These sensitive strategies can be used to evaluate the inhibition efficacy of chemical inhibitors and in combination with CRISPR-Cas9 gene deletion to investigate the drug targets. Instead of using an endpoint assay, parasite growth can be monitored over time to allow calculation of parasite doubling time using a sensitive reporter protein. This assay can potentially be scaled up in 384 or 1, 536 well microplates for the testing of multiple compounds or for the screening of libraries in a high throughput manner.
This strategy can be modified to quantify the growth of other intracellular microbial pathogens and their responses to the drug of interest. To perform a luciferase-based Toxoplasma growth assay, begin by carefully aspirating the medium from pre-seeded 96-well microplate cultures of human foreskin fibroblasts and inoculate 150 microliters of parasite B suspension into the wells in a three column-five row format. After a four-hour incubation in the cell culture incubator, carefully aspirate the medium from each well to remove the non-invaded parasites and fill the wells in each but the first row with room temperature phenol red free medium.
Then add 100 microliters of a 12.5 micromolar luciferase substrate solution in PBS into each well of the top row and incubate the microplates for 10 minutes at room temperature to allow full cell lysis. At the end of the incubation, measure the luciferase activity on a microplate reader. Each reading represents the initial number of invaded parasites at four hours post infection.
Repeat the analysis every 24 hours for the next four days without changing the medium. To calculate the normalized fold-change of parasite growth, each reading can be divided by the initial reading at four hours post infection. The log 2 of the normalized fold-change of parasite growth can then be plotted against each time point and subjected to a linear regression function to obtain the slope which represents the doubling time.
To assess the efficacy of the inhibition of a compound of interest on Toxoplasma growth, culture human foreskin fibroblasts in 96-well microplates for at least seven days before inoculating each well of cells with 150 microliters of parasites as demonstrated for four hours at 37 degrees Celsius and 5%carbon dioxide. During the incubation, prepare the compound of interest at eight different concentrations in a 12-well reservoir by serial dilution. Dilute the compounds in a 12-well reservoir at a one-to-three dilution by pipetting each mixture up and down several times with a one milliliter pipette.
At four hours post infection, replace the medium in each well from columns two through nine with 150 microliters of medium supplemented with each dilution of compound, leaving the first column filled with regular medium to serve as the non-treated control. Then return the cell cultures to the cell culture incubator for 96 hours and measure the luciferase activity for each well to determine the percentage of growth inhibition in the cultures at each concentration of compound. To calculate the normalized luciferase activity as a percentage, divide the average luciferase activity for each compound concentration by the average luciferase activity derived from the non-treated parasites.
Then use an appropriate graphing software program to plot the normalized luciferase activities against the individual compound and to calculate the half maximal inhibitory concentration for each compound. To generate a plasmid construct expressing single-guide RNA and Cas9 for deleting a gene of interest, navigate to the Toxoplasma Genomics Resource page and retrieve the entire gene coding sequence including the introns and exons along with the 1.5 kilobase five and three prime untranslated regions. Copy the retrieved TgCPL sequence into the sequence analysis software and label the five and three prime untranslated regions.
On the Toxoplasma Genomics Resource page, click Download, Data Files, and Current_Release. In the T.gondii GT1 folder, select FASTA. From the data folder, download the Toxoplasma gondii GT1 strain genome sequence file.
Then create a local folder named Toxoplasma Genome in the DNA sequence analysis software and import the Toxoplasma genome sequence file into the folder. Select Tools, Cloning, and Find CRISPR Sites and set three prime Cas9 prime for the PAM site location. Select a single-guide RNA that demonstrates a high specificity score, generally greater than 98%and lacks a G following the NGG.
The selected single-guide RNA is usually located at sites close to the start and stop codons of the gene of interest. Then use a PCR premix with the settings as indicated in the table to perform a PCR reaction to modify the preexisting plasmid expressing single-guide RNA that targets the TgUPRT gene. For Toxoplasma transfection, mix two micrograms of repaired template DNA with 20 micrograms of the single-guide RNA Cas9 expression plasmids and mix 400 microliters of parasite resuspension, DNA, and five microliters of 200 millimolar ATP 500 millimolar reduced glutathione in a 1.5 milliliter centrifuge tube.
Add cytomix buffer to bring the total volume up to 500 microliters as necessary and transfer the parasite DNA mixture to a four millimeter gap with electroporation cuvette. Then electroporate the parasites at two kilovolts and 50 ohms of resistance. To clone knockout parasites, first perform a two-step dilution of the parasites to achieve a 10 parasites per milliliter of D10 medium supplemented with the appropriate antibiotic concentration.
Next, transfer 150 microliters of the resuspended purified parasites to each well of a 96-well microplate pre-seeded with foreskin fibroblasts and incubate the microplate at 37 degrees Celsius and 5%carbon dioxide for seven days. At the end of the incubation, identify the wells containing a single plaque and transfer 75 microliters of the resuspended Toxoplasma-infected human foreskin fibroblasts from each well of the 96-well microplate culture into individual 1.5 milliliter tubes. Collect the cells by centrifugation and resuspend the pellets in 10.25 microliters of lysis buffer containing dilution buffer in DNA release additive.
Then incubate the samples for four minutes at room temperature and two minutes at 98 degrees Celsius before performing PCR to test for the integration of the drug resistance cassette and loss of the gene of interest. The inhibition efficacies of LHVS and wild type in delta CPL strains can be determined by plotting their luciferase activities against different inhibitor concentrations. After PCR, the mutated plasmid is linearized and loaded into a 1%agarose gel for the verification of a successful amplification.
After gel extraction and E.coli transformation, the clones containing the expected plasmids can be screened by restriction endonuclease digestion in DNA sequencing. After repair template amplification, agarose gel electrophoresis can be used to verify the correct size of the PCR product. Generally, seven to eight clones are selected for the initial screening to check for deletion of the coding sequence of the gene of interest, followed by detection of the five and three prime arms to help minimalize the total number of clones to be screened.
Further verification can be completed by immunoblotting if any antibody recognizing the target protein is available. Before obtaining luciferase activity measurements, a clear microplate with a mock infection should be checked under a microscope to ensure that the parasites do not fully lyse the host cells.
