Functionality of the V-type ATPase during asexual growth and development of Plasmodium falciparum

Abstract

V-type ATPases are highly conserved hetero-multi-subunit proton pumping machineries found in all eukaryotes. They utilize ATP hydrolysis to pump protons, acidifying intracellular or extracellular compartments, and are thus crucial for various biological processes. Despite their evolutionary conservation in malaria parasites, this proton pump remains understudied. To understand the localization and biological functions of Plasmodium falciparum V-type ATPase, we employed CRISPR/Cas9 to endogenously tag the subunit A of the V1 domain. V1A (PF3D7_1311900) was tagged with a triple hemagglutinin epitope and the TetR-DOZI-aptamer system for conditional expression under the regulation of anhydrotetracycline. Via immunofluorescence assays, we identified that V-type ATPase is expressed throughout the intraerythrocytic developmental cycle and is mainly localized on the digestive vacuole and parasite plasma membrane. Immuno-electron microscopy further revealed that V-type ATPase is also localized on secretory organelles in merozoites. Knockdown of V1A led to cytosolic pH imbalance and blockage of hemoglobin digestion in the digestive vacuole, resulting in an arrest of parasite development in the trophozoite stage and, ultimately, parasite demise. Using Bafilomycin A1, a specific inhibitor of V-type ATPases, we found that the P. falciparum V-type ATPase is likely involved in parasite invasion but is not critical for ring stage development. Further, we detected a large molecular weight complex in BN-PAGE (∼ 1.0 MDa), corresponding to the total molecular weights of V1 and Vo domains. Together, we show that V-type ATPase is localized on multiple subcellular compartments in P. falciparum, and its functionality throughout the asexual cycle varies depending on the parasite developmental stages.