Molecular mechanisms of sporogonic development in malaria parasites

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New therapies for prevention and treatment of malaria, and for reducing transmission, are urgently needed. This project aims to increase our understanding of the molecular mechanisms underlying the formation and function of the crystalloid - a malaria parasite organelle found uniquely in the ookinete and young oocyst stages that is essential for sporogonic development and sporozoite transmission from mosquito to human. A palmitoyl-S-acyl transferase enzyme named DHHC10 resides in the crystalloid and is essential for crystalloid formation and parasite transmission. DHHC10 catalyses the addition of a palmitoyl lipid to proteins, a reaction known as palmitoylation. This project will focus on the role of palmitoylation in crystalloid biogenesis and function. Using the Plasmodium berghei mouse malaria model, we will determine the substrate range, spatiotemporal dynamics and mechanisms of DHHC10-mediated palmitoylation in crystalloids via three specific objectives: Objective 1: Determine the crystalloid palmitome. Using a method called acyl-biotin-exchange (ABE) the palmitoyls on palmitoylated proteins are chemically exchanged for biotin, which allows their specific capture and purification with microbeads that bind biotin. Following this, the palmitoylated protein fraction (palmitome) is analyzed by mass spectrometry (MS) to determine the identities of the individual protein components. By comparing the palmitomes from ookinetes that express DHHC10 and those that do not (DHHC10-knockouts), the substrates of DHHC10 can be identified, which correspond to the crystalloid palmitome. In parallel, the ookinete palmitome of a DHHC-positive, but crystalloid-negative, parasite line (LAP3-knockout) will be determined to study the spatiotemporal dynamics of DHHC10-mediated palmitoylation. The results will provide a global view of the palmitoylated protein repertoire in the whole ookinete and in the crystalloid, and will identify new crystalloid components.Objective 2: Identify proteins that interact with DHHC10. Proteins that interact with DHHC10 (either subunits of a DHHC10 protein complex, or DHHC10 substrates) will be identified by two approaches. (i) GFP pull-down: ookinetes that express DHHC10 fused to green fluorescent protein (GFP) will be used to capture DHHC10 protein complexes using magnetic beads that can bind to the GFP, and these complexes will then be analyzed by MS to determine their protein composition. (ii) BioID: ookinetes that express DHHC10 fused to a biotin ligase (BirA*) can be used to specifically biotinylate DHHC10 and its neighbour proteins upon addition of excess biotin to the cells. This allows their specific capture with magnetic beads that bind to biotin, followed by MS analysis. BioID analysis of ookinetes expressing a different crystalloid protein (LAP3) fused to BirA* will be carried out to validate the DHHC10 results. The results will identify new protein partners and substrates of DHHC10, as well as new crystalloid components, and provide new insight into the spatiotemporal dynamics and mechanisms of DHHC10-mediated palmitoylation. Objective 3: Validate new candidate crystalloid proteins. To make sure that newly identified proteins of the crystalloid palmitome and DHHC10 interactome are genuinely associated with crystalloid biogenesis and/or function, they will be functionally characterized using fluorescent protein tagging and gene knockout approaches in genetically modified parasites. The combined results obtained from this project will build a better and more comprehensive picture of the molecules that are associated with the crystalloid organelle and are involved in, or subject of, crystalloid-specific S-palmitoylation. They will provide important new insight into the molecular mechanisms that facilitate crystalloid genesis and function, and form a platform for the identification of new drug targets specific for sporogonic development, providing new strategies to prevent transmission.