Title: (Re)solving Mitochondrial Mysteries: Structural and Functional Characterization of Critical Mitochondrial Complexes In Malaria-Causing Parasites

Abstract:

Malaria is a pressing global health challenge, causing about 200 million infections and 600,000 deaths annually. Due to the waning efficacy of frontline antimalarials, there is an increasing need to identify new antimalarial drug targets. Although the mitochondrion of malaria-causing Plasmodium falciparum parasites is a validated target of currently used antimalarial drugs, it remains an enigmatic organelle with many processes poorly understood. Here, we characterize the functions of a recently identified P. falciparum mitochondrial protein, PfATAD3; this is the first study of ATAD3A in unicellular organisms. Genetic knockdown of PfATAD3 revealed its essentiality in asexual stage development as parasites undergo an irreversible growth arrest in their second asexual lifecycle. Assessment of PfATAD3-knockdown parasites via northern blotting, transmission electron microscopy (EM), and live cell super-resolution imaging showed defects in mitochondrial RNA processing and stability, mitochondrial and cellular ultrastructure, as well as mitochondrial membrane potential. Furthermore, we showed that PfATAD3 is present in a multi-megaDalton complex with specific mitochondrial proteins, including inner membrane translocases. These findings highlight the critical role of PfATAD3 in mitochondrial and cellular viability of asexual P. falciparum parasites. To perform a more expansive investigation on the mitochondria of these parasites, we conducted in-situ cryo-correlative light-electron imaging involving vitrification of parasitized red blood cells, cryo-confocal imaging, cryo-focused ion beam milling and scanning EM, and cryo-electron tomography. Using this integrative workflow, we have generated 3D visualizations of the mitochondria in its near-native state. Downstream analyses involve further computational processing and sub-tomogram averaging to resolve the structure of critical mitochondrial machinery including PfATAD3 in asexual P. falciparum parasites.