Plasmodium falciparum Chaperones and Stress Response

Malaria remains a major public health challenge that causes 219 million cases and 435,000 deaths in 2017. During their complex life cycle, Plasmodium parasites (the causative agents of malaria) encounter different cellular stresses due to the changes in the microenvironment, host immune responses and cellular metabolism during rapid parasite growth and expansion. Understanding how Plasmodium reacts to the stresses will provide an opportunity to better control malaria.

Current evidence shows that 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. In Chapters 2 and 3, 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.

In addition to PI(3)P and Hsp70s, parasite’s tolerance against artemisinin also correlates with the expression of the Plasmodium TCP-1 ring complex or chaperonin containing TCP-1 (TRiC/CCT), an essential hetero-oligomeric complex required for de novo cytoskeletal protein folding. In Chapter 4 to 6, we found that the P. falciparum TRiC can be targeted by the antihistamine clemastine by utilizing parallel chemoproteomic platforms. Clemastine destabilized all eight P. falciparum TRiC subunits based on thermal proteome profiling (TPP). Further analysis using stability of proteins from rates of oxidation (SPROX) revealed a clemastine-induced thermodynamic stabilization of the Plasmodium TRiC delta subunit, suggesting an interaction with this protein subunit. We demonstrate that clemastine reduces levels of the major TRiC substrate tubulin in P. falciparum parasites. In addition, clemastine treatment leads to disorientation of Plasmodium mitotic spindles during the asexual reproduction and results in aberrant tubulin morphology suggesting protein aggregation. This clemastine-induced disruption of TRiC function is not observed in human host cells, demonstrating a species selectivity required for targeting an intracellular human pathogen. Our findings encourage larger efforts to apply chemoproteomic methods to assist in target identification of antimalarial drugs, and highlight the potential to selectively target Plasmodium TRiC-mediated protein folding for malaria intervention.