Molecular mechanisms of sporogonic development in malaria parasites

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.

New therapies for prevention and treatment of malaria, and for reducing malaria 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 transmission from mosquito to human. A crystalloid-resident palmitoyl-S-acyl transferase (DHHC10) is essential for crystalloid formation, indicating that S-palmitoylation of crystalloid proteins is critical to these processes. This project aims to study the mechanisms of sporogonic development focussing on the role of palmitoylation in crystalloid biogenesis and function. Using the rodent malaria parasite Plasmodium berghei, we will dissect the substrate range, spatiotemporal dynamics and mechanisms of DHHC10-mediated palmitoylation in crystalloids. The objectives are: (1) Using acyl-biotin-exchange (ABE) and label-free quantitative mass spectrometry (LFQMS), the ookinete palmitome will be determined of DHHC10-positive and DHHC10-negative parasite, allowing the identification of DHHC10 substrates and the crystalloid palmitome; (2) Proteins that interact with DHHC10 (either subunits of a DHHC10 protein complex, or its substrates) will be isolated by GFP pull-down using ookinetes expressing GFP-tagged DHHC10, and identified by LFQMS. In a second approach, interactors for DHHC10 will be identified by BioID using ookinetes that express DHHC10 fused to a biotin ligase; (3) New candidate crystalloid proteins identified will be validated using fluorescent protein tagging and gene knockout in transgenic parasite lines. The results will provide important new insights 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.

The proposed research provides fundamental knowledge of malaria parasite biology that could lead to the development and application of novel transmission-blocking and other anti-parasitic strategies, and hence to improvements in human health.

Benefits to clinical research base / Health service (medium to long-term):
This research has potential impact on the control of malaria, an important human disease. The clinical research base will benefit from this when novel intervention strategies identified require further development and testing (e.g. through clinical trials). The health service will benefit long-term by having new and better chemotherapeutics available for control of the disease, and through the ensuing decrease in malaria incidence.

Benefits to pharmaceutical industry (medium to long-term):
The project will contribute to the pharmaceutical industry through identification of new drug targets of malaria parasite development, particularly sporogonic development. These can be further exploited and developed, ultimately resulting in economic benefits to this industry sector. We will harness intellectual property from our research in the form of patents, which will help forge links with industry.

Benefits to education and training of scientists (short and long-term):
The project will benefit the involved collaborators, postdoc and students (PhD and MSc) through their exposure to a cross-disciplinary research proposal that provides opportunities to acquire a wide range of skills and knowledge of experiments and data analysis, including transferable skills such as communication, writing and time management. Our research is also communicated to the large community of postgraduate students who engage in MSc studies at the LSHTM every year and whose research projects are part of our ongoing research programme.

Benefits to the wider public (medium to long-term):
(A) By contributing to better disease control this project will contribute to a healthier human workforce (particularly in malaria endemic countries), which in turn will improve human wealth, quality of life, creative output, and enhance national and global economic performance.
(B) Intellectual property from this research may be harnessed in the form of patents, which could contribute to the United Kingdom's wealth and economic competitiveness.
(C) The research conducted by us is expected to result in internationally competitive publications, which will impact positively on the academic standing of the LSHTM, which in turn will benefit all LSHTM staff and students, and ultimately the UK's academic and economic competitiveness.
(D) We are actively involved in Public Engagement in Science by communicating our work to pupils from local London schools, for which there exists an active programme at LSHTM, and through contributions at science fairs. Results that have commercial or public interest are publicized via the LSHTM External Relations Office and open access LSHTM websites (such as the LSHTM Malaria Centre websites), informing the general public.