The obligate intracellular parasite Toxoplasma gondii is an important opportunistic pathogen in immunocompromised individuals such as AIDS patients. T. gondii also causes devastating birth defects in neonates. These studies are designed to provide further understanding of the mechanisms by which T. gondii is able to persist and cause disease within its human host. The salary support provided by the K02 award will enable Dr. Kim to have """"""""protected time"""""""" to concentrate upon her efforts to adapt technology and expertise from other areas of biological investigation, specifically eukaryotic cell cycle regulation and signal transduction, to the study of cell cycle and Toxoplasma development. Within human hosts, T. gondii exists as tachyzoites, which are responsible for clinically apparent disease, and as bradyzoites, which are latent encysted forms. With waning immune function, slow-growing bradyzoites can reactivate to the rapidly proliferating tachyzoite forms, leading to Toxoplasma encephalitis. Given the marked difference in proliferation of the tachyzoite compared to the bradyzoite and Toxoplasma's inability to replicate outside host cells, cell cycle regulation is undoubtedly critical in Toxoplasma development and pathogenesis of disease. It is clear from studies of other eukaryotes that responses to signals for development or differentiation occur at specific points within the cell cycle and result in a coordinate modulation of stage-specific gene expression and cell cycle progression. The candidate hypothesizes that unique aspects of T. gondii cell cycle regulation coordinate the processes leading to disease pathogenesis and intracellular survival.
In Aim 1, regulation of cell cycle gene expression in T. gondii will be analyzed focusing on ribonucleotide reductase (RNR) gene expression.
In Aim 2, expression and function of T. gondii cyclin dependent kinases (cdk) will be examined using immunofluorescence, flow cytometry, Northern and Western blotting. Constructs with mutant cdks will be transfected into tachyzoites to determine the role of cdks in cell cycle progression. Finally, in Aim 3, the relationship of cell cycle regulation and T. gondii bradyzoite development will be studied. Cell cycle markers will be used to examine the cell cycle dependence of bradyzoite development. Cell cycle and bradyzoite reporter constructs will be used to test the effects of cell cycle inhibitors and potential inducers or inhibitors of differentiation on gene expression. These will be the initial experiments in a long-term effort to define the role of cell cycle regulation in Toxoplasma tachyzoite-bradyzoite differentiation and virulence.
