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

Our laboratory is interested in understanding how cytoskeletal proteins assemble dynamic structures to regulate functions, such as DNA partitioning and cell division in bacteria. We are also interested in discovering compounds that target these cytoskeletal proteins to develop novel antibiotics. Our protocol is a cell-based assay in which the bacterial cytoskeleton protein polymerizes in a molecularly crowded environment in the eukaryotic yeast, schizosaccharomyces pombe.

With this approach, simultaneously, we can identify molecules that directly affects the bacterial cytoskeleton proteins, polymerization and are potentially toxic. Using this fission yeast platform, we plan to screen libraries of macrocyclic compounds, specifically targeting FtsZ of pathogenic bacteria like helicobacter pylori and pseudomonas aeruginosa. We also plan to expand the screens to target actin-like proteins from clostridium perfringens involved in plasmid segregation.

To begin, aseptically streak a patch of schizosaccharomyces pombe from the glycerol stock onto a fresh yeast-specific plate to obtain enough culture. Incubate the plate at 30 degrees Celsius for 48 hours. After 48 hours, when the colonies appear on the plate, inoculate a small loopful of the inoculum from the streaked patch into five millimeters of yeast-specific EMM-lacking thiamine and incubate for 10 to 12 hours at 30 degrees Celsius.

After the incubation period, use a semi-automated 96-well, multi-channel pipetting instrument and transfer 50 microliters of the grown culture into each well of a 96-well plate containing 150 microliters of fresh yeast EMM. While pipetting in and out for proper mixing, slowly introduce different increasing concentrations of drugs into the culture-added wells each in triplicates. Then, add known bacterial cytoskeletal proteins, targeting drugs as positive control and DMSO as solvent control.

Incubate the controls and drug-treated culture plate at 30 degrees Celsius for six to 10 hours. Start coating the wells of an optically transparent bottom 96-well plate with 20 microliters of concanavalin A and incubate for 20 minutes at room temperature. Then, aspirate the liquid and let it air-dry for 10 minutes.

Transfer 20 microliters of cells from the incubated culture plate into each respective well of the concanavalin A-coated plate and let it stand for 10 minutes. Then, remove the culture from the plate and wash the wells three to four times with sterile EMM. And place the plate in an inverted epifluorescence microscope, controlled by imaging software.

To image the cells, select the desired objective lens. Use the navigator mode in the acquisition panel of the software for 96-well plate alignment and well coverage. Select the carrier option and then, align the well edges of the 96-well plate.

Next, select the well coverage parameters, which include a multi-position selection of the region of interest in the well. Use adaptive auto-focus control with on-demand mode to keep the sample in focus during imaging. Then, acquire the images, using differential interference contrast and fluorescence.

Set the excitation and emission filters to image the GFP-tagged bacterial proteins. Obtain Z-stack images at a step size of 0.2 micrometers through the five-micrometer thick yeast cells. Then, acquire the images at the selected positions across the wells.

In the control wells with DMSO treatment, yeast cells expressing S.aureus FtsZ-GFP, formed polymeric structures as spots or patches throughout the cells after 20 to 24 hours. However, at 15 to 18 hours, cells showed diffused fluorescence or a few patches, indicating insufficient FtsZ-GFP expression for polymerization. The FtsZ-GFP-stabilizing drug PC190723, significantly increased polymeric structures after 17 hours, serving as a positive control.

In contrast, the A22 treatment showed no difference in FtsZ-GFP structures. Likewise, E.coli MreB-GFP formed linear arrays of long filaments along the longitudinal axis of fission yeast, while PC190723 did not affect the polymerization. Treatment of cells with A22 resulted in diffused fluorescence throughout the cytoplasm of the yeast cells.