Toxoplasma gondii infects more than a million individuals in the U.S. each year and is ranked only behind bacterial pathogens, Listeria and Salmonella, as the third-leading cause of food-borne death [1,2]. Mortality from T. gondii infection is high in immuno-compromised patients who suffer chronic cycles of disease, as there is no effective means to prevent tachyzoite-bradyzoite (tissue cyst) stage switching. To stay ahead of Toxoplasma infections in these patients, drug therapies must be administered continuously, however, the side effects of these prescriptions often limits this approach. What is critically needed for HIV infected patients and individuals immuno-suppressed as the result of cancer treatment or organ transplant are treatments that eliminate all parasite stages and/or block tissue cyst formation. Previous research sponsored by this grant established that the course of bradyzoite de-differentiation (tissue cyst recrudescence) and sporozoite-initiated development are identical, which indicates oocyst and tissue cyst infections in the intermediate host share the same cell cycle mechanisms. We have also demonstrated tachyzoites divide by an unusual three-phase cell cycle that is altered in late-S/G2 upon activation of the bradyzoite-switch suggesting tachyzoite-to-bradyzoite development requires changes in the chromosome cycle. We now propose to explore the molecular correlates of the Toxoplasma cell cycle that are relevant to parasite development through the application of forward-genetic strategies--protocols to isolate loss-of-function mutants and a novel recombination-based, genetic complementation method were developed under this grant. We intend to use these approaches to examine: The checkpoints that control the T. gondii cell cycle (or determine these mechanisms are absent?) and the molecular controls of tachyzoite-to-bradyzoite development that lie in the S to M transition. It is our hypothesis that checkpoints present in these parasites regulate 1) S phase entry (START), 2) ensure accurate mitosis (spindle and/or anaphase), 3) coordinate cytokinesis (daughter assembly and mother disassembly), and 4) control bradyzoite development. In this proposal we will; Define the interval of S phase through mitosis in the tachyzoite cell cycle, isolate conditional cell cycle and bradyzoite 'switch-ON' parasite mutants, and validate the phenotype and genetically complement parasite mutants. ? ? ?
