Multifunctionality of 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 eukaryotic organisms. They use ATP hydrolysis to pump protons, acidifying intracellular or extracellular compartments, and are thus crucial for various biological processes. Despite being evolutionarily conserved in malaria parasites, this proton pump remains understudied. To understand the localization and biological function of V-type ATPase in the deadliest human malaria parasite Plasmodium falciparum, we utilized CRISPR/Cas9 to endogenously tag the subunit A of the V1 domain at the C-terminus. V1A (PF3D7_1311900) was tagged with a triple hemagglutinin (3HA) epitope and TetR-DOZI-aptamers for conditional expression under the regulation of anhydrotetracycline. Through immunofluorescence assays, we identified that V-type ATPase was expressed throughout the intraerythrocytic developmental cycle and was mainly localized on the digestive vacuole and plasma membrane. Immuno-electron microscopy further revealed that V-type ATPase was also localized on secretory organelles, such as rhoptries 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 BN-PAGE/Western blot, we detected a large molecular weight complex ([~] 1.0 MDa) corresponding to the total molecular weights of V1 and Vo domains. The complex was readily disrupted by the V-type ATPase specific inhibitor Bafilomycin A1, but not by low glucose conditions or treatment with chloroquine. Together, our data suggest that V-type ATPase is localized on several subcellular compartments in P. falciparum and plays critical roles to support malaria parasites to grow and replicate inside red blood cells.