# PfATP2 is a flippase on the Plasmodium falciparum surface that is important for growth and influences parasite sensitivity to antiplasmodial compounds

## TL;DR

This study shows that PfATP2, a protein on the malaria parasite's surface, is important for its growth and affects how sensitive it is to certain antimalarial drugs.

## Contribution

The study identifies PfATP2 as a phospholipid flippase and potential drug target in Plasmodium falciparum.

## Key findings

- PfATP2 localizes to the parasite surface and is important for growth.
- Overexpression of PfATP2 reduces sensitivity to MMV665794, MMV007224, and MMV665852.
- PfATP2 influences the internalization of phosphatidylserine in parasites.

## Abstract

Antimalarials play a crucial role in the fight against malaria. However, resistance of the most virulent malaria parasite, Plasmodium falciparum, to front-line antimalarials is spreading. To identify new antimalarials, millions of compounds have been screened for their ability to inhibit the growth of blood-stage P. falciparum parasites. To gain insight into the mode of action of novel compounds and the ease by which parasites can acquire resistance to them, many have been tested in ‘in vitro evolution’ experiments, in which parasites are exposed to the compound for a prolonged period of time. In a recent study, parasite resistance to two compounds, MMV007224 and MMV665852, was associated with amplification of the pfatp2 gene, implicating PfATP2, a putative phospholipid flippase, as a parasite drug target or resistance determinant. These two compounds, along with MMV665794 (which is structurally related to MMV007224), had previously been reported to dysregulate pH in parasites. Here, we show that PfATP2 localises to the parasite surface and is important for parasite growth. We demonstrate that parasites genetically engineered to overexpress PfATP2 display reduced sensitivity to MMV665794, MMV007224 and MMV665852 compared to parasites with a normal expression level of the protein, and that parasites in which PfATP2 is knocked down become hypersensitive to the three compounds. We show that PfATP2 expression level does not affect the cytosolic pH of parasites, or the potency by which MMV665794 or MMV007224 dysregulate parasite pH. We show that PfATP2-overexpressing parasites internalise a fluorescent phosphatidylserine analogue (NBD-PS) at a greater rate than parasites with a normal expression level of PfATP2, and that parasites in which PfATP2 is knocked down have a reduced rate of NBD-PS uptake. Further, we provide evidence that MMV665794 and MMV007224 give rise to a reduction in NBD-PS internalisation. Together, our data are consistent with PfATP2 serving as a major ATP-dependent phosphatidylserine internalisation mechanism on the parasite plasma membrane, and being a potential target of MMV665794 and MMV007224.

Recent screens have identified numerous compounds active against the most virulent species of human malaria parasite, Plasmodium falciparum. Previously, researchers exposed parasite cultures to many such compounds, including MMV007224 and MMV665852, for a prolonged time period to select for resistant parasites. Parasites with resistance to MMV007224 and MMV665852 were found to have an increased copy number of the gene encoding a putative phospholipid flippase, PfATP2. Here we sought to understand how PfATP2 affects parasite sensitivity to the compounds. Using genetically modified parasites, we found that PfATP2 is important for parasite growth and localises to the parasite surface. We showed that increasing the expression level of PfATP2 renders parasites less sensitive to MMV007224 (and the structurally similar molecule MMV665794) and MMV665852, and that decreasing its expression level makes parasites more sensitive to the compounds. We found that the rate by which parasites internalise a fluorescent analogue of phosphatidylserine varied with PfATP2 expression level, consistent with PfATP2 serving as a phospholipid flippase. We tested MMV665794 and MMV007224 in a variety of assays and found that they have multiple effects on the parasite, including inhibition of phosphatidylserine internalisation. Together, the data are consistent with the possibility that inhibition of PfATP2 contributes to parasite killing by the compounds.