Chloroquine induces eryptosis in P. falciparum-infected red blood cells and the release of extracellular vesicles with a unique protein profile

Malaria is a vector-borne parasitic disease that affects millions worldwide. In order to reach the objective, set by the World Health Organization to decrease the cases by 2030, antimalarial drugs with novel modes of action are required. Previously, a novel mechanism of action of chloroquine (CQ) was reported involving features of programmed cell death in the parasite, mainly characterized by calcium efflux from the digestive vacuole (DV) permeabilization. Increased intracellular calcium induces the suicidal death of erythrocytes also known as eryptosis. This study aimed to identify the hallmarks of eryptosis due to calcium redistribution and the downstream cellular effects during CQ treatment in iRBCs. Plasmodium falciparum 3D7 at mid-late trophozoites were used for the antimalarial drug treatment. Our results revealed increased phosphatidylserine (PS) exposure, cell shrinkage and membrane blebbing, delineating an eryptotic phenotype in the host RBC. Interestingly, the blebs on the surface of the iRBCs released to the extracellular milieu become extracellular vesicles (EVs) which are essential for intercellular communication due to their cargo of proteins, nucleic acids, lipids and metabolites. The proteomic characterization displayed 2 highly enriched protein clusters in EVs from CQ-treated iRBCs, the proteasome and ribosome. We demonstrated that this unique protein cargo is not associated with the parasite growth rate. Additionally, we found that these particular EVs might activate IFN signaling pathways mediated by IL-6 in THP-1-derived macrophages. Our findings shed new insights into a novel drug-induced cell death mechanism that targets the parasite and specific components of the infected host RBC. IMPORTANCEOur previous studies have shown that chloroquine (CQ) treatment in iRBCs triggers Plasmodium falciparum digestive vacuole (DV) membrane permeabilization leading to calcium redistribution. Interestingly, increased intracellular calcium concentration is the main inducer of the suicidal death of red blood cells (RBCs) called eryptosis. The present study shed new insights into a novel CQ-induced cell death mechanism that targets the parasite and the infected host RBC by inducing key phenotypic hallmarks of eryptosis: PS exposure, cell shrinkage and membrane blebbing. Moreover, the proteomic characterization of the blebs released to the extracellular milieu also known as extracellular vesicles (EVs) revealed a cargo highly enriched in ribosomal proteins and proteasome subunits relevant for host-parasite interactions. These findings highlight CQs effect on calcium homeostasis disruption in infected red blood cells (iRBCs) with cellular and immunological consequences of great significance for malaria pathogenesis and potential clinical implications.