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Exploring the native mitochondrial structural landscape with functional characterization of a critical mitochondrial complex in malaria-causing Plasmodium falciparum parasites
Dissertation

Exploring the native mitochondrial structural landscape with functional characterization of a critical mitochondrial complex in malaria-causing Plasmodium falciparum parasites

Ijeoma Chiamaka Precious Okoye
Doctor of Philosophy (Ph.D.), Drexel University
Aug 2026
DOI:
https://doi.org/10.17918/00011515
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Okoye_Ijeoma_20269.50 MB
PDF Dissertation Embargoed Access, Embargo ends: 31 Aug 2027
mp4
Okoye_Ijeoma_2026_Suppl115.05 MB
Video (supplemental) 3D super-resolution live cell imaging videos demonstrating mitochondrial membrane potential in PfATAD3 (+) parasites Embargoed Access, Embargo ends: 31 Aug 2027
mp4
Okoye_Ijeoma_2026_Suppl242.72 MB
Video (supplemental) 3D super-resolution live cell imaging videos demonstrating mitochondrial membrane potential in PfATAD3(-) parasites Embargoed Access, Embargo ends: 31 Aug 2027
avi
Okoye_Ijeoma_2026_Suppl3235.36 MB
Video (supplemental) 2D animation of tomogram reconstruction showing native intracellular features including the mitochondrion and cytosolic ribosomes in asexual P. falciparum parasites Embargoed Access, Embargo ends: 31 Aug 2027
avi
Okoye_Ijeoma_2026_Suppl432.51 MB
Video (supplemental) 3D segmentation of native intracellular features including the mitochondrion and cytosolic ribosomes in asexual P. falciparum parasites Embargoed Access, Embargo ends: 31 Aug 2027

Abstract

AAA+ ATPases ATAD3 Mitochondria Plasmodium falciparum Malaria Microbiology
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 recently identified P. falciparum mitochondrial protein, PfATAD3, which is homologous to the ATAD3A proteins broadly present in many eukaryotic lineages including Metazoa and plants. While in those multi-cellular organisms ATAD3A has been implicated in essential mitochondrial functions, our study is the first to examine ATAD3 in a unicellular organism, and importantly with medical significance. 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. Altogether, our findings establish PfATAD3 as a critical factor for mitochondrial function in malaria parasites, with ongoing structural analyses expected to yield deeper mechanistic insights and inform future antimalarial drug design.

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