Toxoplasma gondii SUB4 Gene Deletion Mutants and Their Viability in Host Cell Invasion

by John Asmus

Faculty mentor: Professor Swati Agrawal

Toxoplasma gondii is a microscopic parasitic protist. It is responsible for the disease toxoplasmosis which can cause severe health problems in immunocompromised individuals and babies whose mothers become infected while pregnant. T. gondii is an obligate intracellular parasite that can infect almost any warm-blooded mammal. Because of this, host cell invasion is a key function that T. gondii must perform. Vital to this process is a group of secretory organelles including rhoptries, micronemes, and dense granules. Research has shown that subtilisin-like proteases act in processing and cleaving certain sites on proteins associated with these secretory organelles. T. gondii has a set of 12 genes that code for these proteases. While much research has been done on SUB1 and SUB2, little to none has been done on other genes such as SUB4. To determine the vitality of SUB4 in T. gondii host-cell invasion, I am creating gene knockout mutants using CRISPR-cas9 and will observe their ability to invade mammalian fibroblast cells. I created a drug repair cassette by adding SUB4 homology arms to a drug marker amplified from pJET plasmid. I designed and am currently in the process of amplifying two sgRNAs that flank the gene on both 5′ and 3′ ends. Once these are constructed, I will transfect T. gondii ΔKU80 cells, which cannot perform nonhomologous end-joining repairs, with these vectors to knockout the SUB4 gene. Following drug selection and screening to determine if the parasites successfully recombined the SUB4 locus, I will characterize mutant parasites using microscopy and biochemical techniques. Finally, I will conduct invasion and egress assays to determine the ability of Toxoplasma gondii to invade host cells without expression of the SUB4 gene. I expect that they will invade the fibroblast cells but at a slower rate compared to the wild-type parasites, elucidating the role of SUB4.