Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins

Summary

Fission yeast is used here as a heterologous host to express bacterial cytoskeletal proteins such as FtsZ and MreB as translational fusion proteins with GFP to visualize their polymerization. Also, compounds that affect polymerization are identified by imaging using a fluorescence microscope.

Abstract

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB have emerged as important targets for novel antimicrobial discovery. Several assays have been developed to identify compounds targeting nucleotide binding and polymerization of these cytoskeletal proteins, primarily focused on FtsZ. Moreover, many of the assays are either laborious or cost-intensive, and ascertaining whether these proteins are the cellular target of the drug often requires multiple methods. Finally, the toxicity of the drugs to eukaryotic cells also poses a problem. Here, we describe a single-step cell-based assay to discover novel molecules targeting bacterial cytoskeleton and minimize hits that might be potentially toxic to eukaryotic cells. Fission yeast is amenable to high-throughput screens based on microscopy, and a visual screen can easily identify any molecule that alters the polymerization of FtsZ or MreB. Our assay utilizes the standard 96-well plate and relies on the ability of the bacterial cytoskeletal proteins to polymerize in a eukaryotic cell such as the fission yeast. While the protocols described here are for fission yeast and utilize FtsZ from Staphylococcus aureus and MreB from Escherichia coli, they are easily adaptable to other bacterial cytoskeletal proteins that readily assemble into polymers in any eukaryotic expression hosts. The method described here should help facilitate further discovery of novel antimicrobials targeting bacterial cytoskeletal proteins.

Introduction

The widespread resistance to nearly all antibiotics presently employed to combat bacterial infections has created an immediate necessity for novel categories of antibiotics. A 2019 report indicated that antibiotic-resistant infections resulted in the loss of 1.27 million lives, contributing to an overall tally of 4.95 million deaths when considering complications from resistant bacterial infections¹. While still effective in clinical practice, the current arsenal of antibiotics predominantly targets a narrow spectrum of cellular processes, primarily focusing on cell wall, DNA, and protein synthesis. Over the past half-century, fewer than 30 proteins have been commercially exploited as targets for the development of new anti-bacterials^2,3. This limited range of viable targets significantly creates constraints to discovering new antibiotics or their derivatives for combating antibiotic-resistant bacteria. Thus, to overcome the emerging antibiotic resistance problem, there is a need for the development of new antibiotics with novel targets and mechanisms of action.

An antibacterial target should ideally be an essential component of bacterial cell growth, be conserved throughout the phylogenetically diverse species, show least eukaryotic homology, and be accessible to antibiotics⁴. Since the discovery of bacterial cytoskeletal proteins involved in cell division and cell shape maintenance, they have emerged as a promising focal point for developing antibacterial compounds⁵. These proteins are essential for bacterial viability and play a pivotal role in maintaining cell shape (MreB, CreS), division (FtsZ, FtsA), and DNA segregation (ParM, TubZ, PhuZ, AlfA), akin to the cytoskeleton in eukaryotic cells. Notably, FtsZ exhibits a remarkably high level of conservation across a wide range of prokaryotic organisms, while MreB is found in nearly all rod-shaped bacteria. Such wide distribution and relevance in cell viability make these proteins a fascinating target in antibiotic research^5,6,7.

It is crucial to adopt a multi-pronged approach that combines in vivo observations, in vitro interactions, and enzymatic experiments to thoroughly validate bacterial cytoskeleton proteins as the primary target of a potential inhibitor⁷. Laborious procedures or substantial cost implications encumber many available assays for this purpose. These are notable obstacles to their widespread utilization in screening lead compounds that could impact the bacterial cytoskeleton. Among these, microscopy stands out as an exceptionally efficient and rapid method for assessing the effectiveness of compounds by directly examining changes in cell morphology. Yet, the hetero-association of target protein with other cytoskeleton complexes, indirect effects due to off-target binding and changes in membrane potential, difficulty in penetrating the cell efficiently, and presence of efflux pumps, especially in Gram-negative bacteria, make it collectively complex to pinpoint the precise cause of bacterial cell deformation^8,9,10.

Schizosaccharomyces pombe, or fission yeast as it's commonly known, is a rod-shaped unicellular eukaryotic organism. Fission yeast is widely used as a model organism in cellular and molecular biology due to the extraordinary conservation in cellular processes such as the cell cycle and division, cellular organization, and chromosome replication with higher eukaryotes, including humans^11,12. Furthermore, Errington and colleagues expressed a pole-localized bacterial cytoskeletal protein DivIVA in fission yeast to demonstrate that DivIVA accumulated on negatively curved surfaces¹³. Again, Balasubramanian and group had first established fission yeast as a cellular model system to bring new insights into the mechanism, assembly, and dynamicity of the E. coli actin cytoskeleton protein like MreB¹⁴ and tubulin homolog FtsZ¹⁵. They also demonstrate the ability of A22 to efficiently impede the polymerization of MreB through epifluorescence microscopy when expressed in yeast¹⁴. Following this, other groups have also successfully employed fission yeast to study the assembly properties of chloroplast FtsZ1 and FtsZ2 proteins¹⁶. More recently, we have established a proof-of-concept of the viability of the use of fission yeast as a cellular platform to specifically screen for bacterial cytoskeleton inhibitors by conducting a comprehensive assessment of the impact of three known FtsZ inhibitors-sanguinarine, berberine, and PC190723-on FtsZ proteins derived from two pathogenic bacteria, namely Staphylococcus aureus and Helicobacter pylori¹⁷. Additionally, this single-step cell-based assay proves instrumental in minimizing the risk of identifying compounds that may be potentially toxic to eukaryotic cells.

In this report, utilizing the fission yeast system, we propose a systematic workflow using the standard 96-well plate for semi-automated screening and quantification of the effect of small molecule inhibitors targeting FtsZ from Staphylococcus aureus and MreB from Escherichia coli. Here, we set up and optimize the semi-automated workflow using the established inhibitors PC190723 and A22 that specifically target FtsZ and MreB, respectively. This workflow uses an epifluorescence microscope equipped with a motorized high-precision stage and automated image acquisition in a standard 96-well plate to improve upon current standardization. Hence, it can be applied to medium- as well as high-throughput screens of synthetic chemical libraries and circumvents some of the challenges listed above.

Protocol

1. Expression of GFP-tagged bacterial cytoskeleton proteins in S. pombe

NOTE: Please see Table 1 for information on all plasmids and strains used here. Please see Table 2 for all media compositions.

1. Perform cloning of E. coli MreB with an N-terminal GFP fusion (GFP-MreB) and S. aureus FtsZ carrying a C-terminal GFP (SaFtsZ-GFP) into the S. pombe expression vector, pREP42 with a medium-strength thiamine-repressible promoter, nmt41^18,19 as previously described^14,15. Maintain, amplify, and isolate the plasmids from E. coli strains (DH10β) as described in^20,21.
   NOTE: Other E. coli strains such as DH5α, XL1Blue, TOP10, etc. or other commercially available competent cells routinely used for molecular cloning may also be used.
2. Transformation of plasmids pREP42-GFP-EcMreB and pREP42-SaFtsZ-GFP into S. pombe
   1. Transform the plasmids pCCD3 (pREP42-GFP-EcMreB) and pCCD713 (pREP42-SaFtsZ-GFP) into S. pombe strain (h^-leu1-32 ura4-D18) using the lithium acetate method²², as mentioned in the below steps.
   2. Day 1 - Primary culture: Inoculate a loopful of freshly streaked out S. pombe culture in 3 mL of autoclaved yeast extract and supplements (YES) broth. Incubate it in an orbital shaker at 30 °C overnight (O/N).
   3. Day 2:
      1. Secondary culture: Add about 500 µL of primary culture to 30 mL of autoclaved YES broth. Incubate at 30 °C for 3 - 4 h with shaking till the OD₆₀₀ reaches 0.4 - 0.6.
         NOTE: For each transformation, 30 mL is used.
      2. Pellet the 30 mL culture at 2,500 x g for 6-8 min at room temperature. Discard the supernatant and wash the cells with 50 mL of sterile distilled water (D/W). Pellet down again and discard supernatant. Resuspend the cells in 1 mL of sterile D/W and transfer it to a 2 mL centrifuge tube. Centrifuge as above and discard supernatant.
      3. Add 1 mL of 0.1 M lithium acetate, Tris-EDTA (LiAc-TE) solution and centrifuge as above. Discard the supernatant and resuspend in 1 mL of 0.1 M LiAc-TE. Centrifuge and discard supernatant, leaving 100 µL of solution behind.
      4. Add 10 - 20 µg of carrier DNA (Salmon sperm DNA; denatured and flash cooled on ice) and 2 - 3 µg of the plasmid DNA, which needs to be transformed. Mix gently. Incubate at room temperature for 10 min.
      5. Add 260 µL of 40 % PEG/LiAc-TE; mix gently. Incubate for 60 min in the shaker or a thermomixer at 30 °C with gentle mixing. Add 43 µL of DMSO; mix gently.
      6. Heat shock at 42 °C for 10 min in the thermomixer. Pellet at 2,500 x g for 6-8 min and discard supernatant. Wash the pellet 1x with 1 mL of sterile D/W.
      7. Pellet, discard supernatant and resuspend the pellet in 200 µL of sterile D/W. Plate 100 µL on Edinburgh minimal medium (EMM) plates containing 5 µg/ mL thiamine (to represses nmt41 promoter) and amino-acid supplements adenine (0.225 mg/ mL), histidine (0.225 mg/ mL) and leucine (0.225 mg/ mL) but lacking uracil (selection marker for pREP42 plasmid).
         NOTE: Alternately, plate 70 µL and 130 µL in two different plates. Two different volumes (70 µL and 130 µL) are plated to obtain isolated colonies on at least one of the plates depending on the transformation efficiencies, which can vary from experiment to experiment.
      8. Incubate the plates at 30 °C for 2-3 days till colonies appear. Mix a single colony with 100 µL of sterile D/W and spread out on a fresh EMM plate containing thiamine but lacking uracil as described above.
      9. Incubate at 30 °C for 2-3 days until a complete lawn appears. Scrap the grown lawn of cells using an inoculating loop and resuspend it to 1 mL of YES media containing 30% glycerol in a cryo-vial.
      10. Flash freeze the cryo-vial in liquid nitrogen and store at -80 °C to preserve the frozen yeast stocks.
3. Expression of bacterial cytoskeleton proteins in fission yeast
   1. Aseptically streak a patch from the glycerol stock onto a fresh yeast-specific plate to obtain enough culture for further experiments.
      NOTE: In the case of pREP42, we used an EMM agar plate (minimal media) containing adenine, histidine, and leucine (as using S. pombe strain (h- leu1-32 ura4-D18)) without uracil (uracil present in pREP42 as a selection marker). We add 15 - 20 µM thiamine to the agar plates (as pREP42 has thiamine repressible nmt41 promoter) to repress the expression of the gene of interest when growing on the plate.
   2. Inoculate a small loopful of the inoculum from the streaked patch into 5 mL of yeast-specific EMM media lacking thiamine and incubate it for 10 - 12 h at 30 °C.
      NOTE: The expression of FtsZ and MreB from different species under the nmt41 promoter in the S. pombe can vary, typically from 16 to 30 h. The optimal time for protein expression of GFP-EcMreB and SaFtsZ-GFP in S. pombe is 20 - 24 h and 16 - 20 h at 30 °C, respectively, without thiamine.

2. Treatment of S. pombe cultures expressing GFP-tagged bacterial cytoskeletal proteins

NOTE: A number of different drug molecules are tested on the overnight-grown yeast culture in a 96-well plate.

1. Inoculate a fresh culture by transferring 50 µL of the overnight culture into each well of a 96-well plate containing 150 µL of fresh yeast EMM media using the semi-automated, 96-well multichannel pipetting instrument.
2. While pipetting in and out for proper mixing using a semi-automated, 96-well multichannel pipette, slowly introduce different increasing concentrations of drugs into the growing culture, each done in triplicate as represented in the schematic in Figure 1.
3. As a positive control, use the already known drugs, PC190723 for (S. aureus FtsZ) and A22 (for E. coli MreB) in duplicate.
   NOTE: As previously reported, PC190723 and A22 were used at a concentration of 56.2 µM¹⁷ and 72.6 µM^14,17, respectively. Meanwhile, A22 treatment of SaFtsZ and PC190723 treatment of EcMreB served as negative controls, respectively.
4. Use DMSO as a solvent control at three different concentrations according to the lowest and highest concentrations of drugs.
   NOTE: The solvent in which drugs are dissolved is used as a solvent control.
5. Incubate the control and treated cultures at 30 °C for 6-10 h (till the cells show the expression of bacterial fluorescent proteins) and then image using epifluorescence microscopy.

3. Visualization of the polymers

1. Apply a coating of 20 µL of 1 mg/mL concanavalin A to each well of the optically transparent bottom 96-well plate (black-walled for fluorescence imaging) and incubate for 20 min at room temperature. Aspirate liquid and let it air dry for 10 min.
2. Transfer 20 µL of cells from the culture plate into each respective well and allow to sit for 10 min. Wash the cell 3x-4x with the sterile EMM media.
3. With the objective lens to be used in place (see step 3.6), use the navigator mode in the acquisition panel of the image acquisition software for 96-well plate alignment and well coverage. Align well edges on the navigator.
4. Next, select the well coverage parameters, which include a multi-position selection of the region of interest in the well, and use adaptive autofocus control with on-demand mode to keep the sample in focus during imaging.
5. Acquire images using differential interference contrast (DIC) and fluorescence. Set excitation and emission filters of 475/28 nm and 525/48 nm, respectively to image the GFP-tagged bacterial proteins expressed in yeast. Obtain Z-stacks at a step size of 0.2 µm through the thickness of the yeast cells (5 µm).
6. Capture images using an inverted epifluorescence microscope equipped with a 100x, 1.4-NA oil-immersion objective and a 2,000 x 2,000 sCMOS camera (6.5 µm x 6.5 µm pixel size). Use a LED illumination system for excitation of the fluorophore.
   NOTE: Any epifluorescence microscope system that has a motorized stage for a 96-well plate with precise multi-point positioning should be suitable.Acquire all the cell images of control and drug-treated with a similar exposure time (usually in the range of 0.3 - 0.5 s) at a binning of 1 x 1 and illumination at 15% - 20% to minimize the experiment variables and maintain consistency.

4. Quantification of the images using ImageJ

1. Process cell images and measure the number of spots per cell and density for FtsZ¹⁷. In the case of MreB, measure density and anisotropy²³ and compare between control and treated cells using Fiji (v2.0.0-rc-69/1.52p)²⁴.
2. Using the images from the DIC channel, first outline the individual yeast cells using the freehand drawing tool and save as a region of interest (ROI) in the ROI manager. This step is not yet automated, but recently developed tools using machine learning^25,26 could be attempted and incorporated in the near future.
3. Mask the outside area of the cell and fill black as described in²⁷.
4. Use the OPS threshold IJ1 analyse macro, a built-in feature in Fiji, to count the number of spots²⁴.
5. Use the otsu method for auto-thresholding and mask the segmented particles. Run the plug-in analyse particles using a macro.
6. For measuring polymer density (amount of cytoskeleton per unit area in a cell), process images as described, including masking and skeletonization steps^27,28. Use Lpx 2Dfilter in the lpx plug-in to skeletonize the images.
7. For details, check this recent publication¹⁷. Perform EcMreB polymer quantification as previously mentioned in²³, use anisotropy to quantify spatial organization using FibrilTool²⁹ as described previously²³.
   NOTE: These methods of analysis can be used to quantify any other bacterial cytoskeleton proteins which are treated or untreated by the drugs.

Results

Setting up the 96-well plate for the screening of drugs
Use of S. pombe to express a C- terminally GFP tagged S. aureus FtsZ from a vector (pREP42) containing the medium-strength thiamine repressible promoter nmt41has been previously established¹⁷ and similarly, the E. coli MreB tagged with N- terminal GFP was also expressed in S. pombe¹⁴. We have also shown that PC190723, a specific inhibitor of SaFtsZ and A22, a MreB inhibitor, can exert their effects on the respective bacterial cytoskeletal proteins in a specific manner when expressed in yeast^14,17.

Here, we propose to use the yeast expression system to develop a medium or high-throughput screening of antibacterial drugs targeting the bacterial cytoskeleton proteins. An epifluorescence microscope with a motorized stage can be automated to image a 96-well plate, and commercial drug libraries are also readily available in 96-well plate formats. We, therefore, prefer to use a 96-well plate for the proposed screening of drugs. The plates are set as shown in Figure 1. The first row (Figure 1A) consists of SaFtsZ-GFP expressing yeast culture. The second row (Figure 1B) consists of yeast cultures expressing GFP-EcMreB. The first two columns of the first two rows (A1:B2) are set as media blank. The following six columns of the first two rows (A3:B12) are set as DMSO control with three different concentrations according to the drug dilutions in duplicate. Next, PC190723 (56.2 µM) and A22 (72.6 µM) are added to yeast cells expressing SaFtsZ-GFP or GFP-EcMreB. PC190723 and A22 serve as positive controls for FtsZ and MreB, respectively. All other wells (C1:F12) are utilized for adding various drugs (at three different concentrations and in duplicate) used in the screen to identify effectors of the bacterial cytoskeleton proteins, SaFtsZ or EcMreB. Cultures of yeast expressing the GFP-tagged SaFtsZ or MreB are dispensed into these 96-well plates and grown until the effects of the drugs on the assembly of polymers are visualized.

Assessment of growth effects on yeast cells
After incubating the 96-well containing sub-cultured cells at 30 °C for 6 to 10 h, the optical density (OD) of the yeast culture is measured using a microplate reader. The measurement of OD helps assess any growth inhibitory effect of the drugs against the eukaryotic yeast cells and possibly deleterious effects on human cells. Thus, this assay can be used to screen out multiple drugs based on their toxicity in the yeast system. However, neither PC190723 nor A22 exhibits any growth effects or toxicity to yeast cells, and OD₆₀₀ of the cultures expressing SaFtsZ-GFP treated with DMSO, PC190723, or A22 were 0.38 ± 0.03, 0.44 ± 0.02 and 0.43 ± 0.06 (N ≥ 4), respectively. Likewise, the OD₆₀₀ of the cultures expressing GFP-EcMreB and treated with DMSO, PC190723, or A22 were 0.38 ± 0.04, 0.44 ± 0.07 and 0.41 ± 0.08 (N ≥ 4), respectively.

Visualization of the effect of the drugs on the polymeric structures assembled by SaFtsZ and EcMreB expressed in S. pombe
In order to assess the effect of the drugs on polymerization of SaFtsZ or EcMreB, an aliquot of the yeast cells from the above-mentioned 96-well plate containing drugs is transferred to another 96-well plate with optically clear glass bottom 96-well suitable for fluorescence imaging. This 96-well plate is also coated with concanavalin A to allow adhesion of the yeast cells expressing the GFP-tagged SaFtsZ or EcMreB. The 96-well plate is imaged using an epifluorescence microscope covering all the 96-wells of the plate at the predetermined positions as controlled by the image acquisition software (Figure 2). In the control wells (DMSO treated), yeast cells expressing SaFtsZ-GFP showed polymeric structures in the form of spots or patches distributed throughout the cells, after 16 - 18 h of growth in the absence of thiamine. However, at 10 - 12 h after growth in the lack of thiamine, cells only exhibited diffuse fluorescence or few patches, suggesting that FtsZ-GFP expression had not reached the critical concentration required for polymerization (Figure 3A). On the contrary, the FtsZ stabilizing drug, PC190723, showed a considerable increase in the polymeric structures (spots or patches) of SaFtsZ-GFP in comparison to DMSO control after 12 h of growth (Figure 3B), serving as the positive control. In contrast, cells treated with A22 showed no difference in the SaFtsZ-GFP structures assembled (Figure 3C). However, unlike in cultures treated with PC190723, SaFtsZ-GFP assembled into patches in untreated cultures only when induced for longer periods, showing that PC190723 acted to reduce the critical concentration of polymerization of SaFtsZ (Figure 3D). Likewise, GFP-EcMreB expressed in S. pombe formed linear arrays of long filaments along the longitudinal axis of fission yeast (Figure 4A). While PC190723 was found to have no effect on EcMreB polymerization(Figure 4B), treatment of cells with A22 resulted in diffused fluorescence throughout the cytoplasm of the yeast cells (Figure 4C). The images from the rest of the 96-well plates are visually inspected for any stabilization or inhibitory effects of the drugs on the SaFtsZ or EcMreB, as the case may be.

The impact of the drugs on the assembly of bacterial cytoskeletal proteins can then be quantified using image analysis tools and plug-ins in ImageJ or Fiji, as previously reported for SaFtsZ¹⁷ and EcMreB²¹. The automated image acquisition in 96-well plate format and implementation of custom macros and scripts for image processing can accelerate the imaging time and quantification of the effects of the drugs. Thus, using the single-celled eukaryotic yeast, S. pombe, as the host system, we propose that a medium or high-throughput screen for small molecules targeting the bacterial cytoskeletal proteins can be successfully carried out, eventually leading to the discovery of new antibiotics.

Figure 1: Schematic showing the use of fission yeast cells to screen for drugs targeting the assembly of bacterial cytoskeletal proteins SaFtsZ or EcMreB. FtsZ from S. aureus and MreB from E. coli were cloned into the yeast expression vector, pREP42 having GFP tag at C- and N- terminus, respectively and transformed into S. pombe for the phenotypic and drug study. The cultures were grown for 8 - 12 h. Subsequently, the cultures were sub-cultured in 96-well plates with appropriate controls and the different drugs being screened. Further, the plate was incubated at 30 °C for 6 - 8 h till the OD₆₀₀ reaches 0.5-0.6, and the optical density is measured using a microplate reader to assess the toxicity of the drugs towards yeast cells. Another optically clear glass bottom 96-well plate was pre-coated with concanavalin A, for adhering yeast cells. This 96-well plate was used for visualization and image acquisition with an epifluorescence microscope. The images obtained were then analyzed. Please click here to view a larger version of this figure.

Figure 2: Schematic of the image acquisition software for automated imaging of a 96-well plate. Using the navigator in image acquisition software, a 96-well plate was aligned by marking the edges. Well-coverage parameters like the number of images which need to be taken from each well and its location (random or from the center of the well) were selected as desired. Finally, image parameters such as the filter set to be used (DIC and FITC-filter; Ex 475/28 nm and Em 525/48 nm), percentage intensity of illumination light, autofocus and exposure time for image capture were set. Please click here to view a larger version of this figure.

Figure 3: Visualization of the effect of PC190723 and A22 drugs on SaFtsZ-GFP expressed in S. pombe. S. pombe culture expressing SaFtsZ-GFP was grown in the absence of thiamine for 8 - 9 h at 30 °C before DMSO and drug treatment. The culture was further grown in a 96-well plate for 7 - 9 h at 30 °C in the presence or the absence of the drugs. (A) In DMSO control, where an equivalent amount of DMSO was added, cells exhibited a few polymeric structures. (B) In the presence of PC190723 (56.2 µM), SaFtsZ expresses yeast cells showing a considerable increase in the polymeric structures than that of the DMSO control. (C) The MreB inhibitor, A22 (72.6 µM), did not affect SaFtsZ-GFP structures. (D) Longer hours of protein expression were necessary for SaFtsZ-GFP assembly in the absence of PC190723, and therefore, control cultures grown at 30 °C for 12 - 15 h after sub-culturing in 96-well plate containing DMSO also exhibit FtsZ polymers. Scale bar is 5 µm. Please click here to view a larger version of this figure.

Figure 4: Visualization of the effect of PC190723 and A22 drugs on GFP-EcMreB expressed in S. pombe. S. pombe culture expressing GFP-EcMreB was grown in the absence of thiamine for 9 - 11 h at 30 °C prior to adding DMSO or the drugs (A22 or PC190723). The culture was further grown in a 96-well plate for 8 - 10 h at 30 °C with and without drugs as a control. (A) In cultures where an equivalent amount of DMSO was added as a control, cells exhibited linear filaments of EcMreB oriented along the longitudinal axis of the yeast cells. (B) Assembly of GFP-EcMreB was not affected in cultures treated with PC190723 (56.2 µM). (C) The small molecule A22 (72.6 µM), a known inhibitor of MreB polymerization, prevented the assembly of GFP-EcMreB in fission yeast cells. Scale bar is 5 µm. Please click here to view a larger version of this figure.

Table 1: Strains and plasmids used in this study. Please click here to download this Table.

Table 2: Composition of media and buffers used in this study. Please click here to download this Table.

Discussion

Antimicrobial resistance (AMR) is a serious global health threat, and there is an urgent need for new antibiotics with novel targets. The bacterial cytoskeleton has emerged as an attractive target for developing new antibiotics, with small molecule inhibitors of the cell division protein FtsZ, such as TXA709, already in Phase-I clinical trials³⁰. Several methods have been developed to identify inhibitors of FtsZ polymerization^7,31. We have recently used the eukaryotic fission yeast in a cell-based assay to show that the FtsZ stabilizing drug, PC190723, can target HpFtsZ¹⁷. Moreover, we also showed that although drugs such as berberine and sanguinarine affected the polymerization of FtsZ, they also affected the morphology of yeast cells¹⁷. Prompted by these studies and observations, we set out to develop a single-step cell-based assay to screen for small molecule modulators of the bacterial cytoskeleton using the fission yeast expression platform. Since the screen relies upon inhibition of the assembly of GFP-tagged cytoskeletal proteins, small molecules sharing similar fluorophore properties may give rise to false positives. Avoiding long-pass filters and choosing narrow band-pass filters may help reduce these false positives arising from fluorescent small molecules. Here, we show the steps involved in setting up a medium throughput screen using an epifluorescence microscope equipped with a 96-well plate stage holder that can be used to screen for drugs targeting SaFtsZ or EcMreB. The workflow involves semi-automated image acquisition and analysis, which can easily be scaled to high-throughput screens in the future using robotic liquid handling systems and image processing pipelines.

Further, the screen should be useful for other bacterial cytoskeletal proteins, such as the actin-fold containing plasmid maintenance protein ParM³² and against any protein that assembles into polymeric structures in fission yeast. However, optimal expression levels for each protein of interest will need to be standardized prior to use in any drug screens. While for FtsZs, we have found nmt41 to be an optimal promoter, for other proteins of interest, a stronger or weaker version of the nmt1 promoter (Promoter strength (and leakiness): nmt1 > nmt41 > nmt81) or other constitutive promoters such as the yeast adh1 or act1^18,33 may be tried to obtain optimum levels of expression and assembly of polymeric structures. Moreover, while using episomal plasmids, copy number variations from cell to cell also pose problems for uniform gene expression. To circumvent these issues, several vectors that can be integrated into the fission yeast genome (SIVs for stable integration vectors) carrying promoters of various strengths have been recently developed³⁴. The use of these vectors may offer more uniform and quantitative results as compared to the episomal vectors used in this study here. The use of SIVs could avoid the large heterogeneity in expression levels, seen as a variation in the fluorescence intensities across the cells and should be better suited for high-throughput screens.

Furthermore, drugs like PC190723 act to stabilize the FtsZ polymers, resulting in the early assembly of FtsZ filaments compared to untreated cells. While the screen is suitable for screening drugs that inhibit polymerization of bacterial cytoskeletal proteins, because of visual assessment of diffuse fluorescence at the endpoint, screening of drugs stabilizing FtsZ polymers requires further optimization of protein expression levels and the time needed to achieve maximal number of cells having polymers. It is thus critical to have a reasonable estimate of the time needed for protein expression to achieve the critical concentration required for assembly and decide the endpoint for the assay accordingly. In case of drugs that might act to decrease the critical concentration required for polymerization of the protein, the choice of end-point would typically be a time, wherein a considerable population of untreated cells exhibit diffuse fluorescence, but cultures treated with drugs would display polymer assemblies.

One of the limitations which is also a time-intensive step of the workflow is the requirement to identify and segment individual cells for quantitative image analysis. Currently, in the proposed scheme, this is a manual step to identify individual yeast cells and assign the region of interest (ROIs) for the quantification of the effect of the drugs. However, we envisage that the recently developed machine learning algorithms using training sets such as those implementable in ilastik³⁵ should minimize the need for manual intervention.

While our approach here using the yeast platform is useful for identifying hits and potential small molecules targeting the bacterial cytoskeleton, one will eventually need to test their antibacterial activity using the standard minimal inhibitory concentration tests and efficacy in animal models. Nonetheless, the biggest challenge to the global increase in antimicrobial resistance (AMR) still lies in the outer membrane permeability of the Gram-negative bacteria and the numerous efflux pumps in many pathogenic bacterial species. Moreover, efflux pumps in fission yeast may also pose similar problems and higher drug concentrations might be required for the screening process. Alternately, fission yeast strains that are hyper-sensitive to drugs (S. pombe MDR-supML), as a result of the deletion of the seven multi-drug resistance genes, including four transporter genes and a transcription factor, may be used as hosts for screening^32,33. Future developments in technologies that combine the targeted antibacterial discovery and address resistance due to general phenomena such as membrane permeability will be necessary to tackle the growing antimicrobial resistance.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96 Well CC2 Optical CVG Sterile, w/Lid. Black	Thermo Scientific™	160376
96-well plate	Corning	CLS3370
A22 Hydrochloride	Sigma	SML0471	Dissolved in DMSO
Adenine	FormediumTM	DOC0229	225 mg/L of media
Concanavalin A	Sigma	C5275-5MG
DMSO	Sigma	317275
Edinburg minimal medium (EMM Agar or EMM Broth)	FormediumTM	PMD0210	See below for composition
EDTA	Sigma	EDS-500G
epMotion® 96 with 2-position slider	Eppendorf	5069000101
Histidine	FormediumTM	DOC0144	225 mg/L of media
Leica DMi8 inverted fluorescence microscope	Leica Microsystems		German company
Leucine	FormediumTM	DOC0157	225 mg/L of media
Lithium acetate	Sigma	517992-100G
PC190723	Merck	344580	Dissolved in DMSO
Polyethylene glycol (PEG)	Sigma	202398
Thiamine	Sigma	T4625	Filter sterilised
Tris-Hydrochloride	MP	194855
Uracil	FormediumTM	DOC0214	225 mg/L of media, Store solution at 36°C
YES (Yeast extract + supplements) Agar	FormediumTM	PCM0410	See below for composition
YES (Yeast extract + supplements) Broth	FormediumTM	PCM0310	See below for composition
Yeast (S. pombe) media
Yeast extract + supplements (YES)
	Composition	g/L
	Yeast extract	5
	Dextrose	30
	Agar	17
	Adenine	0.05
	L-Histidine	0.05
	L-Leucine	0.05
	L-Lysine HCl	0.05
	Uracil	0.05
Edinburg minimal medium (EMM)
	Composition	g/L	concentration
	potassium hydrogen phthallate	3	14.7mM
	Na2HPO4	2.2	15.5 mM
	NH4Cl	5	93.5 mM
	glucose	2% (w/v) or 20 g/L	111 mM
	Salts (stock x 50)	20 mL/L (v/v)
	Vitamins (stock x 1000)	1 mL/L (v/v)
	Minerals (Stock x 10,000)	0.1 mL/L (v/v)
Salts x 50	52.5 g/l MgCl2.6H20 (0.26 M)	52.5	0.26 M
	0.735 mg/l CaCl2.2H20 (4.99 mM)	0.000735	4.99 mM
	50 g/l KCl (0.67 M)	50	0.67 M
	2 g/l Na2SO4 (14.l mM)	2	14.1 mM
Vitamins x 1000	1 g/l pantothenic acid	1	4.20 mM
	10 g/l nicotinic acid	10	81.2 mM
	10 g/l inositol	10	55.5 mM
	10 mg/l biotin	0.01	40.8 µM
Minerals x 10,000	boric acid	5	80.9 mM
	MnSO4	4	23.7 mM
	ZnSO4.7H2O	4	13.9 mM
	FeCl2.6H2O	2	7.40 mM
	molybdic acid	0.4	2.47 mM
	KI	1	6.02 mM
	CuSO4.5H2O	0.4	1.60 mM
	citric acid	10	47.6 mM
Strains/ Plasmids
Strains	Description	Reference
CCD190	Escherichia coli DH10β	Invitrogen
CCDY4	MBY3532; CCDY346/pREP42- GFP-EcMreB	Srinivasan et al., 2007
CCDY340	CCDY346/pREP42- SaFtsZ-GFP	Sharma et al., 2023
CCDY346	MBY192; Schizosaccharomyces pombe [ura4-D18, leu1-32, h-]	Dr. Mithilesh Mishra (DBS, TIFR)
Plasmids
pCCD3	pREP42-GFP-EcMreB	Srinivasan et al., 2007
pCCD713	pREP42-SaFtsZ-GFP	Sharma et al., 2023