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, 9 (1), 20092

Structure-Guided Design of a Fluorescent Probe for the Visualization of FtsZ in Clinically Important Gram-Positive and Gram-Negative Bacterial Pathogens

Affiliations

Structure-Guided Design of a Fluorescent Probe for the Visualization of FtsZ in Clinically Important Gram-Positive and Gram-Negative Bacterial Pathogens

Edgar Ferrer-González et al. Sci Rep.

Abstract

Addressing the growing problem of antibiotic resistance requires the development of new drugs with novel antibacterial targets. FtsZ has been identified as an appealing new target for antibacterial agents. Here, we describe the structure-guided design of a new fluorescent probe (BOFP) in which a BODIPY fluorophore has been conjugated to an oxazole-benzamide FtsZ inhibitor. Crystallographic studies have enabled us to identify the optimal position for tethering the fluorophore that facilitates the high-affinity FtsZ binding of BOFP. Fluorescence anisotropy studies demonstrate that BOFP binds the FtsZ proteins from the Gram-positive pathogens Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae with Kd values of 0.6-4.6 µM. Significantly, BOFP binds the FtsZ proteins from the Gram-negative pathogens Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii with an even higher affinity (Kd = 0.2-0.8 µM). Fluorescence microscopy studies reveal that BOFP can effectively label FtsZ in all the above Gram-positive and Gram-negative pathogens. In addition, BOFP is effective at monitoring the impact of non-fluorescent inhibitors on FtsZ localization in these target pathogens. Viewed as a whole, our results highlight the utility of BOFP as a powerful tool for identifying new broad-spectrum FtsZ inhibitors and understanding their mechanisms of action.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(a) Chemical structures of the oxazole-benzamide FtsZ inhibitors 1 and 2. The methyl group in 2 that differentiates this compound from 1 is highlighted in red. 2 was prepared as a racemic mixture of R and S enantiomers. (b) Expanded view of the binding site for the R enantiomer of 2 [(R)-2] in complex with SaFtsZ, with the F0 – Fc omit map (cyan) being contoured at 3.0σ. The anomalous difference map (purple) is contoured at 4.0σ. (c) Superposition of the SaFtsZ−(R)-2 complex (orange) with the corresponding SaFtsZ−1 complex (blue). The methyl group in 2 shown in red in (a) is highlighted by the red arrows in (b,c).
Figure 2
Figure 2
(a) Scheme for the synthesis of BOFP by reacting 3 with BODIPY FL-COOH in CH2Cl2 containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP). Both 3 and BOFP were prepared as racemic mixtures of the R and S enantiomers. The tethered hydroxymethyl functionality on 3 and BODIPY functionality on BOFP are both highlighted in red. (b) Fluorescence anisotropy profiles of 0.1 µM BOFP as a function of increasing concentrations of SaFtsZ. The titration experiments were conducted at 15 °C (red), 25 °C (black), or 37 °C (cyan) in solution containing 50 mM Tris-HCl (pH 7.6) and 50 mM KCl. The solid lines reflect non-linear least squares fits of the experimental data points with Eq. 1. (c) Expanded view of the binding site for the R enantiomer of BOFP [(R)-BOFP] in complex with SaFtsZ, with the F0 – Fc omit map (cyan) being contoured at 2.0σ. (d) Superposition of the SaFtsZ−(R)-BOFP complex (green) with the corresponding SaFtsZ−1 complex (blue). (e) Hydrophobic interactions at the surface interface between the BODIPY moiety of BOFP and residues Ile228, Val230, and Val307 of SaFtsZ.
Figure 3
Figure 3
Fluorescence anisotropy profiles of 0.1 µM BOFP as a function of increasing concentrations of EfsFtsZ (a), EfmFtsZ (b), SpyFtsZ (c), SagFtsZ (d), or SpnFtsZ (e). Acquisition and display parameters are as described in the legend to Fig. 2b. Panel (f) shows plots of ln(1/Kd) vs. 1/T for the interaction of BOFP with SaFtsZ (filled circles), EfsFtsZ (filled triangles), EfmFtsZ (open triangles), SpyFtsZ (open diamonds), SagFtsZ (filled diamonds), and SpnFtsZ (open squares). The solid lines reflect linear fits of the experimental data points with Eq. 3.
Figure 4
Figure 4
FtsZ visualization in the Gram-positive bacterial pathogens S. aureus NRS705 (a,b), E. faecalis ATCC 29212 (c,d), E. faecium ATCC 19434 (e,f), S. pyogenes ATCC 19615 (g,h), S. agalactiae ATCC 12386 (i,j), and S. pneumoniae ATCC 49619 (k,l). Differential interference contrast (DIC) and fluorescence micrographs of the indicated bacterial cells treated for 5 minutes with 1 µg/mL BOFP just prior to visualization. The arrows in panels (b,d,f,h,j,l) highlight representative FtsZ Z-rings at midcell labeled by BOFP.
Figure 5
Figure 5
Fluorescence anisotropy profiles of 0.1 µM BOFP as a function of increasing concentrations of EcFtsZ (a), KpFtsZ (b), PaFtsZ (c), or AbFtsZ (d). Acquisition and display parameters are as described in the legend to Fig. 2b. Panel (e) shows plots of ln(1/Kd) vs. 1/T for the interaction of BOFP with EcFtsZ (open circles), KpFtsZ (open squares), PaFtsZ (filled diamonds), and AbFtsZ (filled circles). The solid lines reflect linear fits of the experimental data points with Eq. 3.
Figure 6
Figure 6
FtsZ visualization in the Gram-negative bacterial pathogens E. coli ATCC 25922 (a,b), K. pneumoniae ATCC 13883 (c,d), P. aeruginosa ATCC 27853 (e,f), and A. baumannii ATCC 19606 (g,h). Differential interference contrast (DIC) and fluorescence micrographs of the indicated bacterial cells treated for 5 minutes with 1 µg/mL BOFP in the presence of pentamidine isethionate (at 0.875 mg/mL for E. coli and 3.5 mg/mL for the other three strains) just prior to visualization. The arrows in panels (b,d,f,h) highlight representative FtsZ Z-rings at midcell labeled by BOFP.
Figure 7
Figure 7
Visualization of the impact of treatment with 1 on FtsZ localization in S. aureus NRS705, E. coli N43, and K. pneumoniae ATCC 10031. Differential interference contrast (DIC) and fluorescence micrographs of the indicated bacterial cells treated for 3 hours with either DMSO vehicle (af) or 1 (gl) at 4× MIC (1 µg/mL for S. aureus and 4 µg/mL for E. coli and K. pneumoniae). Just prior to visualization, cells were labeled for 5 minutes with 1 µg/mL BOFP in the absence (for S. aureus) or presence of pentamidine isethionate (at 0.875 mg/mL for E. coli and 3.5 mg/mL for K. pneumoniae). The arrows in panels (b,d,f) highlight representative FtsZ Z-rings at midcell labeled by BOFP.
Figure 8
Figure 8
Comparison of BOFP and induced expression of the FtsZ-mCherry fusion protein for visualization of the impact of treatment with 1 on FtsZ localization in the MRSA LAC FtsZ-mCherry strain. Differential interference contrast (DIC) and fluorescence micrographs of the bacterial cells treated for 3 hours with either DMSO vehicle (ad) or 1 (eh) at 4× MIC (0.25 µg/mL). Just prior to visualization, cells were labeled for 5 minutes with 1 µg/mL BOFP. The arrows in panel panels (b,c,d) highlight representative FtsZ Z-rings at midcell as visualized either by induced expression of FtsZ-mCherry or by labeling with BOFP.

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