The rational to study the interaction network of exported proteins in Plasmodium falciparum lies in the crucial importance of the export of virulence factors to the surface of infected host cells and other modifications allowing the parasite’s survival in its host cell which are the sole cause of morbidity and mortality. P. falciparum, the deadliest form of malaria, exports during its blood cycle over 10% of its proteome into the host cell leading to major host cell modifications such as presentation of adhesins on the surface or change of membrane rigidity and permeability. Why the parasites need to export such large number of proteins is unknown and their interaction is not understood. Only for a small number of proteins a tentative function was determined and hardly any information on exported proteins in the transmission forms, the gametocytes exist. With this project, we aim to elucidate the dynamic interaction network of exported proteins in P. falciparum and their contribution to morbidity during the entire erythrocytic life cycle including gametocytes.
Recent technical advances to genetically manipulate P. falciparum make this project more feasible. Using the CRISPR/CAS9 system we will generate knock out parasites of a number of selected exported proteins including a number of PHIST proteins, which were frequently found in the host cell and for which we have shown that they play an important role in anchoring the virulence factor PfEMP1 to the cytoskeleton. With this system but also the alternative selection linked transfection we will generate inducible knock down clones or introduce endogenous tags. All clones will be used for phenotypical analyses using an array of established and innovative methods (e.g.
microsphiltration membrane rigidity measures, electron-microscopy for ultrastructural analyses). Protein-protein interaction will be identified using conventional immuno-precipitations but also the BioID system which introduces a promiscuous biotin ligase, which subsequently allows the identification of biotinylated proteins that were in close proximity of the protein of interest. Potential interactions will be confirmed and validated by various techniques such as NMR, fluorescent polarization experiments, fluorescence cross correlation spectroscopy, or split GFP system. We finally expect to obtain through this project an interaction network that is important for Maurer’s cleft function and transport and fixation of PfMP1 on the erythrocyte surface. In particular, we expect clarification of the function of PHIST
proteins and their interaction with host cytoskeleton proteins.