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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

Protokół

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.

Wyniki

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...

Dyskusje

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...

Materiały

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