Functional investigations of PfSPM3 – A microtubule associated protein of the malaria parasite Plasmodium falciparum.

• Applicant: Professor Dr. Tim-Wolf Gilberger
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 546727552

The malaria parasite has evolved a complex and unique life cycle alternating between invertebrate vectors and vertebrate hosts. To enable infection of different hosts, malaria parasites have developed several highly specialized and morphologically distinct developmental stages that are adapted to a specific niche within different hosts and tissues. The amazing morphogenesis of this eukaryotic single cell organism is driven by reorganization of cytoskeletal structures, enabling the parasite to infect and exploit the specific physiological requirements of the targeted host cells and tissues. The expansive host range and physiology across the group of 200+ Plasmodium species has led to species specific differences in how they regulate their cytoskeletal structures. An interesting example of inter-species morphological variation is the mosquito transmissible gametocyte stage of development. Gametocytes are the blood-circulating pre-sexual stages critical for malaria transmission. In P. falciparum, the biological agent that causes the most severe form of malaria in humans, the mature gametocyte is banana (falciform) shaped. This distinct morphology is believed to favor the transmigration of mature gametocytes from their bone marrow reservoir to the bloodstream for transmission. We recently identified and characterized a novel subpellicular microtubule associated protein we termed sub-pellicular microtubule protein 3 (SPM3). This previously unknown protein is conserved within the genus Plasmodium but absent in other Apicomplexa. Using a number of reverse genetic approaches, we showed that SPM3 deficiency in P. falciparum leads to round, and not banana shaped, gametocytes with an aberrant sub-pellicular microtubule pattern (SPMT) that support cell structure. To enable a full characterization of SPM3s function in gametocyte develop and support this application we i) used a BioID approach to identify novel SPMT associated proteins and ii) established a cryo-EM workflow that enables ultra-high resolution analysis of overall architecture of the SPMTs and other key structures as well as number of protofilaments per SPMT. We hypothesize that SPM3 either directly - or in concert with other proteins - provides "custom-tailored" scaffolds that links the SPMT to the inner membrane complex for different parasite stages and that these might differ between P. falciparum and related Laveranian species and non-Laveranian species. This application is focused on PfSPM3 function during gametocytogenesis as a model to investigate the varied function of this protein and has three aims: (1) Investigation of the microtubule and cellular architecture in P. falciparum SPM3 deficient parasites using cryo-EM, (2) Interspecies complementation to identify whether SPM3 from different species can recapitulate the P. falciparum gametocyte falciform shape, (3) Functionally characterize novel P. falciparum SPMs identified through BioID.

• DFG Programme: Research Grants