The clinical outcome of Toxoplasma gondii infections in those individuals whose immune system is under-developed or compromised can be severe and ultimately fatal. The ability of tachyzoites to convert to bradyzoites (tissue cysts) is considered to be the underlying cause of Toxoplasma-encephalitis in HIV-1 infection. The molecular events regulating tachyzoite to bradyzoite transition are critical to the course of this disease. Recently, the applicants have demonstrated that tachyzoites emerging from sporozoite infected cells undergo a regulated change to a longer cell cycle that precedes their spontaneous differentiation to the bradyzoite form. The shift to a slower replicative cycle is essential to the tachyzoite developmental program since slow growth is consistently correlated with bradyzoite differentiation. In this proposal the applicants will investigate the mechanisms that regulate the tachyzoite cell cycle in order to ultimately identify new targets for Toxoplasma prevention.
In specific aim I, they will define the major phases of the tachyzoite cell cycle (G1, S, G2+M) and the unique process of cytokinesis associated with apical complex formation.
Specific aim II will address the essential mechanisms that regulate tachyzoite growth. Through genetic complementation of ts cell cycle mutants, they will identify and then characterize genes required for cell cycle progression.
In specific aim III they will introduce thymidine kinase into fast and slow growing VEG strain in order to determine the cell cycle phases which are altered during growth shift. Finally in specific aim IV they will compare the mRNA expression of fast and slow growing TK+ VEG tachyzoites in order to identify molecular changes associated with these distinct cell cycle types.
