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

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