Fluorescence-based bioassay for the detection of changes in mitochondrial physiology during artemisinin-induced growth arrest of blood-stage Plasmodium falciparum

The emergence of artemisinin (ART) resistance in Plasmodium falciparum challenges global malaria treatment programs. To avoid ART-induced death, a proportion of ring-stage parasites arrest and resume cyclic intraerythrocytic growth, leading to recrudescent malaria. However, the direct tracking of mitochondrial physiological and dynamic changes underlying this survival mechanism in P. falciparum remains poorly characterized by traditional mitochondria-based fluorochromes. The aim of this study was to use the mitochondrion specific, pH-sensitive fluorochrome Mtphagy to monitor mitochondrial changes during ART exposure. Mtphagy dye can be detected in the intact mitochondria of all blood-stage parasites. After dihydroartemisinin (DHA) exposure, the fluorescence intensity of Mtphagy dye is greater in the early ring-form trophozoites, indicating a decrease in pH. Despite the presence of undetectable lysosomes, pH-sensitive Mtphagy and mitochondria-specific MitoTracker signals slightly reduced colocalization after DHA-induced growth arrest. Inhibition of mitochondrial fission and the PI3K-mediated pathway significantly reduced the number of Mtphagy-positive parasites and was associated with delayed recrudescence in the DHA-treated parasites. Taken together, these data demonstrate that the Mtphagy-based fluorescence assay serves as a useful tool for detecting mitochondrial changes during growth arrest. Therefore, targeting changes in mitochondrial dynamics may offer a promising strategy for identifying, monitoring, and potentially overcoming ART resistance in malaria parasites.