <b>(C)</b> Gal8⁺ Ubq^<b>−</b>LC3⁺, <b>(E)</b> Gal8⁺ Ubq⁺ LC3⁺, <b>(G)</b> Gal8^<b>–</b> Ubq⁺ LC3⁺, <b>(I)</b> Gal8^<b>–</b> Ubq⁺ LC3^<b>–</b>, <b>(K)</b> Gal8^<b>–</b> Ubq^<b>−</b>LC3⁺. hBMECs stably expressing Gal8-YFP were infected with WT SPN (blue) and stained with anti-LC3 Ab (pink) and anti-Ubq Ab (red). Fluorescent line scans across the yellow lines in the merged insets in “<b>A, C, E, G, I</b> and <b>K</b>”, depicting association of SPN with Gal8, Ubq and LC3 are given in “<b>B, D, F, H, J</b> and <b>L</b>”, respectively.</p

Differential Ply expression governs the spatio-temporal location, survival and trafficking of SPN across BBB.

<p><b>A.</b> Flow cytometry of a recombinant SPN strain with <i>ply</i> gene transcriptionally fused with <i>gfp</i> to monitor variation in Ply expression among isogenic SPN population. <b>B.</b> Confocal micrographs depicting heterogeneous Ply expression within a population of <i>in vitro</i> grown SPN culture. GFP expressing SPN (<i>hlpA</i>-GFP integrated in genome) was probed with Anti-Ply Ab. Scale bar, 1 μm. <b>C—D.</b> Quantification of different population subsets of intracellular WT:Ply-High and WT:Ply-Low SPN strains at 3 h <b>(C)</b> and 9 h <b>(D)</b> p.i. for association with Gal8, Ubq and LC3. n ≥ 100 bacteria per coverslip. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using two-way ANOVA (Bonferroni test). *<i>p</i><0.05, **<i>p</i><0.005, ***<i>p</i><0.001. <b>E.</b> Intracellular location of WT:Ply-High and WT:Ply-Low SPN strains at 3 and 9 h p.i. in hBMECs. Gal8⁺ Ubq^<b>−</b>LC3^-, Gal8⁺ Ubq^<b>−</b>LC3⁺, Gal8⁺ Ubq⁺ LC3⁺ and Gal8^<b>–</b> Ubq^<b>−</b>LC3⁺ constituted vacuolar SPN population, while Gal8^<b>–</b> Ubq⁺ LC3⁺ and Gal8^<b>–</b> Ubq⁺ LC3^<b>–</b> constituted vacuolar escaped SPN population. n ≥ 100 bacteria per coverslip. Data are presented as mean ± SD of triplicate hBMEC cultures. Statistical analysis was performed using two-way ANOVA (Bonferroni test). *<i>p</i><0.05, **<i>p</i>< 0.005. <b>F.</b> Comparison of intracellular survival efficiencies between WT:Ply-High and WT:Ply-Low strains in hBMECs expressed as percent survival at indicated time points relative to 0 h. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using two-way ANOVA (Bonferroni test); *<i>p</i> <0.05, ***<i>p</i> <0.001. <b>G.</b> Comparison of egression efficiencies of WT:Ply-High and WT:Ply-Low SPN strains across confluent hBMEC monolayers at indicated time points. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using two-way ANOVA (Bonferroni test); ns, non-significant, **<i>p</i>< 0.005.</p