Phosphatidylinositol 3-phosphate and Hsp70 protect Plasmodium falciparum from heat-induced cell death

Phosphatidylinositol 3-phosphate (PI(3)P) levels in Plasmodium falciparum correlate with tolerance to cellular stresses caused by artemisinin, a first-line malaria treatment, and environmental factors. However, the functional role of PI(3)P in the Plasmodium stress response and a possible mechanism of protection were unknown. Here, we used multiple chemical probes including PI3K inhibitors and known antimalarial drugs to examine the importance of PI(3)P under thermal conditions that recapitulate malaria fever. Live cell microscopy using both chemical and genetic reporters revealed that PI(3)P stabilizes the acidic and proteolytic digestive vacuole (DV) under heat stress. We demonstrate that heat-induced DV destabilization in PI(3)P-deficient P. falciparum precedes cell death and is reversible after withdrawal of the stress condition and the PI3K inhibitor. These phenotypes are not observed with an inactive structural analog of the PI3K inhibitor. A chemoproteomic and biochemical approach identified PfHsp70-1 as a parasite PI(3)P-binding protein. Targeting PfHsp70-1 with a small molecule inhibitor phenocopied PI(3)P-deficient parasites under heat shock. Furthermore, tunable knockdown of PfHsp70-1 showed that PfHsp70-1 downregulation causes DV destabilization and hypersensitizes parasites to heat shock and PI3K inhibitors. Our findings underscore a mechanistic link between PI(3)P and PfHsp70-1, and present a novel PI(3)P function in stabilizing the DV compartment during heat stress.