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

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.


Results and Discussion
Structure-guided identification of a suitable site on the oxazole-benzamide FtsZ inhibitor for conjugation of a fluorophore. A clue to a potential site on the oxazole-benzamide FtsZ inhibitor 1 for conjugation of a fluorophore came from the crystal structure we determined for the complex of SaFtsZ  (the enzymatic domain of SaFtsZ, residues 12-316) with 2 13 , a methyl analog of 1 whose chemical structure is shown in Fig. 1a. The only difference between the two compounds is the presence of a methyl group (shown in red in Fig. 1a) on the linker connecting the oxazole and difluorobenzamide rings of 2 that is absent in 1. This difference makes 2 a chiral molecule, while 1 is achiral. A racemic mixture of the R and S enantiomeric forms of 2 was dissolved in DMSO and introduced into crystals of SaFtsZ 12-316 by soaking. The structure of the R enantiomer of 2 [(R)-2] in complex with SaFtsZ 12-316 was determined at 1.4 Å resolution. An extra electron density was clearly observed in the cleft between the N-and C-terminus domains, which enabled us to determine the position and orientation of (R)-2 with an occupancy of 1.0 (Fig. 1b). We have previously reported the structure of 1 in complex with SaFtsZ 12-316 21 . (R)-2 binds the same protein cleft as 1 in a similar orientation to that observed in the structure of the 1-SaFtsZ  complex (see the overlay of 1 and (R)-2 depicted in Fig. 1c). Note that no SaFtsZ complexes were observed with the S enantiomer of 2, suggesting that SaFtsZ is selective for the R over the S enantiomer.
Inspection of the crystal structure of (R)-2 in complex with SaFtsZ  reveals that the methyl group distinguishing (R)-2 from 1 does not engage in significant interactions with SaFtsZ, but rather is oriented away from the cleft of the FtsZ molecule (as highlight by the red arrows in Fig. 1b,c). This observation suggested to us that a bulky fluorescent moiety could be conjugated to the same site on the linker in 1 as the methyl group in 2 without disrupting the FtsZ binding interaction to a significant degree.   Table 1, which ranged from 0.88 ± 0.08 µM at 15 °C to 3.14 ± 0.13 µM at 37 °C. These gratifying results indicate that BOFP can bind SaFtsZ with a robust affinity in the sub-to low-micromolar K d range.
the binding of Bofp to SaftsZ does not require the presence of Gtp or magnesium. The filamentation of SaFtsZ requires the presence of both GTP and magnesium 28 . Note that neither of these reagents was present in the fluorescence anisotropy binding studies depicted in Fig. 2b, indicating that the binding of BOFP to SaFtsZ does not require the presence of GTP or magnesium. This observation markedly contrasts the fluorescence anisotropy studies previously reported by Artola et al. 27 , which demonstrated that the fluorescent analogs of the benzamide FtsZ inhibitor PC190723 required both GTP and magnesium in order to bind SaFtsZ. For comparative purposes, we sought to determine whether the presence of a non-hydrolyzable analog of GTP (GMPCPP) and magnesium exerted an impact on the binding of BOFP to SaFtsZ, as reflected by a change in fluorescence anisotropy. At identical concentrations of BOFP (0.1 µM) and SaFtsZ (10 µM), the presence of neither GMPCPP alone (at 0.1 mM) nor MgCl 2 alone (at 10 mM) has a significant effect on the anisotropy of SaFtsZ-bound BOFP (Fig. S2), confirming that the binding of BOFP to SaFtsZ is independent of either GTP or magnesium. The presence of both GMPCPP and MgCl 2 results in a modest increase in the anisotropy of bound BOFP (Fig. S2), which likely reflects the filamentation of BOFP-bound SaFtsZ induced by the combination of GMPCPP and magnesium.
BOFP binds in the same cleft of SaFtsZ as 1 and 2. We next sought to determine the crystal structure of BOFP in complex with SaFtsZ  . Toward this end, a racemic mixture of the R and S enantiomeric forms of BOFP in DMSO was introduced into crystals of SaFtsZ 12-316 by soaking. The structure of the R enantiomer of BOFP [(R)-BOFP] in complex with SaFtsZ 12-316 was determined at 1.6 Å resolution. Although the electron density of (R)-BOFP was not quite as robust as that of (R)-2 due to a lower occupancy of 0.6, we were able to confirm that (R)-BOFP does indeed bind in the same cleft (compare Figs. 1b and 2c). The conjugated BODIPY extends toward the outside of the FtsZ molecule, away from the binding cleft. As hypothesized, interactions between the 1 portion of (R)-BOFP and SaFtsZ are similar to those exhibited by 1 (see the overlay of 1 and (R)-BOFP depicted in Fig. 2d). In addition to these interactions, the complex of (R)-BOFP with SaFtsZ is further stabilized by hydrophobic interactions between the BODIPY moiety and the hydrophobic surface formed by residues Ile228, Val230, and Val307 (Fig. 2e), with these interactions being unlikely to hamper the ability of SaFtsZ to self-associate into filaments (Fig. S3).
As seen with 2, no SaFtsZ complexes were observed with the S enantiomer of BOFP, further suggestive of the selectivity of SaFtsZ for the R versus the S enantiomeric form. Previous studies by Stokes et al. have shown that the R enantiomeric form of 3 has significantly greater activity against methicillin-sensitive S. aureus (MSSA) than the S enantiomeric form 13 . This enhanced antistaphylococcal activity of the R enantiomer likely reflects the corresponding selectivity of SaFtsZ for the R enantiomeric form.
Note that the FtsZ targeting of BOFP confers the compound with antistaphylococcal activity, though this activity is somewhat reduced relative to the parent compounds 1 and 3 (MIC versus MRSA NRS705 = 0.25, 0.5, and 1.0 µg/mL for 1, 3, and BOFP, respectively). The reduced activities of both 3 and BOFP relative to 1 may be due in part to 3 and BOFP being racemic mixtures of active R and weakly active S enantiomers. In the aggregate, our collective fluorescence anisotropy, crystallographic, and antibacterial results for BOFP serve to validate our structure-guided design approach. www.nature.com/scientificreports www.nature.com/scientificreports/ Bofp can target the ftsZ proteins from a broad range of clinically important Gram-positive bacterial pathogens, including enterococcal and streptococcal species. In addition to SaFtsZ, we also sought to determine whether BOFP can target the FtsZ proteins from other Gram-positive bacterial pathogens, including E. faecalis (EfsFtsZ) E. faecium (EfmFtsZ), S. pyogenes (SpyFtsZ), S. agalactiae (SagFtsZ), and S. pneumoniae (SpnFtsZ). Fluorescence anisotropy studies conducted at 15, 25, and 37 °C reveal that addition of each of the five target FtsZ proteins increases the anisotropy of BOFP significantly (Fig. 3a-e), indicative of a binding interaction between the probe and each of the host proteins. Significantly, no such binding interactions were observable with BODIPY FL-COOH (as exemplified by the SpyFtsZ results shown in Fig. S1). Thus, BOFP can target not only SaFtsZ, but also the FtsZ proteins from a broad range of other clinically important Gram-positive pathogens.
Analysis of the anisotropy isotherms in Fig. 3 with Eq. 1 yielded outstanding fits of the experimental data points (as depicted by the solid curves), with the K d values derived from these fits being listed in Table 1 Fig. 4b,d,f,h,j,l), consistent with the labeling of FtsZ Z-rings. Additional fluorescence staining (though weaker than that at midcell) is also evident along the periphery of each cell, suggesting that FtsZ is also localized throughout the cell membrane. These results indicate that brief exposure to a low concentration of BOFP affords outstanding visualization of FtsZ and its localization patterns in live Gram-positive bacterial cells.
To ensure that the ester linkage connecting the BODIPY fluorophore in the BOFP conjugate was not being hydrolyzed by the bacterial cells, we examined the potential, if any, of BODIPY FL-COOH to label FtsZ in S. aureus cells. Significantly, no fluorescence staining of the S. aureus cells was detectable upon treatment with 1 µg/  www.nature.com/scientificreports www.nature.com/scientificreports/ mL BODIPY FL-COOH for 5 minutes (Fig. S4). This behavior markedly contrasts the clear staining patterns observed when the cells are treated similarly with BOFP (Fig. S4). These results confirm that BOFP is not hydrolyzed by the cells during the treatment regimen and that the staining patterns observed were not the result of nonspecific interactions with a fluorescent product of hydrolysis.
BOFP targets FtsZ proteins from Gram-negative bacterial pathogens with an even higher affinity than ftsZ proteins from Gram-positive pathogens. In addition to targeting Gram-positive FtsZ proteins, we sought to determine whether BOFP could also target Gram-negative FtsZ proteins. To this end, we used fluorescence anisotropy to explore the interactions of BOFP with the FtsZ proteins from the four Gram-negative pathogens, E. coli (EcFtsZ), K. pneumoniae (KpFtsZ), P. aeruginosa (PaFtsZ), and A. baumannii (AbFtsZ), with the resulting anisotropy profiles acquired at 15, 25, and 37 °C being depicted in Fig. 5a-d. Inspection and analysis of these anisotropy profiles reveals that BOFP binds to all four Gram-negative FtsZ proteins with sub-micromolar affinity. At 25 °C, the K d values for EcFtsZ, KpFtsZ, PaFtsZ, and AbFtsZ are 0.28 ± 0.02, 0.58 ± 0.04, 0.36 ± 0.06, and 0.55 ± 0.02 µM, respectively (Table 1). A comparison of the K d values at 25 °C listed in Table 1 indicates that BOFP binds the Gram-negative FtsZ proteins with an approximately 2-to 12-fold higher affinity than the Gram-positive FtsZ proteins. Thus, in striking contrast to the early-generation fluorescent FtsZ inhibitors reported by Artola et al. 27 , BOFP can target a broad range of Gram-negative FtsZ proteins with a high degree of affinity. As observed with the Gram-positive FtsZ proteins, no binding interactions with BODIPY FL-COOH were detectable with the Gram-negative FtsZ proteins (as exemplified by the KpFtsZ and PaFtsZ results shown in Fig. S1).
For the majority of target FtsZ proteins studied, enthalpy provides a significant driving force for the binding of Bofp. We used the temperature dependence of the K d values for the binding of BOFP to the Gram-positive and Gram-negative FtsZ proteins to derive the thermodynamic parameters associated with the binding reactions. Free energy changes (ΔG) at 37 °C (310 K) were derived from the corresponding K d values using Eq. 2, while enthalpy and entropy changes (ΔH and ΔS, respectively) were determined from linear fits of the ln(1/K d ) vs. 1/T plots shown in Figs. 3f and 5e with Eq. 3. The resulting thermodynamic parameters are listed in Table 1. For seven of the ten FtsZ proteins studied (SaFtsZ, EfsFtsZ, SpyFtsZ, SagFtsZ, EcFtsZ, KpFtsZ, and PaFtsZ), ΔH contributes >50% to the observed ΔG of binding, with the enthalpic contribution to binding being 100% for two of those seven FtsZ proteins (SaFtsZ and EfsFtsZ). As suggested by our crystal structure of BOFP in complex with SaFtsZ, these favorable enthalpic contributions to binding likely stem from the extensive array of favorable van der Waals contacts between the host protein and both the 1 and BODIPY portions of the probe (Fig. 2c-e). For the remaining three FtsZ proteins (EfmFtsZ, SpnFtsZ, and AbFtsZ), ΔS contributes >50% to the observed ΔG of binding. These favorable entropic contributions to binding may reflect favorable binding-induced changes in hydration and/or conformation of the host proteins.

In addition to Gram-positive bacterial cells, BOFP also labels FtsZ effectively in live Gram-negative cells of E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii. Armed with knowledge that
BOFP binds to the Gram-negative FtsZ proteins EcFtsZ, KpFtsZ, PaFtsZ, and AbFtsZ with an even higher affinity than any of the Gram-positive FtsZ proteins, we further explored the potential of the probe to label FtsZ in live Gram-negative bacterial cells. When exposed to BOFP in a similar manner (1 µg/mL for 5 minutes) to that described above for the Gram-positive bacteria, little or no FtsZ labeling in the Gram-negative cells was observable by fluorescence microscopy (as exemplified by the K. pneumoniae results shown in Fig. S5b). Previous studies have indicated that the large size of fluorescent antibiotics resulting from the conjugation of bulky fluorophores restricts the passage of the agents across the outer membrane of Gram-negative cells 29 . Stokes et al. have shown that pentamidine can effectively permeabilize the outer membrane of Gram-negative bacterial cells to large antibiotics that would normally be unable to cross the membrane 30 . When co-treating K. pneumoniae cells with 1 µg/mL BOFP and 3.5 mg/mL pentamidine isethionate for 5 minutes, bright fluorescence staining becomes visible both at midcell and along the cell periphery (compare Fig. S5b,d). Co-treatment of E. coli, P. aeruginosa, and A. baumannii cells results in a similar fluorescence staining pattern to that observed in K. pneumoniae cells (Fig. 6b,d,f,h). The fluorescent bands visible at midcell are consistent with the labeling of FtsZ Z-rings (highlighted by the arrows in Figs. S5d and 6b,d,f,h), with the peripheral staining reflecting the presence of FtsZ in the cell membrane as well. Note that no such FtsZ staining was detectable in K. pneumoniae cells co-treated for 5 minutes with 1 µg/mL BODIPY FL-COOH and 3.5 mg/mL pentamidine isethionate (Fig. S6), thus confirming that BOFP is not being hydrolyzed by the Gram-negative cells during the labeling procedure. Viewed as a whole, our results indicate that BOFP is useful for visualizing FtsZ not only in live Gram-positive cells, but also in live Gram-negative cells.

BOFP can also be used to visualize the impact of non-fluorescent FtsZ inhibitors on the localization of ftsZ in both Gram-positive and Gram-negative bacterial cells.
For BOFP to be useful as a tool for identifying new FtsZ inhibitors in a live cell-based assay, it must facilitate the detection of changes in FtsZ localization induced by non-fluorescent test compounds with the potential for FtsZ inhibition. Toward this end, we tested the ability of BOFP to visualize the impact of the oxazole-benzamide FtsZ inhibitor 1 on FtsZ localization in live S. aureus, E. coli, and K. pneumoniae cells, with the results being shown in Fig. 7. As expected with a known FtsZ inhibitor, treatment with 1 (at 4x MIC for 3 hours) induces a significant change in cell morphology consistent with the impairment of cell division (compare Fig. 7a,c,e with Fig. 7g,i,k). This morphological change takes the form of cell enlargement in cocci like S. aureus and filamentation in rods like E. coli and K. pneumoniae. Significantly, BOFP effectively labels FtsZ in the cells treated with 1, showing clear mislocalization of FtsZ and an absence of Z-rings in any of the treated cells (Fig. 7h,j,l). In addition, the presence of FtsZ in the membranes of cells treated with 1 is reduced relative to that in the membranes of untreated cells. In S. aureus, we observed similar results for cells treated with the benzamide FtsZ inhibitors PC190723 and TXA707 (Fig. S7).   compound synthesis. 1, 2, 3, and TXA707, were synthesized as previously described [11][12][13]31 . BOFP was synthesized as detailed in the Supplementary Information.
Crystallization and structure determination of SaFtsZ in complex with 2 and BOFP.  was cloned, expressed, and purified as described previously 21  www.nature.com/scientificreports www.nature.com/scientificreports/ (pH 7.8), 45% (w/v) pentaerythritol propoxylate 629 (PEP629), and 300 mM KCl. After 3 weeks, the crystal was soaked in the same reservoir supplemented with 5 mM 2 for 3 days. For the BOFP complex, the protein was crystallized at 20 mg/mL under conditions of 100 mM HEPES (pH 8.0) and 39% (w/v) PEP629. After 7 days, the crystal was soaked in the same reservoir supplemented with 1.4 mM BOFP for 3 days.
The crystal of the 2-SaFtsZ complex was flash-frozen in a nitrogen gas stream at −180 °C without cryoprotectants. The crystal of the BOFP-SaFtsZ complex was briefly soaked in a cryoprotectant solution containing 100 mM HEPES (pH 8.0), 39% (w/v) PEP629, and 25% (v/v) glycerol and then flash-frozen in the same manner as described above. X-ray diffraction data from the crystals of the 2-SaFtsZ and BOFP-SaFtsZ complexes were collected at SPring-8 BL44XU (Hyogo, Japan) under a cryogenic nitrogen gas stream at 100 K. Diffraction data were processed and scaled with HKL2000 32 and XDS 33 , respectively. The phases for both complexes were determined by molecular replacement with Phaser in the CCP4 suite 34 using the previously determined structure of the SaFtsZ-GDP complex (PDB entry: 3VOA) 35 as a search model. Both models were refined with REFMAC5 36 and PHENIX 37 , with manual modification using Coot 38 . The refined structures were validated with MolProbity 39 . Data collection and refinement statistics are summarized in Table 2. The final atomic coordinates and structure factor amplitudes have been deposited in the RCSB Protein Data Bank (PDB entries: 6KVP and 6KVQ). Figures were prepared with PyMOL (Schrödinger). fluorescence anisotropy assays for the binding of Bofp to ftsZ proteins. Fluorescence anisotropy experiments were performed using an AVIV model ATF105 spectrofluorometer at 15, 25, or 37 °C. In these experiments, bandwidths were set to 4 nm in both the excitation and emission directions, with the excitation and emission wavelengths being set at 488 nm and 510 nm, respectively. BOFP (0.1 µM) was titrated with increasing concentrations (ranging from 0 to 12 µM) of FtsZ in 120 µL of buffer containing 50 mM Tris-HCl (pH 7.6) and 50 mM KCl. After each protein addition, the samples were equilibrated for 3 minutes, whereupon the fluorescence anisotropy was measured.
Plots of the fluorescence anisotropy (r) of BOFP as a function of FtsZ concentration (as shown in Figs. 2, 3, and 5) were analyzed by non-linear least squares regression using the following 1:1 binding formalism: In this equation, r 0 is the anisotropy of the protein-free compound, r ∞ is the anisotropy of the compound in the presence of an infinite concentration of FtsZ, [C] tot is the total concentration of the compound, and [P] tot is the total concentration of protein with each addition. These analyses yielded the equilibrium dissociation constant (K d ) for each binding reaction.
The binding free energy (ΔG) at temperature T was derived from the corresponding K d value determined at T using the following relationship: The binding enthalpy (ΔH) and entropy (ΔS) were derived from linear fits of the ln(1/K d ) vs. 1/T plots shown in Figs. 3f and 5e using the following relationship: The impact of guanosine nucleotide and magnesium on the binding of BOFP to SaFtsZ was assessed at 37 °C in the same buffer described above. In these studies, the anisotropy of 0.1 µM BOFP alone or in the presence of 10 µM SaFtsZ was measured, with the latter also being measured in the presence of 0.1 mM GMPCPP (a non-hydrolyzable GTP analog), 10 mM MgCl 2 , or both.
A quartz ultra-micro cell (Hellma) with a 2 × 5 mm aperture and a 15 mm center height was used for all measurements. The pathlengths in the excitation and emissions directions were 1 and 0.2 cm, respectively. All steady-state anisotropy experiments were conducted in at least triplicate, with the reported anisotropies reflecting the average values.
Minimum inhibitory concentration (MIC) Assays. MIC assays of 1, 3, and BOFP were conducted by standard broth microdilution in TH media. Briefly, log-phase S. aureus NRS705 (MRSA) cells were added to 96-well microtiter plates (at 5 × 10 5 CFU/mL) containing 2-fold serial dilutions of each test compound in 0.1 mL of TH broth, with each compound concentration being present in duplicate. The MIC is defined as the lowest compound concentration at which growth is ≥90% inhibited after 18-24 hours of aerobic growth.
For visualizing FtsZ in Gram-positive bacteria using BOFP, the bacterial cells were grown to log-phase in media suitable for each individual pathogen. Specifically, S. aureus NRS705 (MRSA) was grown in tryptic soy broth (TSB), E. faecalis ATCC 29212 and E. faecium ATCC 19434 were grown in lactobacilli MRS broth, S. agalactiae ATCC 12386 and S. pneumoniae ATCC 49619 were grown in TH broth, and S. pyogenes ATCC 19615 was grown in CAMH broth supplemented with 3% (v/v) LHB. For each Gram-positive bacterial strain, a total of 1 mL of cell culture was centrifuged at 15,000 × g for 1 minute and washed 2-3 times with 1 mL of PBS. After the final wash, the pelleted cells were resuspended in 500 µL of PBS containing 1 µg/mL of BOFP and incubated in the dark for 5 minutes at room temperature. The cells were then centrifuged at 15,000 × g for 1 minute, washed twice with 1 mL of PBS, and subsequently resuspended in 200 µL of PBS. 8 µL of this final cell suspension was then spread on a 0.25 mm layer of 1.5% high-resolution agarose (Sigma) in PBS, which was mounted on a standard 75 × 25 × 1 mm microscope slide (Azer Scientific) using a 1.7 × 2.8 × 0.025 cm Gene Frame (ThermoFisher). A 24 × 40 mm cover slip (Azer Scientific) was then applied to the agarose pad to prepare the slide for microscopic visualization. Comparative control experiments with 1 µg/mL BODIPY FL-COOH were conducted in S. aureus NRS705 cells as described above for BOFP.
For visualizing FtsZ in Gram-negative bacteria using BOFP, E. coli ATCC 25922, K. pneumoniae ATCC 13883, P. aeruginosa ATCC 27853, and A. baumannii ATCC 19606 were grown to log-phase in CAMH broth. For each Gram-negative bacterial strain, a total of 1 mL of cell culture was centrifuged at 15,000 × g for 1 minute and washed twice with 1 mL of Tris-buffered saline (TBS) composed of 50 mM Tris-HCl (pH 7.6) and 150 mM NaCl. After the final wash, the pelleted cells were resuspended in 500 µL of TBS containing 1 µg/mL of BOFP and  www.nature.com/scientificreports www.nature.com/scientificreports/ pentamidine isethionate (at 0.875 mg/mL for E. coli and 3.5 mg/mL for the other three strains). The resuspended cells were then incubated in the dark for 5 minutes at room temperature, centrifuged at 15,000 × g for 1 minute, washed twice with 1 mL of TBS, and subsequently resuspended in 200 µL of TBS. This final cell suspension was then prepared for microscopy as described for the Gram-positive bacterial strains. Comparative control experiments with 1 µg/mL BODIPY FL-COOH were conducted in K. pneumoniae ATCC 13883 cells as described above for BOFP.
To visualize the impact of treatment with the FtsZ inhibitor 1 using BOFP, S. aureus NRS705 (MRSA), E. coli N43, and K. pneumoniae ATCC 10031 were grown to log-phase in CAMH broth and diluted to an OD 600 of 0.1. Each cell culture was then treated with either DMSO vehicle or 1 at 4× MIC (1 µg/mL for S. aureus or 4 µg/mL for E. coli and K. pneumoniae) for 3 hours at 37 °C. Following this treatment, 1-5 mL of each culture was centrifuged at 15,000 × g for 1 minute and washed twice with 1 mL of PBS (for S. aureus) or TBS (for E. coli and K. pneumoniae). The resulting S. aureus cell pellets were further processed and labeled with BOFP as described above for the Gram-positive bacterial strains and the resulting E. coli and K. pneumoniae cell pellets were further processed and labeled with BOFP as described above for the Gram-negative bacterial strains. The impact of treating S. aureus NRS705 cells with the FtsZ inhibitors PC190723 and TXA707 at 4× MIC (2 µg/mL for PC190723 and 4 µg/mL for TXA707) was also examined using BOFP as described above.
To visualize the impact of treating MRSA LAC FtsZ-mCherry with 1, cells were grown to log-phase in CAMH broth and diluted to an OD 600 of 0.1. The cells were then treated with 10 µM IPTG and either DMSO vehicle or 1 at 4× MIC (0.25 µg/mL) for 3 hours at 37 °C. Following this treatment, 1-5 mL of each culture was centrifuged at 15,000 × g for 1 minute and washed twice with 1 mL of PBS. The resulting cell pellets were further processed and labeled with BOFP as described above for the Gram-positive bacterial strains.