Molecular cloning, functional complementation in Saccharomyces cerevisiae and enzymatic properties of phosphatidylinositol synthase from the protozoan parasite Toxoplasma gondii

摘要: The obligate intracellular parasite Toxoplasma gondii, the causative agent of toxoplasmosis, switches between rapidly dividing tachyzoite and slowly replicating bradyzoite in intermediate hosts such as humans domestic animals. We have recently identified a cDNA encoding putative phosphatidylinositol (PtdIns) synthase using subtractive library [Yahiaoui, B., Dzierszinski, F., Bernigaud, A., Slomianny, C., Camus, D., Tomavo, S. (1999) Mol. Biochem. Parasitol. 99, 223-235]. Here, we report cloning another PtdIns that is exclusively expressed stage. two transcripts are encoded by different genes, which stage-specifically regulated. deduced amino-acid sequence (258 amino acids with calculated total molecular mass 27.8 kDa) tachyzoite-specific shares significant degree identity (between 26.5 30.1%) to synthases from human, rat, Arabidopsis thaliana yeast. Interestingly, protein encompasses an N-terminal extension approximately 40 amino-acids longer than other organisms. Functional complementation realized tetrad analysis segregants Saccharomyces cerevisiae synthase-deficient mutant (PIS1/pis1:kanMX4) showed only T. gondii truncated at its able restore viability cells. demonstrate this yeast transformants functionally active membrane preparation requires manganese magnesium ions for activity. To our knowledge, first on functional gene protozoan parasites.