Characterization of the ATP4 ion pump in Toxoplasma gondii

The Plasmodium falciparum ATPase PfATP4 isthetarget of a diverse range of antimalarial compounds, including the clinical drug candidate cipargamin. PfATP4 was originally annotated as a Ca2 transporter, but recent evidence suggests that it is a Na efflux pump, extruding Na in exchange for H. Here we demonstrate that ATP4 proteins belong to a clade of P-type ATPases that are restricted to apicomplexans and their closest relatives. We employed a variety of genetic and physiological approaches to investigate the ATP4 protein of the apicomplexan Toxoplasma gondii, TgATP4. We show that TgATP4 is a plasma membrane protein. Knockdown of TgATP4 had no effect on resting pH or Ca2 but rendered parasites unable to regulate their cytosolic Na concentration ([Na]cyt). PfATP4 inhibitors caused an increase in [Na]cyt and a cytosolic alkalinization in WT but not TgATP4 knockdown parasites. Parasites in which TgATP4 was knocked down or disrupted exhibited a growth defect, attributable to reduced viability of extracellular parasites. Parasites in which TgATP4 had been disrupted showed reduced virulence in mice. These results provide evidence for ATP4 proteins playing a key conserved role in Na regulation in apicomplexan parasites.