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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Clinical Investigator Award (CIA) (K08)
Project #
1K08AI001286-01
Application #
2057539
Study Section
Microbiology and Infectious Diseases B Subcommittee (MID)
Project Start
1994-09-30
Project End
1997-08-31
Budget Start
1994-09-30
Budget End
1995-08-31
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Stanford University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Ortega-Barria, E; Boothroyd, J C (1999) A Toxoplasma lectin-like activity specific for sulfated polysaccharides is involved in host cell infection. J Biol Chem 274:1267-76
Boothroyd, J C; Black, M; Bonnefoy, S et al. (1997) Genetic and biochemical analysis of development in Toxoplasma gondii. Philos Trans R Soc Lond B Biol Sci 352:1347-54