Toxoplasma gondii infects up to 50% of the world's population. The organism has a remarkable ability to invade a broad range of cells within its mammalian host, by mechanisms that are poorly understood at the molecular level. The recent development of techniques to introduce DNA directly and stably into T. gondii, has opened the possibility of identifying gene products that are only characterized by the gross phenotype to mutants. As a first step toward the isolation of genes that play a key role in controlling Toxoplasma infectivity, temperature- conditional mutants defective in their ability to infect mammalian cells have been isolated in this laboratory. When tested under restrictive conditions, three of the mutants displayed a particularly clear phenotype with about 4-5 fold less infectivity that the parental cell type. We propose to clone and characterize the gene(s) responsible for the attenuated phenotype by functional complementation, and study the structure/function relationship of the gene products by a combination of genetic, immunological and biological tools. For this purpose, additional temperature sensitive mutants will be isolated. A genomic DNA library from the parental parasite strain will be constructed in the SAG1/CAT vector, and used to transform the mutants. Mutants that become phenotypically wild type will be expended and their DNA introduced back into E. coli under ampicillin selection. Then, selected plasmids will be used again to rescue the wild type phenotype. Precise identity will be established by using subclones of the plasmid containing the gene of interest. In addition, recombinant protein and monoclonal antibodies will be produced and tested for their ability to modify parasite infectivity in vitro and in vivo. These studies will strengthen our understanding of Toxoplasma-host cell interaction and may ultimately lead to the development of novel reagents useful for ligand and immunotherapy of toxoplasmosis.
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