Exocytosis of Plasmodium egress and invasion organelles

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Project Summary Human malaria caused by the intracellular parasite Plasmodium falciparum is responsible for nearly 600,000 deaths every year. The clinical symptoms of malaria are caused by the exponential asexual growth of parasites within human red blood cells. This cycle begins with the invasion of P. falciparum into the erythrocyte, where it hides within a vacuole (parasitophorous vacuole or PV) to divide into daughter merozoites and ends with the rapid release of merozoites that invade a red blood cell (RBC) to start the cycle anew. The egress and invasion of merozoites requires the signal-dependent exocytosis of specialized vesicles known as exonemes into the PV during egress and organelles known as rhoptries into the RBC during invasion. Data show that intracellular calcium oscillations lead to exocytosis of both exonemes and rhoptries within minutes of each other. But it is not known how the exocytic machinery on exonemes and rhoptries differentiate between the two intracellular calcium oscillations. It is likely that the proteins responsible for exocytosis are differentially sensitive to calcium and hence, respond differentially to the varied calcium signals during egress and invasion. However, the proteins localized in the secretory pathway required for signal dependent exocytosis remain mostly unknown. Therefore, we took a bioinformatic approach to identify several proteins in the secretory pathway with calcium binding domains that we hypothesized to function in organelle discharge during Plasmodium egress and invasion. This approach led to the identification of a calcium binding protein (PfERC) with an essential role in P. falciparum egress. Based on these published data, we wanted to test if PfERC functions in exoneme exocytosis but organelle discharge during egress or invasion has never been observed in live parasites. In the first aim, we developed fluorescence microscopy-based assays to investigate signal-dependent exoneme exocytosis and we will develop assays to assess calcium oscillations during egress of live P. falciparum. In PfERC conditional mutants, these live microscopy assays show that PfERC knockdown inhibits exoneme exocytosis. Using a quantitative proteomic approach, we have identified PfERC interactors and generated conditional mutants for prioritized candidates. Another candidate, rhoptry neck protein 11 (RON11), is essential for merozoite invasion. Therefore, in the second aim, we will focus on P. falciparum invasion and the proposed research will lead to the development of reporters to study rhoptry discharge as well as calcium oscillations in live merozoites during invasion. Using these assays, we will test the function of RON11 in rhoptry discharge, calcium oscillations as well as merozoite invasion into the RBC. The proposed research will identify several putative targets for antimalarial drug development. Since organelle secretion is required for egress and invasion of Plasmodium parasites during all stages of their lifecycle, this research may lead to the identification of pan-active antimalarials.