This article details the methodology for assessing the efficacy of antiparasitic compounds on Toxoplasma gondii within host cells. The study utilizes a luminescence-based assay to measure the impact of drug treatments on parasite growth.
Toxoplasma gondii — a protozoan parasite — reproduces exclusively within nucleated, animal host cells. It invades host cells through the endocytic process, forming a parasitophorous vacuole. Inside the vacuole, the parasite ingests host cytosolic proteins, acquiring nutrients for growth.
To screen antiparasitic chemicals' efficacy on parasites inside host cells, take an assay plate containing an adherent culture of human foreskin fibroblasts in multiple wells.
Dispense equal amounts of Toxoplasma parasites expressing luciferase — a luminescence-producing enzyme — into each well. Incubate, allowing the parasites to invade the host cells and grow inside the parasitophorous vacuole.
Post-incubation, aspirate the medium, removing the non-ingested parasites. Supplement the wells with different concentrations of cysteine protease-targeting antiparasitic compound, and incubate.
Drug molecules enter the host cell to reach the parasites. Once inside, the drug inhibits the parasite's cysteine protease enzyme, disrupting the host-bound parasite's nutrient acquisition and arresting its growth.
Aspirate the drug-containing media. Add an assay buffer containing a substrate specific to the luciferase enzyme. The buffer components lyse the cells, allowing the parasite's luciferase enzyme to oxidize the substrate, producing a bright luminescence.
Measure the luminescence for all chemical inhibitor concentrations. Reduction in luminescence intensity with increasing drug concentrations indicates the chemical inhibitor's antiparasitic efficacy.
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 7 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-walled reservoir by serial dilution.
Dilute the compounds in a 12-well reservoir at a 1:3 dilution by pipetting each mixture up and down several times with a 1-milliliter pipette.
At 4 hours post-infection, replace the medium in each well from columns 2 through 9 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.
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