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Abstract
Malaria is a deadly disease caused by Plasmodium falciparum parasites. The clinical manifestations of malaria are closely tied to parasite proliferation within human red blood cells (RBCs), initiated by merozoite invasion of RBCs. Intracellular parasites then begin a process of remodeling host cells by protein export to create a favorable environment for development. Secretion of parasite proteins is essential for the success of both invasion and remodeling. In this manuscript we describe the study of these two processes by screening interactors of proteins involved in protein export, and studying the role of a protein essential for merozoite invasion. Protein export is essential for remodeling human RBCs. However, the process by which membrane proteins are extracted from the parasite's plasma membrane for export remains a mystery. In one study, we addressed this question by fusing the exported membrane protein SBP1 with TurboID, a highly efficient biotin ligase (SBP1TbID). Through time-resolved proximity biotinylation and quantitative proteomics, we identified two groups of SBP1TbID interactors: early (pre-export) and late (post-export) interactors. Significantly, two membrane-associated proteins emerged as pre-export interactors, one harboring a predicted translocon domain, potentially facilitating the export of membrane proteins. Conditional mutants of these candidates were found essential for asexual growth and localized at the host-parasite interface during early intraerythrocytic stages, suggesting their role in guiding membrane proteins from the parasite plasma membrane for export to the host RBC.
Invasion of RBCs involves effectors secreted from specialized organelles like the rhoptries. Our research focused on Rhoptry Neck Protein 11 (RON11), containing seven transmembrane domains and a calcium-binding EF-hand domain. RON11 knockdown mutants inhibited parasite growth by preventing merozoite invasion of RBCs. Utilizing ultrastructure expansion microscopy (U-ExM), we observed unique phenotypes, with RON11 depletion leading to fully developed merozoites featuring single rhoptries. Surprisingly, RON11 loss did not affect attachment or rhoptry effector release but blocked merozoite internalization. Furthermore, RON11 participated in forming the second rhoptry pair in the final schizogony stages, coinciding with merozoite segregation. In summary, RON11 is a key player in generating two rhoptries and is critical for merozoite internalization into RBCs, shedding light on the invasion mechanisms of P. falciparum.