Design of a two-fluorophore pH biosensor targeted to the digestive vacuole of Plasmodium falciparum

Abstract

The maintenance of pH homeostasis is critical for biological processes in all organisms, with the malaria parasite, Plasmodium falciparum, being no exception. The parasite food vacuole is a major acidic digestive organelle, where the pH is thought to be regulated by proton pumps in its membrane. The acidic environment promotes the action of enzymes involved in hemoglobin degradation, and polymerization of toxic heme into hemozoin. This biological process is vital for the parasite and a proven chemotherapeutic target. Thus, determining the pH dynamics along the parasite life cycle and in the context of antimalarial drugs might provide unique information about the parasite pathophysiology, cell metabolism and function, shedding light into drugs mechanism of action and possibly target(s) disclosure. To reliably measure the pH of P.falciparum digestive vacuole along its life cycle, we designed a genetically encoded fluorescent sensor strategy, consisting of fusing the ratiometric pHLuorin2 with emiRFP670 to the falcipain-2 protein, a cysteine protease located in this compartment.This work was supported by Fundação para a Ciência e Tecnologia (FCT) national funds, under the national support to R&D units grant, through the reference project UIDB/04436/2020 and UIDP/04436/2020. V. Baptista thanks FCT for the SFRH/BD/145427/2019 grant. Susana Catarino thanks FCT for her contract funding provided through 2020.00215.CEECIND. Maria Isabel Veiga thanks FCT for her contract funding provided through 2020.03113.CEECIND