Encyclopedia of Experiments: Biology
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Transcript
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.
