Molecular processes essential for parasite sporogony.

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Apicomplexan parasites are widespread protozoan parasites of animals, which include major pathogens of humans, domestic animals and livestock. Many existing measures against these parasites remain insufficient for disease control, and new strategies for prophylaxis, treatment and control of transmission are urgently needed. Sporogony is defined as the production of sporozoites by repeated divisions of a zygote. It is an essential part of the life cycles of many apicomplexan parasites including Plasmodium, the causative agent of malaria, where sporogony takes place in the mosquito within extracellular encysted forms named oocysts. In Plasmodium, sporogony is critically dependent on a unique organelle named the crystalloid, which is exclusively found in the ookinete (a motile form of the zygote) and young oocyst stages. Many of the crystalloid proteins thus far identified in Plasmodium are conserved in and unique to the Apicomplexa, supporting the hypothesis that the molecular processes underlying sporogony are at least partly conserved across the phylum. In this proposal, we will use an integrated cross-disciplinary approach to study the crystalloids of Plasmodium berghei aimed at increasing our fundamental knowledge of the essential cellular and molecular processes underlying sporogony. This will be achieved via the following specific objectives:Objective 1: Study crystalloid loss in the oocyst by microscopy. Using various parasites lines expressing different crystalloid proteins tagged with fluorescent protein as markers of the crystalloid organelle, combined with superresolution confocal microscopy, the destination of the crystalloid and its protein cargo will be investigated in live oocysts during sporogony. This will help elucidate the processes involved in loss of the organelle at the subcellular level, and our understanding of crystalloid function.Objective 2: Refine the crystalloid protein interactome. Three transgenic parasite lines expressing different crystalloid proteins (LAP3, NTH and TPM2) each fused to a twin fluorescent protein/affinity tag (GFP/Strep) will be generated and used to harvest protein complexes from ookinetes by Strep-based affinity purification, followed by quantitative mass spectrometry-based protein identification. These analyses will produce a high confidence crystalloid protein interaction network (interactome).Objective 3: Determine the crystalloid proteome. Parasite lines expressing the GFP/Strep-tagged crystalloid membrane proteins NTH and TPM2 will be used to purify intact organelles from mechanically lysed ookinetes by Strep-based affinity purification, followed by quantitative mass spectrometry-based protein identification. This analysis will help determine the entire crystalloid protein repertoire (proteome) irrespective of protein interactions.Objective 4: Establish the crystalloid metabolome. Whole ookinetes and Strep-based affinity-purified crystalloid organelles from parasites expressing GFP/Strep-tagged NTH (generating NADPH), and a parasite line expressing an enzymatically inactive NTH version, will be subject to mass spectrometry-based global metabolite analysis. These analyses will allow differences in the metabolite repertoire (metabolome) of NTH-deficient parasites to be identified, which will help inform which NADPH-dependent biosynthetic processes are taking place in the organelle. Objective 5. Validate newly identified crystalloid proteins. Select proteins identified from the above objectives will be validated by GFP tagging and gene knockout studies in transgenic parasite lines to make sure they are genuinely linked with crystalloid function and sporogony.The collective results will provide new basic knowledge of the essential functions of the crystalloid in sporogony, and help to identify new molecules and molecular pathways for rational intervention strategies against development and transmission of malaria and related apicomplexan parasites