This video illustrates a new phenotypic screening method to isolate mutants with a temperature sensitive egress phenotype. Mutagen and parasites are applied to cultures of toxoplasma infected human foreskin fibroblasts. For two hours, the non invading parasites are removed and the flask is transferred to 40 degrees Celsius to induce phenotype development.
After 26 hours, the vacuoles contain eight to 16 parasites. Next cells are stimulated with egress inducing compounds. Mutant parasites with a defect in egress will remain intracellular, whereas wild type parasites will egress into the medium.
The wild type parasites are removed by washing and the remaining cells are again incubated at 35 degrees Celsius to enrich the mutant population. Finally, the mutant phenotype is verified and single parasite clones are isolated from the enriched population by limiting dilution. The main advantage of this technique over existing methods, like the bio inhalation method, is that the specific enrichment of egress mutants is far more efficient.
We first had the ID for this method when we identified an egress mutant from a panel of mutants with growth defects. Using that mutant, we optimize the screening procedure by mixing in various ratios with wild type parasites. Demonstrating the procedure will be Bradley Coleman, a postdoc for my lap.
This procedure uses highly mutagenic compounds and liquids as well as the infectious parasite. Toxoplasma Gandhi use of appropriate protective gear, including a lab coat, double gloves, and a biological safety cabinet is essential. Any liquid waste generated should be collected separately for proper disposal.
Begin by inoculating confluent, human foreskin fibroblast or HFF cells with one milliliter of freshly lysed tachy Lloyds in ed one medium. Then place the flask in a humidified 37 degrees Celsius incubator with 5%CO2 for 18 to 25 hours. The next day, replace the medium with 10 milliliters of 0.1%fetal bovine serum medium, incubate at 37 degrees Celsius for 10 minutes.
Following the incubation add zero, 12.5 25 50, or 100 microliters of ENU mutagen per flask. The working dilutions will be 1.25, 2.55, and 10 millimolar respectively. Add up to 100 microliters of DMSO to each flask.
Incubate for four hours in a 37 degrees Celsius incubator. Rinse the monolayer with 10 milliliters, cold PB S3 times for 10 seconds. Add five milliliters, PBS and use a cell scraper to loosen the monolayer.
Then using heavy duty scissors, clip off the end of the needle cap so that the needle is not exposed. Next, transfer the cells to a syringe and pass them through a 26.5 gauge needle. To physically remove the parasite from the fibroblasts, multiple needle passages will increase the efficiency of releasing parasites from the host cell.
Attach a 3.0 micrometer polycarbonate filter to a syringe. Load the needle past cells into the syringe and push them through the filter into a tube pipe at the parasites onto a hemo cytometer for counting. The single most difficult aspect of this procedure is distinguishing parasites from debris.
To assure success, it's important that you let the parasites settle before counting. After allowing the parasites to settle for five minutes, count them. Once the concentration of toxoplasma has been determined, dilute them to 3, 333 parasites per milliliter in ED one for 10 milliliters.
33, 333 parasites are needed. Add three milliliters to the first well of a six well plate of HFF cells to inoculate them with 10, 000 parasites. Then serially dilute the parasites tenfold over the next three wells by transferring 300 microliters from one well to the next.
Leave the plates undisturbed in the 37 degree Celsius incubator for seven days. Then after a week has passed, aspirate the medium and fix the cells by adding three milliliters of 100%ethanol to each well for 15 minutes. After removing the ethanol, stain the cells by adding three milliliters of crystal violet solution to each well for 15 minutes.
Finally, rinse each well with three milliliters PBS for one minute. Once the plate is dry, count the plaques and select the concentration of mutagen needed to achieve survival of 30%of the exposed parasites. A dosage of 70%killing has been used historically and induces less than 100 point mutations per genome.
The next step of the procedure is to enrich the egress mutants in the population. Once the population has been expanded, add 120, 000 freshly lyed parasites to confluent HFF cells in 10 milliliters. ED one incubate for two hours at 35 degrees Celsius.
This will synchronize the population by limiting the invasion time. Following the incubation, aspirate the medium and rinse the cells for 10 seconds With 10 milliliters cold PBS add 10 milliliters. ED one medium supplemented with 25 milligrams per milliliter dextran sulfate.
This will saturate the glycan binding surface proteins on the parasites so they can reattach and reinve the host cell. Incubate the flask for 26 hours in a humidified incubator at 40 degrees Celsius with 5%CO2. During this time, normally folded protein will be replaced with misfolded temperature sensitive protein.
The next day dilute the egress inducer of choice to the working concentration in a 15 milliliter tube containing 10 milliliters. H-B-S-S-C supplemented with 25 milligrams per milliliter dextran sulfate. Place it in a water bath to prewarm the solution for 30 minutes.
Aspirate the medium from the parasite infected flasks and add the prewarm egress inducer solution. Incubate at 37 degrees Celsius as indicated in the accompanying document. Next, aspirate the medium and rinse for 10 seconds.
With 10 milliliters of cold PBS, add 10 milliliters of ED one supplemented with 25 milligrams per milliliter, dextran sulfate and 50 micromolar pyridine DIO carbamate then incubate for five hours at 35 degrees Celsius. Following the incubation. Aspirate the medium rinse once with PBS for 10 seconds.
Then add 10 milliliters. EED one medium, return the flask to the 35 degree Celsius incubator. Shake daily to disperse the egress parasites.
Continue incubating until the parasites destroy the monolayer. About seven days following the enrichment screen, the population is propagated for one passage. Then the phenotypes are confirmed by an egress assay.
Inoculate 20, 000 parasites into each well of a 24 well plate containing confluent HFF cells grown on cover slips, incubate for eight hours in a 35 degree Celsius incubator to allow invasion of all parasites in the medium at the permissive temperature. Then transfer the plate to a 40 degree Celsius incubator for 24 hours to induce the phenotype and grow oles containing eight to 24 parasites after washing each well with one milliliter PBS for 10 seconds. At room temperature, add one milliliter egress inducer pre prewarm and diluted in H-B-S-S-C incubate for the times described in the accompanying written protocol.
Aspirate the medium and fix by adding one milliliter of 100%methanol to each well and incubate for 15 minutes at room temperature. Then wash with one milliliter of room temperature PBS After washing and mounting the slides use a microscope with a 40 to 60 x objective to count the percentage of S egress versus the intracellular vacuoles. After validation of the phenotype of the enriched egress mutant population, the polyclonal population is cloned to obtain single parasite clones.
After determining the parasite concentration diluted to 500 parasites per milliliter in ED one medium in a 3 84 well plate containing a confluent monolayer of HFF cells. Replace the medium with 40 microliters per well of ed, one medium using a multi-channel pipette. Four polyclonal populations represented as red, blue, yellow, and green.
In this illustration can be cloned on a plate to achieve this pipette. 40 microliters per well of diluted mutant one into wells C3 to C 12 mutant two into wells C 13 to C 22 mutant three into wells H three to H 12, and mutant four into wells H 13 to H 22. The total volume in each well should now be 80 microliters.
Then using a multi-channel pipette, pipette the solution up and down five times, transfer 40 microliters to the row below the starting row. Continue these twofold serial dilutions through row H mutants one and two or row N mutants three and four. Discard the extra 40 microliters from the last row.
Incubate in a 35 degrees Celsius incubator for seven to 10 days without disturbing the plate. The next week, check the wells on an inverted microscope with a 10 to 20 x objective for the presence of single plaques visible as holes in the monolayer. Pick four wells per mutant with a single plaque and transfer the parasites into a T 12.5 Tissue culture flask confluent with HFF cells and filled with five milliliters.
ED one medium grow the parasites up in a 35 degree Celsius incubator. Shake the flasks daily to disperse the extracellular parasites. This typically takes seven days.
Plaque assays were performed in a six well plate using various EMS mutagen concentrations and various numbers of parasites per well. As shown in this video, the white spots on the wells shown here are parasite plaques formed in the HFF monolayer on plates treated with EMS. The results from these plaque assays were used to generate survival curves of parasites exposed to increasing dosages of EMS and ENU.
The dosage inducing 70%killing was then chosen for mutagenesis experiments. In a different experiment, the percentage of egress mutant phenotype in the population following enrichment was assessed by flow cytometry as shown here. When the egress mutant to wild type parasites start ratio was one in 10, 000, 80%purity was achieved through enrichment.
However, when the start ratio was one in 100, 000 parasites, the isolated population made up only one to 2%This data suggests that the enrichment power of the screen is 1000 fold. In a third experiment, egress was induced by the calcium ion four A 2 3 1 8 7. In these images, the arrows mark four different VAEs containing four to eight parasites that express YFP in the controls.
However, the arrows mark four groups of scattered parasites reflecting four independent egress VAs. Thus, the ratio of mutant to wild type phenotypes can easily be determined by counting Following this procedure. Additional methods like cosmic complementation or whole genome sequencing can be performed to identify the causative mutation and therefore the gene of interest.
After watching this video, you should have a good understanding of how to titrate a mutagen of interest, mutagen a population of toxoplasma parasites, and isolate individual temperature sensitive egress mutants from that mutagen population. Don't forget that working with param methymine resistant toxoplasma parasites can be extremely hazardous, and precautions such as safety goggles should always be taken while performing this procedure.
