https://orcid.org/0000-0003-1063-5571
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Figures
Abstract
Malaria remains an urgent threat to global health as the mortality and infection rates keep rising annually and our frontline antimalarials are becoming less effective due to the emergence and spread of resistance-conferring mutations. Although the mitochondrion of P. falciparum parasites is a validated drug target, there remain many uncharacterized mitochondrial proteins. The goal of this study was to investigate the essentiality and functions of a recently identified mitochondrial protein - PF3D7_0707400. Our results show that PF3D7_0707400 is an ATAD3A homolog that is essential to parasite survival and is present in a megadalton complex that is critical for multiple mitochondrial processes such as mitochondrial RNA stability, membrane potential, ultrastructure, and protein import. ATAD3A has been previously studied in multicellular eukaryotes and has been implicated in several childhood mitochondrial diseases, with suggested functions in mitochondrial nucleoid stabilization, mitochondrial RNA translation, and mitochondrial inner membrane integrity. This study is the first characterization, to our knowledge, of ATAD3A in unicellular organisms. Our findings here expand our knowledge on apicomplexan mitochondrial biology and our arsenal of potential antimalarial drug targets.
Author summary
Each year, malaria is responsible for about 200 million infections and 600,000 deaths across the world. Thus, it constitutes a huge global health crisis. Increasing rates of antimalarial resistance necessitates the identification and characterization of novel parasitic proteins that can be exploited for the development of new antimalarial therapeutics. To this end, the mitochondrion of P. falciparum parasites has been studied as a validated target for effective antimalarials. However, much remains to be understood about critical mitochondrial processes and proteins that are essential for mitochondrial viability and parasite survival. Our study details the first characterization of an ATAD3 protein in a unicellular eukaryote, specifically in an apicomplexan parasite. The conservation of this protein in these deep-branching organisms highlights the importance of its biological functions, further emphasizing the significance of our study. By employing advanced molecular biology techniques, we show the presence of PfATAD3 in a giant molecular complex and its essentiality in asexual P. falciparum parasites. Conditional knockdown of PfATAD3 resulted in defects in critical mitochondrial processes such as mitochondrial RNA stability, mitochondrial membrane potential, and mitochondrial morphology. Divergence of PfATAD3 from the host allows for exploitation of this protein as a target for new antimalarials.
Citation: Okoye IC, Lamb IM, Cheung Y-W, Morrisey JM, Sharma M, Kumar R, et al. (2026) ATAD3 megadalton complex in Plasmodium falciparum is essential for mitochondrial and cellular viability. PLoS Pathog 22(6): e1014317. https://doi.org/10.1371/journal.ppat.1014317
Editor: Sean T. Prigge, Johns Hopkins Bloomberg School of Public Health, UNITED STATES OF AMERICA
Received: October 3, 2025; Accepted: May 29, 2026; Published: June 3, 2026
Copyright: © 2026 Okoye et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Dataset for proteomics is included in the Supplemental Information. Whole genome sequencing data are deposited at the NCBI Sequence Read Archive under project PRJNA1444484.
Funding: ABV; R01 AI028398; National Institutes of Health; https://www.nih.gov; The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Y-WC; R35 GM156396; National Institutes of Health; https://www.nih.gov; The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Y-WC; 1022785; Burroughs Wellcome Fund; https://www.bwfund.org; The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Malaria has plagued humanity for millennia. Even today, more than half the world’s population is at risk of malaria infection. Hence, malaria remains a significant global health problem. Malaria is caused by unicellular apicomplexan parasites, Plasmodium spp., transmitted by infected female Anopheles mosquitoes [1]. Amongst the Plasmodium spp. that cause human malaria, P. falciparum is the species that causes the most fatalities [1,2]. The prevalence and incidence of malaria have increased in the last decade due to multiple factors including increased resistance to existing antimalarial therapies [3–10]. Thus, there is a continued need to develop new and effective antimalarial drugs to counter the ever-present threat of drug resistance.
The mitochondrion of P. falciparum is a validated antimalarial drug target [11,12]. Some antimalarials already in use, such as atovaquone, target mitochondrial components [13]. Each asexual Plasmodium parasite possesses a single mitochondrion that differs significantly from the human mitochondrion, having multiple divergent features and reduced metabolic function [14,15]. The P. falciparum mitochondrion houses about 30 copies of a 6 kb mitochondrial DNA element. This 6 kb DNA encodes mitochondrial ribosomal RNA fragments and three proteins that are components of Complexes III and IV of the Plasmodial electron transport chain – cytochrome c oxidase I and III (COX I/COXIII), and cytochrome b (Cyt b) [16–19]. Aside from the three proteins made in the mitochondrion, there are more than 400 proteins that are encoded by the parasite’s nuclear genome but are imported into the mitochondrion to perform an array of critical functions including mitochondrial DNA replication/transcription, mitochondrial mRNA translation, ubiquinone biosynthesis, pyrimidine biosynthesis, and Fe-S cluster synthesis [12]. However, several essential mitochondrial processes, such as tRNA import into the mitochondrion and mitochondrial ribosomal assembly, are still poorly understood. Therefore, much remains to explore and understand about mitochondrial biogenesis and functioning in P. falciparum parasites, with a view to identify potential targets for selective inhibition by new antimalarials.
Previously, through proximity biotinylation with mitochondrially targeted biotin ligase TurboID, we identified 122 putative mitochondrial proteins of P. falciparum parasites [20]. One of these proteins was PF3D7_0707400, annotated as ATPase family AAA+ (ATPases associated with diverse cellular activities) domain containing protein 3 (PfATAD3). PfATAD3 is distantly related to a human protein, ATAD3A, which is a nuclearly encoded protein that has been demonstrated to have or modulate multiple mitochondrial functions including mitochondrial nucleoid stabilization, mitochondrial protein synthesis, mitochondria-endoplasmic reticulum inter-organellar interactions, and mitochondrial inner membrane protein scaffolding [21–31]. Mutations in ATAD3 are implicated in many childhood mitochondrial diseases and are responsible for a variety of disorders in humans, ranging from neurological diseases to cancers [32–38]. Until now, there has been no experimental characterization of this protein in P. falciparum or other apicomplexan parasites. This study represents the first characterization, to our knowledge, of ATAD3 in a unicellular system. Here, we demonstrate that PfATAD3 is essential for the growth of P. falciparum parasites and is present in a large multi-megadalton complex involved in critical mitochondrial processes.
Results
PfATAD3 is a homolog of human ATAD3A and is found in the P. falciparum mitochondrion
Human ATAD3A is a 586-amino-acid protein classified as belonging to the AAA+ ATAD3 family of proteins with conserved ATAD3 and ATPase domains at its N and C termini, respectively. It has additional features such as a proline rich motif (PRM) and coiled-coil regions (CC1 and CC2) at its N-terminus, which extend into the cytoplasm of the cell, as well as transmembrane domains (TM1 and TM2), which span the inner and outer mitochondrial membranes (Fig 1A). PfATAD3 is a 663-amino-acid protein that possesses conserved N-terminal ATAD3 and C-terminal AAA+ family ATPase domains, as well as potential TM helices, but lacks the PRM (Fig 1A). As AAA + ATPases are known to exist as hexamers, we carried out a comparison of the predicted 3D structures of the hexameric human ATAD3A and PfATAD3 protein complexes, revealing a probable structural homology with a TM-score of 0.557 and RMSD of 0.964 (Fig 1A, S1A Fig). A pairwise global alignment between human ATAD3A and PfATAD3 showed 29.59% sequence identity between the two proteins (Fig 1B). We performed a phylogenetic analysis to determine the evolutionary relationships of ATAD3A in apicomplexan parasites and other eukaryotes (S1B & S1C Figs). Homologs of PfATAD3 exist in other apicomplexan parasites and Myzozoans, including Cryptosporidium parvum which has no mitochondrial DNA but has a mitosome (a mitochondrion-derived organelle that does not produce ATP). Plants and algae also possess ATAD3A homologs (S1B & S1C Fig). Interestingly, there are no ATAD3A homologs in fungi. A multiple sequence alignment of the ATAD3 homologs across apicomplexan parasites revealed a significant conservation in sequence similarity, including the Walker A, Walker B, and Arginine Finger domains that are crucial for ATPase activity and oligomerization (S1D Fig). Plasmodium spp. parasites tend to have distinctly long C-terminal extensions in addition to the conserved domains, indicating potential specific functions of ATAD3 in Plasmodium (S1D Fig).
(A) Sequence and structural homology analysis of human ATAD3A (Above) and Pf3D7_0707400 (Below). Sideview orientations on the left side and top-down view on the right side. Domains are distinguished by colors: Coiled-coil domains in purple, Transmembrane 1 (TM1, Outer Membrane) domain in yellow, Transmembrane 2 (TM2, Inner Membrane) domain in red, ATPase domain in dark blue. (B) Pairwise sequence alignment between Pf3D7_0707400 and human ATAD3A indicating 29% sequence identity and a conservation of critical domains including the Walker A domain (highlighted in green), the Walker B domain (highlighted in light blue), and the conserved Arginine finger (highlighted in yellow).
Next, we sought to determine the localization of PfATAD3 through immunofluorescence microscopy. To facilitate this, we generated endogenously 3xHA-tagged parasites using CRISPR/Cas9 homology-directed recombination (S2A-S2E Fig). The transgene was engineered to conditionally express PfATAD3 under TetR-DOZI aptamer regulation. Immuno-electron microscopy of these transgenic PfATAD3-TetR-3xHA parasites demonstrated PfATAD3 localization to the mitochondrion of asexual P. falciparum parasites (Fig 2A, S3A Fig). Localization of PfATAD3 to the mitochondrion has been previously validated through an immunofluorescence assay with MitoTracker using ectopically expressed PfATAD3 [20]. Another transgenic parasite line was generated in which PfATAD3-3xHA-TetR parasites also express an mScarlet fluorescent tag targeted to the mitochondrion via the leader sequence of the mitochondrial chaperone protein PfHSP70–3 [39]. Mitochondrial localization of the PfHSP70–3-mScarlet has been previously validated in [39], and integration of the mScarlet fluorescent tag in our transgenic line was shown via whole-genome sequencing (NCBI Sequence Read Archive (SRA) [PRJNA1444484]). Integration PCR demonstrated proper integration of gene modification cassettes and consequently confirmed the transgenic PfATAD3-3xHA-mitomScarlet parasite line (S2E Fig). Immunofluorescence assay using these PfATAD3-3xHA-mito-mScarlet parasites showed significant colocalization of PfATAD3 with the mitochondrion across the ring, trophozoite, and early schizont asexual stages of the parasite (Fig 2B).
(A) Immuno-electron microscopy of PfATAD3-HA-TetR parasites demonstrating localization of PfATAD3 to the mitochondrion of asexual P. falciparum parasites using gold colloidal anti-mouse coated beads and mouse anti-HA antibodies. To the right is a zoomed-in view of PfATAD3 in the double-membraned mitochondrion. [RBC – Red Blood Cell; DG – Digestive Vacuole; N – Nucleus; ER – Endoplasmic Reticulum]. Representative image from three biological replicates. (B) Immunofluorescence Assay of PfATAD3-HA/mito-mScarlet parasites demonstrating localization to the mitochondrion across asexual stages of P. falciparum parasites. Mander’s Correlation Coefficient (MCC) – quantitative measure of colocalization between PfATAD3-HA and mito-mScarlet.
PfATAD3 is essential for the growth and development of asexual P. falciparum parasites
We generated a transgenic PfATAD3-3xHA-TetR parasite line also ectopically expressing PfTOM22 (Translocase of Outer Mitochondrial Membrane 22) fused to a fluorescent mNeonGreen tag at its N-terminus to assess essentiality and functions of PfATAD3 in asexual stage P. falciparum parasites (S2 Fig, S3B Fig). Proper mitochondrial localization of PfTOM22-mNeonGreen has been previously validated in [40], and integration of the mNeonGreen fluorescent tag in our transgenic line was shown via whole-genome sequencing (NCBI Sequence Read Archive (SRA) [PRJNA1444484]). Proper integration of gene-modification cassettes was demonstrated by integration PCR, thereby confirming the transgenic PfATAD3-3xHA-TOM22-mNeonGreen parasite line (S2E Fig). Removal of aTc from the culture medium at ring stages resulted in ~95% knockdown of PfATAD3 expression as early as 24 hours later (Fig 3A,and 3B, S4 Fig). The deterioration of parasite morphology visualized by Giemsa staining (Fig 3C), together with measurement of the declining parasite growth rate via flow cytometry (Fig 3D), demonstrated that the parasites in the absence of PfATAD3 were in a state of growth arrest by 72 hours post aTc withdrawal and effectively died in the second intraerythrocytic asexual cycle. We further assessed the viability of PfATAD3(-) parasites by adding back aTc at different time points following the knockdown induced at ring stages. We observed that a significant proportion of 24 h and 48 h PfATAD3(-) parasites remained viable, as their growth resumed following aTc addition at these timepoints (Fig 3E). However, after 72 h and 96 h, a significant proportion of PfATAD3(-) parasites were no longer viable, indicating minimal growth recovery relative to the earlier timepoints as they were unable to resume their growth after addback of aTc at these timepoints (Fig 3E). Thus, knockdown of PfATAD3 results in a growth arrest and eventual death of parasites within the second asexual cycle, indicating that PfATAD3 is essential for asexual P. falciparum parasite viability, growth and development.
(A) Western blot and (B) Quantification of anhydrotetracycline (aTc)-mediated conditional regulation of PfATAD3 expression. PfAldolase was used as the loading control. The bar plot is an average of three independent biological replicates (mean ± SD). Statistical analyses were conducted between normalized + /- PfATAD3 conditions via a multiple unpaired t-test on Prism. (C) Visualization of Giemsa-stained + /-PfATAD3 parasites by light microscopy indicating normal morphology and growth of PfATAD3(-) knockdown parasites for the first asexual cycle but the development of a growth arrest, aberrant morphology, and eventual death by the second asexual cycle. (D) Growth curve via flow cytometry of cumulative parasitemia for each condition (+/-PfATAD3) and each timepoint (every 24 hours for six days). 100,000 red blood cells were counted for each sample. The blue curve represents PfATAD3(+) parasites while the red curve represents PfATAD3(-) knockdown parasites. The growth curve is an average of three independent biological replicates. Statistical analyses were conducted via a two-way ANOVA on log-transformed values on Prism. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001. (E) Growth curves demonstrating the loss of cell viability by 72h of PfATAD3 knockdown. Arrows indicate the timepoint that aTc was added back, i.e., 24h, 48h, 72h, and 96h. Solid lines represent +aTc control, dash lines represent the aTc addback condition, and the red line represents continuous –aTc knockdown condition. N = two independent biological replicates. Statistical analyses were conducted via a two-way ANOVA on log-transformed values on Prism. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.
PfATAD3 is critical for mitochondrial function in asexual P. falciparum parasites
To assess the morphology and functionality of the mitochondrion in PfATAD3(-) parasites, we used the PfATAD3-3xHA-TetR/PfTOM22 parasite line. PfTOM22 imaging permitted visualization of the outer mitochondrial membrane morphology of the parasite, while staining of these parasites with Tetramethylrhodamine ethyl ester (TMRE) and MitoTracker dyes enabled assessment of the mitochondrial membrane potential. Quantitative measurement of the TMRE mean fluorescence intensity demonstrated a decline in mitochondrial membrane potential in PfATAD3(-) parasites within the first cycle of PfATAD3 knockdown, albeit non-statistically significant, with a nearly total loss by the second cycle following the knockdown (Fig 4A and S5A Fig). Furthermore, super-resolution live cell imaging of PfATAD3(+) parasites stained with MitoTracker showed colocalization with PfTOM22 as expected due to a stable mitochondrial membrane potential (S5B and S5C Fig). However, a loss of mitochondrial membrane potential was observed in PfATAD3(-) parasites 48 h after initiation of the knockdown, as revealed by the appearance of parasites with dispersed MitoTracker staining (S5B and S5C Fig, S1 Movie and S2 Movie). Meanwhile, outer mitochondrial membrane morphology in PfATAD3(-) parasites remained unaltered as indicated by PfTOM22 organization in a normal tubular morphology after 48 hours of PfATAD3 knockdown (S5B and S5C Fig). These observations suggest the importance of PfATAD3 for the maintenance of mitochondrial membrane potential, which contributes to mitochondrial physiology in asexual P. falciparum parasites. Notably, the continued retention of MitoTracker in the parasite cytoplasm suggests that the plasma membrane potential in the parasites remained intact at 48 h after knockdown initiation, even as the mitochondrial membrane potential was disrupted. This is consistent with our observation that the parasites were viable at 48 h following PfATAD3 knockdown in the aTc complementation growth assays.
(A) Quantification of normalized TMRE mean fluorescent intensity to assess mitochondrial membrane potential. N = three independent biological replicates (mean ± SEM). (B) Absolute quantification of cytochrome b copy number in +/- PfATAD3 parasites; average of two independent biological replicates (mean ± SD). (C) Northern blot of mitochondrial messenger and ribosomal RNA transcripts in +/- PfATAD3 parasites. (D-H) Quantification of northern blot in (C) using FIJI software. Average of three independent biological replicates (mean ± SEM). (I) Denaturing RNA gel electrophoresis demonstrating equivalent loading of total RNA isolated from +/- PfATAD3 samples as well as stability of cytosolic ribosomal RNA in +/- PfATAD3 parasites. (J) Northern blot of nuclear-encoded PfATP4 messenger RNA in +/- PfATAD3 samples. Statistical analyses were conducted via an unpaired T-test on Prism. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.
Next, we assessed the effect of PfATAD3 knockdown on the mitochondrial DNA and its transcription. Digital PCR was used for absolute quantification of the mitochondrial DNA, which showed no significant change in its copy number upon the knockdown of PfATAD3 (Fig 4B). Furthermore, we used northern blot analysis and denaturing RNA agarose gel electrophoresis to assess mitochondrial and cytoplasmic RNA levels respectively following PfATAD3 knockdown (Fig 4C and Fig 4I). At 24 h following PfATAD3 knockdown, the levels of mitochondrial mRNAs encoding Cyt b, COX I/III and of small mitochondrial rRNA transcripts were noticeably reduced (Fig 4C-4G). Notably, a statistically significant decrease was observed in the levels of Cyt b mRNA and ribosomal RNA transcripts. After 72 hours of PfATAD3 knockdown, minimal mitochondrial mRNA and rRNA transcripts were detected (Fig 4C-4G). However, cytosolic 18S and 28S RNAs were relatively stable irrespective of PfATAD3 knockdown (Fig 4I). Nuclear-encoded PfATP4 mRNA also remained relatively stable upon PfATAD3 knockdown (Fig 4J). The stability of the cytosolic RNAs and PfATP4 mRNA serves as a control for any effects of long-term aTc withdrawal. Interestingly, a 2.3 kb RNA, which is likely an unprocessed precursor of mitochondrial ribosomal RNA, remained mostly unchanged in its abundance during the knockdown (Fig 4H). Overall, these results show that PfATAD3 plays a crucial role in maintaining the stability and abundance of processed mitochondrial RNA transcripts in asexual P. falciparum parasites. We note that our data do not distinguish between direct or indirect consequences of PfATAD3 knockdown on mitochondrial RNA profiles.
PfATAD3 is important for functions beyond pyrimidine biosynthesis/ubiquinone regeneration
The primary function of the mitochondrial electron transport chain (mtETC) in blood stage P. falciparum parasites is to support de novo pyrimidine biosynthesis due to the parasite’s inability to salvage pyrimidines [41]. Mitochondrially localized dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in this synthetic pathway and requires ubiquinone as the electron acceptor. The reduced ubiquinone needs to be re-oxidized to continue to serve as an electron acceptor for DHODH, a step catalyzed by Complex III of the mtETC. Treatment of parasites with Complex III inhibitors kills the parasites since ubiquinone regeneration is essential for parasite survival. However, supplementation with decylubiquinone can partially rescue the pyrimidine biosynthesis pathway and consequently the growth of the parasites even when Complex III is inhibited [42]. To assess whether PfATAD3 has roles in addition to maintaining the mtETC activity, we induced knockdown of PfATAD3 in parasites supplemented with 25 mM or 50 mM decylubiquinone. In contrast to the PfATAD3(+) atovaquone-treated parasites whose growth defect was partially reversed upon supplementation with decylubiquinone, PfATAD3-knockdown parasites were not rescued by the addition of decylubiquinone (Fig 5A and 5B). Therefore, the functions of PfATAD3 appear to extend beyond maintaining the essential mitochondrial process of ubiquinone regeneration and pyrimidine biosynthesis.
(A) Growth curve of cumulative parasitemia against time after drug (Atovaquone, Decylubiquinone) treatment. Statistical analyses were conducted between the Atovaquone-treated vs decylubiquinone-rescue conditions via a two-way ANOVA on log-transformed values on Prism. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001. (B) Growth curve of cumulative parasitemia against time after PfATAD3 knockdown/ Decylubiquinone treatment. N = three independent biological replicates (mean ± SEM). Statistical analyses were conducted between the PfATAD3-knockdown vs decylubiquinone-rescue conditions via a two-way ANOVA on log-transformed values on Prism. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.
PfATAD3 is present in a multi-megadalton complex in asexual P. falciparum parasites
AAA + ATPases typically exist in a hexameric complex and are associated with additional proteins [43,44]. To determine if PfATAD3 exists within a complex, we proceeded to conduct native gel electrophoreses. Although a parasitophorous vacuolar membrane protein complex, PfEXP2, migrated on a standard Blue-Native polyacrylamide gel (BN-PAGE) at the expected size of about 720 kilodalton, no band for PfATAD3 could be detected in the western blots of the BN-PAGE (S6A Fig). Treatment with minimal amounts of SDS separated the giant complex into its subcomplexes that could then be seen on a commercial BN-PAGE (S6A Fig). We reasoned that the native complex containing PfATAD3 was too large to enter the standard 4–12% BN-PAGE gel. Hence, we employed composite agarose-polyacrylamide native gel electrophoresis with a larger pore size than the standard BN-PAGE. On this gel, the PfATAD3 complex migrated at a position greater than 1 megadalton as judged from the migration pattern of PfEXP2 complex (Fig 6A, S6B and S6C Fig).
(A) Large-pore composite agarose-polyacrylamide gel showing specific presence of >1MDa PfATAD3 complex in PfATAD3-HA (+) parasites (B) Volcano plot of fold change (log base 2) against the p-value illustrating proteins significantly enriched with PfATAD3. Points are color-coded based on predicted localization: mitochondrial proteins are denoted in green, an ER-localized protein is denoted in purple, and the others are denoted in pink. N = three independent biological replicates.
To determine what proteins are present in this multi-megadalton complex, we carried out affinity isolation of PfATAD3 complex using anti-HA magnetic beads followed by mass spectrometry and proteomic analysis. As a negative control, HA affinity isolation was carried out with wildtype P. falciparum parasites lacking tagged proteins. Using a cutoff of a log2fold change enrichment of 20 and a p-value of 5% (p < 0.05), we generated a list of proteins that were significantly enriched with tagged PfATAD3 relative to untagged controls (Fig 6B, S1 Table). We included annotated cellular localization and mutagenesis index scores of these proteins obtained from PlasmoDB, the Plasmodium bioinformatic database (S1 Table). Some of these enriched proteins have also been assessed in published literature to be mitochondrially localized. As expected, PfATAD3 was the most enriched mitochondrial protein closely followed by inner membrane protein complexes such as another AAA + ATPase protein PF3D7_1119600 (a putative PfFTSH zinc metalloprotease) and Translocases of the Inner Mitochondrial Membrane, PfTIM50 and PfTIM23 (critical components of the mitochondrial import machinery). Mitochondrial DNA repair proteins such as DNA (apurinic or apyrimidinic site) endonuclease (PF3D7_0305600) and flap endonuclease I (PF3D7_0408500) were also significantly enriched in the HA-pulldown. The significance of other proteins enriched with PfATAD3 remains to be investigated.
Loss of PfATAD3 results in defects in the mitochondrial and cellular ultrastructure of asexual P. falciparum parasites
As ATAD3 proteins have been shown to be crucial for inner membrane proteins scaffolding as well as cristae formation [21,45], we sought to assess the morphology of the mitochondrion as well as the cellular ultrastructure of parasites within the first (36 h) and second cycle (84 h) of PfATAD3 knockdown using transmission electron microscopy. Close examination of the mitochondrion in the parasites lacking PfATAD3 for 36 h reveals a marked increase in the width and area, as well as a reduction in the electron intensity in the mitochondrial matrix compared to their wildtype counterparts (Fig 7A-7D). Furthermore, we observed significant aberrations in the intracellular membrane integrity and organization in the parasites without PfATAD3 for 84 h (Fig 7A-7D). This finding is consistent with our observation of a loss of mitochondrial RNA and cellular viability at 72 h post PfATAD3 knockdown. Hence, PfATAD3 is important for maintaining proper mitochondrial morphology and, consequently, cellular viability in asexual P. falciparum parasites.
(A) Transmission electron micrographs of +PfATAD3 (control) parasites – left panels and –PfATAD3 (knockdown) parasites (right panels) at 36 h (top panels) and 84 h (bottom panels). Mitochondrion is marked as ‘M’. Scale bar is 200nm. (B) Quantification of the width (um), (C) area (um2), and (D) pixel intensity of the mitochondrion on FIJI illustrated via Box and Whiskers plot (Tukey). Statistical analyses were conducted via unpaired t-tests.
Discussion
In this report we characterize ATAD3, a mitochondrial AAA + ATPase, from the unicellular eukaryote P. falciparum. All previous studies on ATAD3 have been performed from multicellular eukaryotes [31]. In multicellular eukaryotes, ATAD3 has been associated with a myriad of functions such as mitochondrial inner membrane protein assembly and structure, mitochondrial DNA nucleoid dynamics, mitochondrial protein synthesis, lipid metabolism, and cholesterol trafficking [21,22,25,27,30,35,36,46]. Its deletion results in embryonic lethality in worms, fruit flies, and mice [24,45,47,48]. In humans, multiple mutations in ATAD3 gene have been associated with neurological and developmental disorders including various forms of cancer [32,34,37,49,50]. Interestingly, the ATAD3 gene is absent among the fungal lineage, yet it is present in much more evolutionarily distant organisms such as apicomplexan parasites. Indeed, ATAD3 was initially thought to be confined only to multicellular eukaryotes [24]. Therefore, our investigation represents the first exploration of its apparent homolog in a unicellular eukaryotic mitochondrion.
As is characteristic of all AAA + ATPases, ATAD3 proteins have the conserved ATPase domain comprising of Walker A and B domains that allow for the binding and hydrolysis of ATP, respectively. The energy derived from ATP hydrolysis drives molecular processes attributed to AAA + ATPases such as translocation of macromolecules, including nucleic acids and proteins, enabling DNA unwinding and replication, as well as protein unfolding and transport [33,44,51]. Previous studies on mammalian ATAD3A have demonstrated a fascinating topology of human ATAD3A as a membrane protein complex with its C-terminal ATPase domain localized in the mitochondrial matrix, its transmembrane domains I and II anchoring the complex in the inner and outer mitochondrial membranes, and its N-terminal coiled-coil domain extending into the cytoplasm to mediate interactions with other organelles such as the endoplasmic reticulum (ER) [24,28]. Although all ATAD3 homologs across humans and apicomplexan parasites have the conserved Walker A and Walker B ATPase motifs as well as the Arginine Finger, there are some features that are unique to either human ATAD3A or P. falciparum ATAD3. Mammalian ATAD3A has a lineage-specific proline-rich motif (PRM) at its N-terminus that has been suggested to be involved in distinct functions such as mitochondria-ER inter-organellar interactions [22]. In contrast, P. falciparum ATAD3 lacks a PRM but has a C-terminal extension of about 100 amino acids that may confer its own specific functions in the parasites. Additionally, it is particularly notable that Cryptosporidium species, with a minimal mitochondrial remnant lacking a genome, also maintain ATAD3 genes. Conservation of ATAD3 over this vast evolutionary distance indicates essential, but possibly distinct, functions that this mitochondrial protein may serve in different lineages of multicellular and unicellular eukaryotes.
Using a robust conditional knockdown system, we show here that PfATAD3 is an essential protein for development of asexual stages of P. falciparum. Parasites appear to remain in a growth arrested stage in the absence of PfATAD3 up to 72 h. Remarkably, we observed dramatic reduction of mitochondrially transcribed RNA as early as 24 h following the ATAD3 knockdown. Messenger RNAs as well as ribosomal RNAs encoded by the parasite mtDNA were barely detectable at 72 h following the knockdown. Cytoplasmic RNAs, however, did not seem to be affected. Interestingly, a 2.3 kb RNA moiety that is likely to be a precursor of mitochondrial rRNAs according to previous reports remained unchanged during the same period of PfATAD3 knockdown [52,53]. Similarly, the copy number of mitochondrial DNA also remained unchanged during this period. These data suggest that PfATAD3 is necessary for accumulation and/or processing of mitochondrial RNA transcripts. Absence of PfATAD3 would therefore result in reduced production of mitochondrially encoded mtETC components and diminished respiration. As expected, the arrested parasites underwent disruption of their mitochondrial membrane potential. This phenocopies previous observations with atovaquone treatment of asexual P. falciparum parasites where the parasites remain viable for a duration of time until eventual death despite an inhibited mtETC [54]. However, the growth arrest does not appear to be solely due to a lack of ubiquinone regeneration (the main function of mtETC in the blood stage of the parasite to serve pyrimidine biosynthesis [41]), since addition of decyl-ubiquinone failed to rescue parasite growth in the absence of PfATAD3 [42]. Thus, the absence of PfATAD3 appears to have a pleiotropic effect on mitochondrial functions in a manner that leads to parasite growth arrest and subsequent demise. This is consistent with existing literature on metazoan systems describing altered mitochondrial functions including reduced expression of mtETC complexes and dysregulated respiratory activity upon loss or mutation of ATAD3 [21,37,46]. However, we remain limited in our ability to distinguish which of these mitochondrial defects are because of direct or indirect effects of PfATAD3 knockdown, similar to the functions of ATAD3A in metazoan systems.
This defect of mitochondrial functions in PfATAD3 (-) parasites was also observed in tandem with defects in the mitochondrial ultrastructure of these parasites. Transmission electron microscopy revealed significant morphological changes in the mitochondrion of PfATAD3 (-) parasites. As early as 36 hours post PfATAD3 knockdown, the mitochondrion appeared swollen and with reduced internal electron density. This may be because of the reduced expression of mtETC complex protein components seen at 24 hours post PfATAD3 knockdown. This would inhibit critical processes such as protein import into the mitochondrion or assembly of essential mitochondrial inner membrane complexes. By 84 hours post PfATAD3 knockdown, the mitochondrion looked degenerated, coinciding with the data demonstrating loss of cellular and mitochondrial viability at this time. Changes in mitochondrial ultrastructure upon knockout or mutations of mammalian ATAD3 such as altered cristae structure, disrupted inner-outer mitochondrial membrane contact sites, electrolucent matrix areas, and overall degeneration have also been observed in mice and humans [35,55,56]. Although asexual P. falciparum mitochondrion largely lacks cristae, our data shows that PfATAD3 still plays a critical role in maintaining normal mitochondrial morphology and ultrastructure in a unicellular eukaryotic organism.
To further understand the molecular functions of PfATAD3, we studied its native complex. Typically, AAA + ATPase proteins oligomerize into hexameric complexes which can form pores [51,57]. ATAD3 proteins are predicted to form hexameric complexes that have been suggested to traverse the inner and outer mitochondrial membranes [22,43,51,57]. Although PfATAD3 is predicted to also form similar hexameric complexes, we found it to be present in a much larger megadalton complex that could only be displayed by composite large-pore native gel electrophoresis. This apparent size far exceeds six times the molecular mass of PfATAD3 that would be expected of a PfATAD3 hexamer (i.e., 468kDa), suggesting the presence of additional candidate interacting proteins.
Proteomic analysis of immuno-isolated PfATAD3 complex revealed the identity of several associated protein candidates, including the components of the TIM23 complex (Translocase of Inner Membrane; TIM23, TIM50), which mediate import of nuclearly-encoded inner mitochondrial membrane and matrix proteins critical for essential mitochondrial processes such as heme biosynthesis, Fe-S cluster biosynthesis, DNA/RNA replication, protein synthesis, cardiolipin synthesis, ubiquinone biosynthesis, and electron transport chain activity [58]. Furthermore, another hexameric AAA + ATPase, PfFTSH, involved in protein homeostasis within the inner mitochondrial membrane was also pulled down with PfATAD3. PfFTSH1 is annotated as an AAA + ATP-dependent zinc metalloprotease, mediating mitochondrial protein processing and mitochondrial protein degradation. Experimentally, PfFTSH1 has been shown to also localize to the mitochondrion of asexual parasites and oligomerize to form a hexameric complex >700 kDa, characteristic of AAA+ proteins [59]. Higher order complexes were also observed suggesting the interaction of PfFTSH1 with other proteins, which until now have not been identified [59]. Our pulldown experiments also revealed a possible interaction of PfATAD3 with DNA repair enzymes, PfApe1 and PfFen1; suggesting a role of PfATAD3 in mitochondrial genome maintenance, which may impact abundance or expression of stable mitochondrial RNA transcripts.
The specificity of these associations is emphasized by the lack of other integral mitochondrial membrane proteins such as cytochrome b, COX I/III, and mitoribosomal proteins in the pulldown. The interaction of PfATAD3 with specific inner membrane and matrix proteins indicates a possible role in various critical mitochondrial processes such as mitochondrial protein import, mitochondrial nucleoid stabilization, mitochondrial membrane potential, and mitochondrial ultrastructure. Other proteins annotated to localize in the endoplasmic reticulum and symbiont-containing vacuoles such as shewanella-like protein phosphatase and syntaxin, respectively, are also present in the PfATAD3-HA pulldown, indicating possible roles of PfATAD3 beyond the aforementioned mitochondrial functions. Limitations of this study include a lack of evidence of a direct effect of PfATAD3 knockdown in import or localization of the nuclearly-encoded proteins or tRNAs into the mitochondrion. Isolation of mitochondria from parasites to directly and quantitatively assess impact of PfATAD3 knockdown on import and localization of mitochondrial proteins in P. falciparum is notoriously challenging due to contamination by hemozoin, the sticky crystalline by-product of hemoglobin degradation. Hence, we are currently limited to indirect assessments of mitochondrial protein import.
As this study serves as the first characterization of PfATAD3 in unicellular eukaryotes, we provide initial insights into the roles of ATAD3 in P. falciparum parasites but also generate new outstanding questions for subsequent investigation. Mutagenesis studies on metazoan ATAD3A have previously demonstrated the essentiality of the conserved Walker A and B domains to the mitochondrial and cellular functions of ATAD3A. As these domains are highly conserved in PfATAD3, we anticipate that they are also critical for PfATAD3 mitochondrial and cellular functions. However, the role of these domains in PfATAD3 activity remain unknown. Future directions of our study include generating parasite lines with various ATAD3A variants including those with mutations in the Walker A and B domains, as well as the Arginine finger motif to assess the importance of features in PfATAD3 function. Furthermore, additional experiments include proteinase K assays to determine the topology of PfATAD3 across the mitochondrial membranes and co-immunoprecipitations to assess the stability of the interactions between PfATAD3 and the identified candidate interacting proteins. Structural determination of the native PfATAD3 complex is also underway. Findings from structural, topological, and mutagenesis analysis of PfATAD3 would provide us with more details on the key residues and domains of PfATAD3 that can be exploited for rational structure-based drug design.
In conclusion, as is characteristic of AAA + ATPases, PfATAD3 is involved in many important biological processes that are critical for mitochondrial biogenesis and functioning in asexual P. falciparum parasites. This paper provides the foundation for more in-depth and expansive analyses on the role of AAA + ATPases, specifically ATAD3 proteins, in apicomplexan parasites and exploration of PfATAD3 as a potential target for novel antimalarial therapeutics.
Materials and methods
Plasmid construction
To generate a line in which PfATAD3A was endogenously tagged and expressed only in the presence of aTc, a gene containing homology regions 3’ and 5’ to the PF3D7_0707400 CRISPR/Cas9 induced double break site was synthesized by Genewiz (Azenta Life Sciences) (S2A Fig). This gene was digested with AflII and BstEII and ligated into the pMGBKRML-PfATAD3-HA plasmid which contained the TetR and aptamer sequences (S2B and S2C Fig). Prior to transfection, pMGBKRML-PfATAD3-HA vector was linearized by EcoRV digestion. For guide RNA construction, the pMKCas9 was linearized by EcoRI digestion and gel purified (S2D Fig). Guide RNA inserts were selected using analysis from the Eukaryotic Pathogen CRISPR guide RNA design tool (RRID: SCR_018297). Guide RNAs were cloned into the EcoRI-linearized pMKCas9 vector by DNA assembly reaction (NEBuilder 59 HiFi DNA Assembly Master Mix, New England Biolabs, Inc.). The PF3D7_0707400gRNA1/2 primer sequences used in this reaction are listed in S2 Table.
Parasite lines, culture, and transfections
NF54 attB P. falciparum parasites were used in this study. Asexual P. falciparum parasites were cultured in human O+ red blood cells with RPMI 1640 medium supplemented with 0.5% w/v Albumax II (Fischer Scientific), sodium bicarbonate (2.1 g/L, Corning by ThermoFisher Scientific), HEPES (15 mM, ThermoFisher), hypoxanthine (10 mg/L, Fisher Scientific), and gentamycin (50 mg/L, VWR). To generate the PfATAD3-TetR-3xHA parasite line and the PfATAD3-TetR-3xHA/TOM22-mNeonGreen parasite line, wildtype NF54 parasites and PfNF54-attB-TOM22-strepmNeongreen parasites generated as previously described [40] were transfected with 40 µg of linearized pMG75BB[3HA-10spApt-noP]-PfATAD3A plasmid as well as 20 µg of each gRNA plasmid. Transfections were performed on cultures containing 5–6% rings by electroporation via a Bio-Rad gene pulser (0.31 kV, 960 μFD). After electroporation, parasites were cultured under standard conditions for 48 hours before the addition of blasticidin (1.25 μg/mL, InvivoGen). The PfATAD3-TetR-3xHA parasites were grown in RPMI media containing 1.25 μg/mL blasticidin and 250 nM aTc while the PfATAD3-TetR-3xHA/TOM22-mNeonGreen were grown in RPMI media containing 1.25 μg/mL BSD, 250nM aTc, and 5 nM WR. To generate the parasite line where PfATAD3 was conditionally expressed and mScarlet was targeted to the mitochondrion, the PMG75-PfATAD3-tetRDOZI construct described above was transfected into the PfNF54/iGPglmS line as previously described [60]. The newly established PfNF54iGP-PfATAD3 line was then transfected with a plasmid integrating the mitochondrial targeting sequence of Hsp70–3 fused to mScarlet into the SIL7 locus as previously described [39]. This PfATAD3-3xHA-mitomScarlet line was maintained in RPMI media containing 2.5 mM glucosamine, 1.25 μg/ml blasticidin, and 250 nM aTc.
Genotyping and confirmation of transgenic parasite lines
Integration PCR to demonstrate proper integration of gene modification cassettes and consequently confirm the transgenic parasite lines – PfATAD3-HA/TOM22-mNG and PfATAD3-3xHA-mitomScarlet – was conducted (S2E Fig). Additionally, whole genome sequencing (WGS) of both PfATAD3-HA/TOM22-mNG and PfATAD3-3xHA-mitomScarlet was performed using the Illumina DNA PCR-Free library preparation kit, following the manufacturer’s instructions, and sequenced on a NextSeq 2000 platform. Raw sequencing reads were quality-filtered and adapter-trimmed using fastp (v1.3.0) [61]. Trimmed paired-end reads were aligned to a composite reference genome comprising the Plasmodium falciparum 3D7 reference genome (PlasmoDB v68) supplemented with relevant plasmid sequences using BWA-MEM (v0.7.19) [62] with default parameters. Alignments were sorted and indexed using SAMtools (v1.22.1) [63], and duplicate reads were marked using Picard (v3.4.0) [64].
Validation of gene modifications and fluorescent tags (TOM22-mNG and mScarlet) was performed by identifying discordant read pairs and soft-clipped reads, followed by clustering breakpoint positions based on soft-clipping coordinates. Regions of interest were further evaluated using SAMtools (v1.22.1) coverage profiling [63], and candidate junction-supporting reads were assembled de novo using SPAdes (v4.2.0) [65] to reconstruct integration structures. Consensus sequence reconstruction and variant calling were performed using bcftools (v1.22) [66] and GATK (v4.6.2.0) [67] respectively, with variants filtered using thresholds of minimum read depth (DP ≥ 10) and variant quality (QUAL ≥30). WGS data are available at the NCBI Sequence Read Archive (SRA) [PRJNA1444484]. Colocalization of Pfmito-mScarlet and PfTOM22-mNG with mitochondria has also been previously validated in [39] and [40] respectively. No subcloning was done in the generation of these transgenic parasite lines.
Immunofluorescence assay
50–100 µL of packed parasite cultures were pre-labeled with 60 nM MitoTracker Red CMXRos (Life Technologies by ThermoFisher Scientific) for 30 minutes at 37 ºC. The samples were washed three times with 1X PBS. The cells were fixed with 4% v/v paraformaldehyde and 0.0075% v/v glutaraldehyde in 1X PBS for an hour at 37 ºC with agitation and incubated overnight at 4 ºC. The fixed cells were washed with PBS, permeabilized with 0.25% TritonX-100 for 10 minutes, and reduced with 0.1 mg/mL NaBH4 for 5 minutes at room temperature. The cells were blocked with 3% w/v bovine serum albumin (BSA) for 1 hour, incubated in primary mouse anti-HA antibodies 1:300 in BSA/PBS;(sc-7392 Santa Cruz Biotechnology) overnight at 4 ºC, washed 3X in PBS and incubated in secondary Alexa Fluor 488 conjugated anti-mouse IgG 1:300 in BSA/PBS; (A32723 Invitrogen) overnight at 4 ºC. Cells were also stained with DAPI. The cells were washed and resuspended in equilibration buffer and antifade (S2828 ThermoFisher Scientific) to preserve the samples and prevent photobleaching. 3 µL of each sample was mounted on a slide and imaged using a conventional inverted Nikon-Ti fluorescent microscope.
Immuno-electron microscopy
NF54 PfATAD3-3xHA parasites were synchronized with alanine (0.3 M in 10 mM HEPES, pH 7.6) and enriched by a MACS Cell Separation Column (MiltenyiBiotec) at the trophozoite stage. 25–50 µL of the enriched trophozoites were fixed with 1 mL of 2% paraformaldehyde, 2.5% glutaraldehyde, and 100 mM sodium cacodylate for 1 hour on a rotator at room temperature. The cells were washed in sodium cacodylate buffer, embedded in 10% gelatin, and infiltrated overnight with 2.3 M sucrose and 20% polyvinyl pyrrolidone in PIPES/MgCl2 at 4 ºC. The samples were then trimmed, frozen in liquid nitrogen, and sectioned using a Leica Ultracut UCT7 cryo-ultramicrotome. 50 nm ultrathin sections were then blocked with 5% fetal bovine serum and 5% normal goat serum for 30 minutes followed by incubation with primary mouse anti-HA antibodies (sc-7392, Santa Cruz Biotechnology) for 1 hour at room temperature. After washing in blocking buffer, the sections were incubated with 18 nm colloidal gold-conjugated secondary goat anti-mouse IgG (H + L) antibodies for 1 hour. The sections were stained with 0.3% uranyl acetate and 2% methylcellulose. The stained sections were viewed on a JOEL 1200 EX transmission electron microscope with an AMT 8-megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques). All labeling experiments were done in parallel with controls lacking the primary antibody. These controls were consistently negative at the concentration of colloidal gold-conjugated secondary antibodies used in these experiments. Immuno-electron microscopy was conducted at the Molecular Microbiology Imaging Facility at Washington University, St. Louis, MO.
Parasite growth assay
The parasite culture was synchronized with 0.3 M alanine for two cycles to ensure all parasites were at the ring stage. Alanine was used for synchronization in place of sorbitol as both work under the same principle, utilizing the new permeation pathways only in late-stage parasites to induce selective osmotic lysis of these parasites [68,69]. After 48 hours, the parasites were washed three times with regular media to remove the aTc. The culture was split into two flasks where one was maintained with WR, aTc, blasticidin media serving as a control culture while the other was maintained with only WR99210 and blasticidin media serving as the knockdown culture. A 30–50 µL cell pellet was collected from each flask every 24 hours, fixed with 4% paraformaldehyde and 0.0075% glutaraldehyde for 1 hour at 37 ºC and overnight at 4 ºC. The fixed cells were washed with 1X PBS, stained with SYBR Green at a 1:10000 dilution, and then resuspended in filtered deionized water at a 1:100 pellet to water ratio. The samples were then assessed for parasitemia via flow cytometry. Thin blood smears were also made from each flask 24 hours after washout of aTc and stained with GIEMSA to manually calculate the parasitemia but also observe any morphological changes in the parasites after PfATAD3 knockdown.
Western blotting
Parasite cultures were lysed with 0.14% w/v saponin in 1X PBS supplemented with 1X protease inhibitor cocktail (P8215, MilliporeSigma). The parasite pellet was resuspended in 1X SDS/sample buffer and heated at 65 ºC for five minutes. Samples were centrifuged at 12,000rpm for five minutes and the supernatant from each sample was run on a 10% BioRad Tris-glycine polyacrylamide gel. The proteins were electroblotted onto a methanol activated PVDF membrane. The membrane was blocked in 5% fat-free milk for 1.5 hours and washed with 1X TBS/Tween (TBST) before being incubated in primary mouse anti-HA antibodies (sc-7392, Santa Cruz Biotechnology) at 1:10000 in 1% fat-free milk overnight. The membrane was washed four times with TBST for 15 minutes each and incubated in a horseradish peroxidase-conjugated (HRP) secondary goat anti-mouse antibody (A16078, ThermoFisher Scientific) overnight at a 1:10000 dilution in 2% fat-free milk. The membrane was developed with SuperSignal Dura substrate (Pierce) and imaged. As a loading control, the blots were later re-blocked, washed, and probed with a rabbit anti-PfAldolase at a 1:30000 dilution followed by a HRP secondary mouse anti-rabbit antibody at 1:30000. All other steps followed the standard protocol.
Live cell imaging
Glass bottom culture dishes (35 mm) were coated with 0.1% poly-L-lysine overnight and washed 3 times with PBS. Parasite culture (250 µL at 2.5% hematocrit) was added to the dishes and incubated for 30 min at 37 ºC to allow attachment of parasites to the culture dish. Culture dishes were washed 3 times with PBS followed by addition of phenol red-free RPMI culture medium into which MitoTracker at 61 nM final concentration and Hoechst at 10 µg/mL final concentration were added and incubated for 30 and 20 minutes at 37 ºC respectively to stain the mitochondrion and DNA respectively. Imaging was done using the Nikon Ti fluorescent microscope with the stage heater set to 37 ºC. Hoechst was visualized using a DAPI (49,6-diamidino-2-phenylindole) filter set, mNeonGreen with (fluorescein isothiocyanate) FITC, and MitoTracker was visualized using tetramethyl rhodamine isocyanate (TRITC) filter set.
Mitochondrial membrane potential assay
61 nM of TMRE was incubated with 25 ul of packed RBCs resuspended in 1ml of phenol-free media for 20 minutes. Cells were then treated with 200 nM of Bafilomycin to allow for specific accumulation of the TMRE dye exclusively in the mitochondria based on stable mitochondrial membrane potential. TMRE fluorescent intensity was then measured quantitatively across 10,000 cells and divided by the corresponding TOM22-mNG fluorescent intensity (indicative of parasitemia). These were assessed using the PE and the FITC lasers respectively. The TMRE values of the control and knockdown conditions were then normalized using the TMRE/FITC values of the CCCP-treated condition, as that was used to determine complete loss of membrane potential.
DNA and RNA Isolation
Red blood cells were lysed using 0.14% w/v Saponin to isolate parasites and washed three times with 1X PBS. DNA was isolated according to the DNA isolation from blood protocol in the QIAGEN kit. For RNA isolation, the parasite pellet was resuspended in TriZol and then RNA was extracted using the phenol-chloroform phase separation method. Extracted RNA was run on a 1% denaturing RNA agarose gel to assess RNA integrity and the concentration/purity of RNA was determined via Nanodrop.
Digital PCR
Custom QuantStudio Absolute Q DNA Digital PCR Assays were developed to determine the absolute copy number of Cytochrome b and GAPDH as measures of mitochondrial DNA and nuclear DNA copy number at 24 hours and 72 hours post PfATAD3 knockdown. Each assay consists of custom designed primers that are linked to FAM and VIC fluorescent probes to allow detection of Cyt b and GAPDH amplicons respectively (S3 Table). A 9 µL reaction mix was prepared for each sample, consisting of 1.8ul of Absolute Q DNA Digital PCR Master Mix (5X), 0.45ul of Cyt b – FAM Digital PCR Assay (20X), 0.45ul of GAPDH – VIC Digital PCR Assay (20X), 1 µL of 0.1ng/µL of genomic DNA from +/- PfATAD3-HA/TOM22-mNG parasites, and 5.3ul of nuclease-free water. Each 9 µL reaction was loaded to the bottom of the well and 15 µL of the Absolute Q Isolation buffer was loaded on top in the same well. All columns of the micro-array plate (MAP16) were sealed and loaded onto the Quant Studio Digital PCR machine. PCR thermal parameters were set to preheat to 96 ºC for 10 minutes, denature at 95 ºC for 3 seconds, anneal and extend at 60 ºC for 30 seconds (20 cycles). Both FAM and VIC channel dyes were selected to detect copies of Cytochrome b and GAPDH genes respectively. Genomic DNA from wildtype NF54 parasites was used as a positive control and nuclease-free water was used as a negative control.
Northern blotting
20 µg of total RNA was mixed with RNA loading dye, 6% formaldehyde, 50% formamide, and 1X MOPS electrophoresis buffer. The samples were incubated in a 68 ºC water bath for 5 minutes. The samples were loaded on a 1.2% agarose-2.2 M formaldehyde gel in 1X MOPS/DEPC buffer and run at 35 V at room temperature for 18–20 hours. One half of the gel was stained with ethidium bromide and visualized using a UV transilluminator to assess the presence of intact ribosomal RNA bands. The remaining gel half was rinsed 4x with DEPC-treated water to remove formaldehyde. RNA was transferred via capillary action 18–20 hours using 10xSSC onto a Genescreen PLUS nylon membrane, prewet with DEPC water and equilibrated with 10XSSC. After transfer the membrane was rinsed briefly in 2XSSC, air dried, and baked at 80 C in a vacuum oven for 2hrs. Ethidium Bromide staining and visualization of the gel post transfer indicated successful transfer of nucleic acids to the membrane as indicated by the lack of nucleic acids in the gel. The baked membrane was briefly rinsed in 2X SSPE buffer and prehybridized in 0.05 mL/cm2 hybridization solution containing 2X SSPE, 50% deionized formamide, 1% SDS, 5X Denhardts, and 10% Dextran Sulfate in a sealed bag for 2–4 hours at 42 ºC with gentle agitation. The blots were probed with gel purified 6kb PCR of P. falciparum mitochondrial DNA labeled with Easytides dATP a-p32 (Perkin Elmer BLU512Z 6000 Ci/mmol) using the Random Primer labelling method. The labelled probes were purified using an Illustra ProbeQuant G-50 micro column (GE Healthcare 28-0934-28) and labelling confirmed using Cerenkov counting in a Packard Tricarb LSC. Sheared calf thymus DNA at 100 µg/mL and probe at 1x106 cpm/mL were heated together for 5 minutes at 95 ºC and placed on ice for 15 minutes. The probe was added to the blot in the bag. The bag was resealed and incubated at 42 ºC for 18–24 hours with gentle agitation. The blot was removed from the bag and rinsed with 100–200 mL 2xSSC buffer at room temperature for 30 minutes, washed twice with 200 mL 2XSSC, 1% SDS at 50 ºC for 30 minutes each, and washed with 0.2XSSC at room temperature for 30 minutes. The signal on the blot was checked after each wash using a Ludlum model 3 Geiger counter. The blot was drained, placed on a damp Whatman filter paper, covered with plastic wrap, exposed to X-ray film for 24–72 hours at -80 ºC, and developed.
Blue-Native Page Electrophoresis
Saponin isolated Parasite pellets were solubilized with solubilization buffer (200 mM 6-aminocaproic acid, 50 mM Bis-Tris, pH 7, 1 mM EDTA, 2% digitonin, protease inhibitor cocktail 1:1000 Sigma P1812,1 mM AEBSF) overnight at 4 ºC. The samples were centrifuged at 12000 rpm for 10 minutes and the supernatants were collected. Supernatants were run on a 4–12% gradient BN-PAGE gel as described in manufacturer’s standard protocol (Invitrogen). The ladder was fixed with 40% methanol/10% acetic acid for 30 minutes, stained with colloidal Coomassie, and then destained with 8% acetic acid. The proteins in the gel were transferred to a PVDF membrane and visualized by the methods described previously.
Large pore composite agarose-polyacrylamide gel electrophoresis
6 mL of deionized water, 1 mL of 30% bis/acrylamide, 1 mL of 20X Native Buffer (Invitrogen), 1.6 mL of 30% glycerol, 200 µL of 1M MgCl2, 200 µL of 10% ammonium persulfate (APS), 10 mL of 1% melted agarose in deionized water, and 20 µL of TEMED were mixed in order. The gel was cast using BioRad minigel 1.5 mm glass plates with a 6-well 1.5 mm comb. After solidifying for 1 hour at room temperature, the gel was submerged in 1X Native Running buffer and stored at 4 ºC overnight. Samples were prepared in 1X Native Sample buffer (from Invitrogen NativePAGE Sample Prep Kit)/ 5% G-250 and loaded in the wells. The gel was run at 140 V for 30 minutes and then at 100 V until the loading dye ran off. The gel was submerged in 1X SDS running buffer for 3 minutes before transferring proteins onto a PVDF membrane and visualizing as described previously.
Immunoprecipitation and proteomic analysis
NF54 wildtype parasites and PfATAD3-HA/TOM22-mNG parasites were cultured in large volume in triplicate and then lysed with 0.14% Saponin/1x Protease Inhibitor Cocktail. Parasite pellet was solubilized with 2% digitonin, protease inhibitor cocktail (1:1000), 200 mM AEBSF, and solubilization buffer overnight at 4 ºC. The samples were centrifuged at 12000 rpm for 10 minutes and the supernatant was collected. 100 µL of an anti-HA magnetic bead solution (Invitrogen) were captured via a chilled magnetic stand and washed with 1X PBS/1x Protease Inhibitor Cocktail two times. The solubilized protein supernatant was added to the beads and incubated at 4 ºC on a rotator overnight to allow binding of the native PfATAD3 complex to the anti-HA beads. The beads were collected using the magnetic stand and washed three times with Native Wash buffer (25 mM HEPES, 10 mM MgCl2, 50 mM KCl, 100 mM NaCl, 0.2% digitonin, 1x Protease Inhibitor Cocktail). The beads were then resuspended in Native Wash buffer/0.2% digitonin/1x Protease Inhibitor Cocktail and shipped to University of South Florida Mass spectrometry & Proteomic Core Facility for further processing and analysis. Data obtained from NF54 wildtype parasites was used as a negative control. LFQ intensity values were used for quantification and statistical analysis. Visualization of the data on a volcano plot was done on GraphPad Prism.
Transmission electron microscopy
NF54 PfATAD3-3xHA/TOM22-mNG parasites were synchronized with alanine (0.3 M in 10 mM HEPES, pH 7.6). Knockdown of PfATAD3 was induced at the ring stage by washout of aTc and the parasite culture was split into control and knockdown flasks. Late-stage parasites were then enriched using MACS Cell Separation Column (MiltenyiBiotec) at 36 hours and 84 hours after induction of PfATAD3 knockdown. The enriched parasites were then resuspended in their respective fresh culture media and maintained at proper culture conditions for about 1 hour to recover. The parasites were gently spun down at 100–300 × g for 10 minutes in 15 mL conical-bottom polypropylene tubes. The pellet was resuspended in 3 mL of freshly prepared fixative (2% glutaraldehyde in 0.1 M Na-Cacodylate buffer), incubated for 5 minutes at room temperature, and kept at 4 ºC. Further processing was performed by the Electron Microscopy Technician at Thomas Jefferson University, Philadelphia, PA including secondary fixation with 1% tannic acid, further staining with 2% OsO4 and 1% UA followed by agarose embedding, dehydration, and Spurr’s embedding. Thin sections were prepared on electron microscopy grids and imaged using a FEI Tecnai 12 120 keV digital Transmission Electron Microscope. Images were acquired at magnifications from 4kx to 42kx.
Bioinformatic analyses
Amino acid sequences were retrieved using BLASTP software (https://00blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) and protein family databases to find ATAD3A-like sequences from different phylogenetic groups. Multiple sequence alignment of the chosen FASTA complete sequences was done using MAFFT (Multiple Alignment using Fast Fourier Transform) on European Bioinformatics Institute online server [70]. Low quality alignment positions were trimmed from the alignment based on Transitive Consistency Score (TCS) scoring [71] calculated on the T-Coffee server (http://tcoffee.crg.cat/tcs). Positions in which a majority of the taxa phenotypes (amino acid residues) had TCS scores 7 or higher were retained in the trimmed MSA, which was then used for the maximum likelihood phylogenetic analysis. Phylogenetic trees were generated via the Phyml server using either the full length protein sequences or only the conserved P-loop NTPase regions from ATAD3-like proteins and additional 6 AFG3-like AAA+ mitochondrial matrix metalloproteases as the outgroup. The phylogenetic trees were inferred using maximum likelihood inference software accessed at NGPhylogeny.fr [72]. Pairwise global alignment using BLOSUM62 as the scoring matrix was calculated between Human ATAD3 (UniProt ID: Q9NVI7) and PfATAD3 (UniProt ID: C0H4M1_PLAF7) and feature annotation was done using Jalview Software [73]. Predictions of localization and essentiality (Mutagenesis Index Scores) of PfATAD3 and associated proteins were obtained from the Plasmodium bioinformatic database, PlasmoDB [74,75]. Predicted 3D structures alongside the associated confidence metrics were retrieved from the AlphaFold 3.0 Server [76]. TM-scores and RMSDs were calculated using US Align [77] and USCF Chimera [78].
Statistical analyses
Unpaired t-tests were used to calculate statistical significance for the bar graphs. Two-way ANOVA tests on log-transformed values were used to calculate statistical significance for the growth curves. Fold change for the volcano plot was calculated using the median of the independent triplicate LFQ intensity raw values and a paired two-tailed distribution t-test analyses were conducted to determine statistical significance with a p-value cutoff of 0.05. 16-bit images were loaded onto FIJI and three lines of 50-pixel thickness each were drawn across the mitochondrial membranes to determine intensity measurements across the membrane. A region of interest within the mitochondrial matrix (lumen) was drawn to determine the intensity measurements within the matrix (lumen). Mean intensity measurements were subtracted from the maximum intensity measurements to calculate the inverted pixel values as the electron dense regions had lower pixel values. Calculated intensity measurements for the corresponding membrane were used to normalize the calculated lumen measurements, and an unpaired t-test was used to determine statistical significance on GraphPad Prism. To measure the width and corresponding area of mitochondrial TEM ultra sections, a 50-pixel thickness line was drawn across the smallest ends of the mitochondria. Unpaired t-test was used to calculate statistical significance on GraphPad Prism. Number of cells included for TEM statistical analyses: 36 h + PfATAD3, n = 11, 36 h -PfATAD3, n = 9, 84 h + PfATAD3, n = 12, 84 h -PfATAD3 = 12.
Supporting information
S1 Fig. (A) Confidence and similarity metrics for structural assembly modelling.
(B) Phylogenetic analyses showing the evolutionary relationship between homologs of ATAD3 amongst alveolates and other eukaryotes including Homo sapiens, Arabidopsis thaliana, Caenorhabditis elegans, Plasmodium falciparum, Toxoplasma gondii, and Cryptosporidium parvum. AFG3-like proteins were chosen for the putative outgroup as selected members of the AFG3-like proteins exhibited the highest similarity to PfATAD3 and this analysis is limited to the NTPase domain because no other AAA+ protein subclass has detectable similarity with the ATAD3 domain. (C) Phylogenetic analyses showing the evolutionary relationship between homologs of ATAD3 amongst alveolates and other eukaryotes including Homo sapiens, Arabidopsis thaliana, Caenorhabditis elegans, Plasmodium falciparum, Toxoplasma gondii, and Cryptosporidium parvum using the full-length ATAD3 sequence. (D) Multiple sequence analysis of ATAD3 homologs in apicomplexan parasites.
https://doi.org/10.1371/journal.ppat.1014317.s001
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S2 Fig. (A) Sequence of synthetic gene used to create the endogenously tagged PfATAD3A parasite line.
The sequences are color coded and identified in a key below the synthetic construct. (B) CRISPR-Cas9 homologous recombination integration cloning strategies to generate endogenously tagged PfATAD3-HA-TetR, PfATAD3-HA-TetR/TOM22-mNG and PfATAD3-HA-TetR/mito-mScarlet transgenic parasite lines using corresponding parental lines. (C) Plasmid map of the pMGBKRML-PfATAD3-HA plasmid for endogenous tagging and conditional expression. Restriction sites and the identity of each region are indicated. Created with SnapGene. (D) Plasmid map of the pMKCas9 plasmid for guide RNA construction. Restriction sites and the identity of each region are indicated. Created with SnapGene. (E) Integration PCR confirming presence of HA-tagged ATAD3 construct with TetR aptamers in both PfATAD3-HA-TetR/TOM22-mNG and PfATAD3-HA-TetR/mito-mScarlet transgenic parasite lines. Band sizes are denoted in blue while marker sizes are in black.
https://doi.org/10.1371/journal.ppat.1014317.s002
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S3 Fig. (A) Immuno-electron Micrographs showing localization of PfATAD3 to mitochondria of asexual parasites via mouse anti-HA or rabbit anti-HA primary antibodies and corresponding anti-mouse or anti-rabbit 18 nm colloidal gold particles.
(B) Immunofluorescence Assay (IFA) demonstrating colocalization of PfATAD3 to PfTOM22-mNeonGreen (Translocator of Outer Mitochondrial Membrane 22) in the mitochondria of asexual P. falciparum parasites.
https://doi.org/10.1371/journal.ppat.1014317.s003
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S4 Fig. Full gel image representative of the growth assay western blot.
https://doi.org/10.1371/journal.ppat.1014317.s004
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S5 Fig. (A) Live cell imaging of +/-PfATAD3 (48h and 96h) TetR/TOM22-mNG parasites stained with TMRE dye to assess mitochondrial membrane potential upon loss of PfATAD3 (B) Live Cell Scanning Confocal Super-resolution Microscopy of +/-PfATAD3 (48h) TetR/TOM22-mNG parasites [PfDNA – DAPI (Blue); Outer mitochondrial membrane – PfTOM22-mNG (Green); Mitochondrion – MitoTracker (Red); Host red blood cell – Wheat Germ Agglutinin (Purple).
Scale bar is 10 µm (C) An enlarged view of a representative cell from (B). (D) Representative full northern blot (left) and denaturing agarose RNA gel (right) demonstrating reduction of processed mitochondrial RNA transcripts upon knockdown of PfATAD3.
https://doi.org/10.1371/journal.ppat.1014317.s005
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S6 Fig. (A) Blue-Native PAGE of control and SDS-treated solubilized extract demonstrating the presence of multiple sub-complexes of PfATAD3.
PfEXP2 was used as a loading control. (B) Full large pore composite native gel showing PfATAD3 is present in a mega-Dalton hetero-oligomeric complex. (C) Large pore composite native gel showing aTc washout for 24h induces knockdown of the giant megaDalton PfATAD3 complex.
https://doi.org/10.1371/journal.ppat.1014317.s006
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S1 Table. Enriched interacting protein partners of PfATAD3-HA in asexual P. falciparum parasites.
https://doi.org/10.1371/journal.ppat.1014317.s007
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S2 Table. Primer sequences used in parasite line generation and validation.
https://doi.org/10.1371/journal.ppat.1014317.s008
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S3 Table. Digital PCR custom designed taqman gene expression assay details.
https://doi.org/10.1371/journal.ppat.1014317.s009
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S1 Movie. 3D super-resolution live cell imaging videos demonstrating mitochondrial membrane potential in PfATAD3 (+) parasites.
PfDNA – DAPI (Blue); Outer mitochondrial membrane – PfTOM22-mNG (Green); Mitochondrion – MitoTracker (Red); Host red blood cell – Wheat Germ Agglutinin (Purple). Scale bar is 10 µm.
https://doi.org/10.1371/journal.ppat.1014317.s010
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S2 Movie. 3D super-resolution live cell imaging videos demonstrating mitochondrial membrane potential in PfATAD3(-) parasites.
PfDNA – DAPI (Blue); Outer mitochondrial membrane – PfTOM22-mNG (Green); Mitochondrion – MitoTracker (Red); Host red blood cell – Wheat Germ Agglutinin (Purple). Scale bar is 10 µm.
https://doi.org/10.1371/journal.ppat.1014317.s011
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S1 Appendix. Full list of proteomics data of pulled down proteins using PfATAD3-HA immunopurification.
https://doi.org/10.1371/journal.ppat.1014317.s012
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Acknowledgments
We thank Hangjun Ke and Julie Verhoef for sharing the PfTOM22-mNG and PfMito-mScarlet plasmids used in the generation of parasite lines in this study. We thank Gordon Ruthel at University of Pennsylvania PennVet Imaging Core for super-resolution laser scanning confocal imaging services, Dale Chaput at University of South Florida Mass Spectrometry and Proteomics Core Facility for proteomics services, and the Jefferson University Imaging Core for Transmission EM services. We also acknowledge the Huck Institutes’ Genomics Core Facility (RRID:SCR_023645) at The Pennsylvania State University for whole genome sequencing and Ashley Price for library preparation. We acknowledge the support of the VEuPathDB resource in our work.
This work was supported by National Institutes of Health grant R01AI028398 to A.B.V., National Institute of Health R35 Award (R35GM156396) and a Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Disease Program Award (1022785) to Y.-W.C.
References
- 1. Carter R, Mendis KN. Evolutionary and historical aspects of the burden of malaria. Clin Microbiol Rev. 2002;15(4):564–94. pmid:12364370
- 2.
Zekar L, Sharman T. Plasmodium falciparum malaria. Treasure Island (FL): StatPearls; 2023.
- 3. Asua V, Conrad MD, Aydemir O, Duvalsaint M, Legac J, Duarte E, et al. Changing prevalence of potential mediators of aminoquinoline, antifolate, and artemisinin resistance across uganda. J Infect Dis. 2021;223(6):985–94. pmid:33146722
- 4. Balikagala B, Fukuda N, Ikeda M, Katuro OT, Tachibana SI, Yamauchi M. Evidence of artemisinin-resistant malaria in Africa. New England J Med. 2021;385(13):1163–71.
- 5. Bergmann C, van Loon W, Habarugira F, Tacoli C, Jäger JC, Savelsberg D, et al. Increase in Kelch 13 polymorphisms in Plasmodium falciparum, Southern Rwanda. Emerg Infect Dis. 2021;27(1):294–6. pmid:33350925
- 6. Ehrlich HY, Bei AK, Weinberger DM, Warren JL, Parikh S. Mapping partner drug resistance to guide antimalarial combination therapy policies in sub-Saharan Africa. Proc Natl Acad Sci U S A. 2021;118(29):e2100685118. pmid:34261791
- 7. Ndwiga L, Kimenyi KM, Wamae K, Osoti V, Akinyi M, Omedo I, et al. A review of the frequencies of Plasmodium falciparum Kelch 13 artemisinin resistance mutations in Africa. Int J Parasitol Drugs Drug Resist. 2021;16:155–61. pmid:34146993
- 8. Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM, et al. Evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med. 2008;359(24):2619–20. pmid:19064625
- 9. Oguche S, Okafor HU, Watila I, Meremikwu M, Agomo P, Ogala W, et al. Efficacy of artemisinin-based combination treatments of uncomplicated falciparum malaria in under-five-year-old Nigerian children. Am J Trop Med Hyg. 2014;91(5):925–35. pmid:25246693
- 10. Uwimana A, Legrand E, Stokes BH, Ndikumana J-LM, Warsame M, Umulisa N, et al. Emergence and clonal expansion of in vitro artemisinin-resistant Plasmodium falciparum kelch13 R561H mutant parasites in Rwanda. Nat Med. 2020;26(10):1602–8. pmid:32747827
- 11. Krungkrai J. The multiple roles of the mitochondrion of the malarial parasite. Parasitology. 2004;129(Pt 5):511–24. pmid:15552397
- 12. van Esveld SL, Meerstein-Kessel L, Boshoven C, Baaij JF, Barylyuk K, Coolen JPM, et al. A prioritized and validated resource of mitochondrial proteins in plasmodium identifies unique biology. mSphere. 2021;6(5):e0061421. pmid:34494883
- 13. Srivastava IK, Morrisey JM, Darrouzet E, Daldal F, Vaidya AB. Resistance mutations reveal the atovaquone-binding domain of cytochrome b in malaria parasites. Mol Microbiol. 1999;33(4):704–11. pmid:10447880
- 14. Divo AA, Geary TG, Jensen JB, Ginsburg H. The mitochondrion of Plasmodium falciparum visualized by rhodamine 123 fluorescence. J Protozool. 1985;32(3):442–6. pmid:3900366
- 15. Lamb IM, Okoye IC, Mather MW, Vaidya AB. Unique Properties of Apicomplexan Mitochondria. Annu Rev Microbiol. 2023;77:541–60. pmid:37406344
- 16. Feagin JE, Harrell MI, Lee JC, Coe KJ, Sands BH, Cannone JJ, et al. The fragmented mitochondrial ribosomal RNAs of Plasmodium falciparum. PLoS One. 2012;7(6):e38320. pmid:22761677
- 17. Feagin JE, Mericle BL, Werner E, Morris M. Identification of additional rRNA fragments encoded by the Plasmodium falciparum 6 kb element. Nucleic Acids Res. 1997;25(2):438–46. pmid:9016576
- 18. Feagin JE. The 6-kb element of Plasmodium falciparum encodes mitochondrial cytochrome genes. Mol Biochem Parasitol. 1992;52(1):145–8. pmid:1320735
- 19. Vaidya AB, Akella R, Suplick K. Sequences similar to genes for two mitochondrial proteins and portions of ribosomal RNA in tandemly arrayed 6-kilobase-pair DNA of a malarial parasite. Mol Biochem Parasitol. 1989;35(2):97–107. pmid:2549417
- 20. Lamb IM, Rios KT, Shukla A, Ahiya AI, Morrisey J, Mell JC, et al. Mitochondrially targeted proximity biotinylation and proteomic analysis in Plasmodium falciparum. PLoS One. 2022;17(8):e0273357. pmid:35984838
- 21. Arguello T, Peralta S, Antonicka H, Gaidosh G, Diaz F, Tu Y-T, et al. ATAD3A has a scaffolding role regulating mitochondria inner membrane structure and protein assembly. Cell Rep. 2021;37(12):110139. pmid:34936866
- 22. Baudier J. ATAD3 proteins: brokers of a mitochondria-endoplasmic reticulum connection in mammalian cells. Biol Rev Camb Philos Soc. 2018;93(2):827–44. pmid:28941010
- 23. Gerhold JM, Cansiz-Arda Ş, Lõhmus M, Engberg O, Reyes A, van Rennes H, et al. Human mitochondrial DNA-protein complexes attach to a cholesterol-rich membrane structure. Sci Rep. 2015;5:15292. pmid:26478270
- 24. Gilquin B, Taillebourg E, Cherradi N, Hubstenberger A, Gay O, Merle N, et al. The AAA+ ATPase ATAD3A controls mitochondrial dynamics at the interface of the inner and outer membranes. Mol Cell Biol. 2010;30(8):1984–96. pmid:20154147
- 25. Goel D, Kumar S. Advancements in unravelling the fundamental function of the ATAD3 protein in multicellular organisms. Adv Biol Regul. 2024;93:101041. pmid:38909398
- 26. He J, Cooper HM, Reyes A, Di Re M, Sembongi H, Litwin TR, et al. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis. Nucleic Acids Res. 2012;40(13):6109–21. pmid:22453275
- 27. He J, Mao C-C, Reyes A, Sembongi H, Di Re M, Granycome C, et al. The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization. J Cell Biol. 2007;176(2):141–6. pmid:17210950
- 28. Hubstenberger A, Merle N, Charton R, Brandolin G, Rousseau D. Topological analysis of ATAD3A insertion in purified human mitochondria. J Bioenerg Biomembr. 2010;42(2):143–50. pmid:20349121
- 29. Hughes DT, Brar KK, Morris JL, Subramanian K, Krishna S, Gao F. PERK-ATAD3A interaction protects mitochondrial proteins synthesis during ER stress. bioRxiv. 2022.
- 30. Ishihara T, Ban-Ishihara R, Ota A, Ishihara N. Mitochondrial nucleoid trafficking regulated by the inner-membrane AAA-ATPase ATAD3A modulates respiratory complex formation. Proc Natl Acad Sci U S A. 2022;119(47):e2210730119. pmid:36383603
- 31. Waters ER, Bezanilla M, Vierling E. ATAD3 proteins: unique mitochondrial proteins essential for life in diverse eukaryotic lineages. Plant and Cell Physiol. 2023.
- 32. Chen L, Li Y, Zambidis A, Papadopoulos V. ATAD3A: a key regulator of mitochondria-associated diseases. Int J Mol Sci. 2023;24(15):12511. pmid:37569886
- 33. Cooper HM, Yang Y, Ylikallio E, Khairullin R, Woldegebriel R, Lin K-L, et al. ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia. Hum Mol Genet. 2017;26(8):1432–43. pmid:28158749
- 34. Huang KC-Y, Chiang S-F, Yang P-C, Ke T-W, Chen T-W, Lin C-Y, et al. ATAD3A stabilizes GRP78 to suppress ER stress for acquired chemoresistance in colorectal cancer. J Cell Physiol. 2021;236(9):6481–95. pmid:33580514
- 35. Issop L, Fan J, Lee S, Rone MB, Basu K, Mui J, et al. Mitochondria-associated membrane formation in hormone-stimulated Leydig cell steroidogenesis: role of ATAD3. Endocrinology. 2015;156(1):334–45. pmid:25375035
- 36. Teng Y, Ren X, Li H, Shull A, Kim J, Cowell JK. Mitochondrial ATAD3A combines with GRP78 to regulate the WASF3 metastasis-promoting protein. Oncogene. 2016;35(3):333–43. pmid:25823022
- 37. Brügel M, Kiesel A-S, Haack TB, Peralta S. Mutations in mitochondrial ATAD3 gene and disease, lessons from in vivo models. Front Neurosci. 2024;18:1496142. pmid:39605788
- 38. Planté-Bordeneuve P, Bérat C-M, Hanein S, Gitiaux C, ATAD3 Study Group, Steffann J, et al. ATAD3 duplications bridge mitochondrial diseases and Aicardi-Goutières syndrome. Dev Med Child Neurol. 2026;68(2):287–94. pmid:40665566
- 39. Verhoef JMJ, Boshoven C, Evers F, Akkerman LJ, Gijsbrechts BCA, van de Vegte-Bolmer M, et al. Detailing organelle division and segregation in Plasmodium falciparum. J Cell Biol. 2024;223(12):e202406064. pmid:39485315
- 40. Morano AA, Xu W, Navarro FM, Shadija N, Dvorin JD, Ke H. The dynamin-related protein PfDyn2 is essential for both apicoplast and mitochondrial fission in Plasmodium falciparum. mBio. 2025;16(1):e0303624. pmid:39611847
- 41. Painter HJ, Morrisey JM, Mather MW, Vaidya AB. Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum. Nature. 2007;446(7131):88–91. pmid:17330044
- 42. Ke H, Morrisey JM, Ganesan SM, Painter HJ, Mather MW, Vaidya AB. Variation among Plasmodium falciparum strains in their reliance on mitochondrial electron transport chain function. Eukaryot Cell. 2011;10(8):1053–61. pmid:21685321
- 43. Jessop M, Felix J, Gutsche I. AAA+ ATPases: structural insertions under the magnifying glass. Curr Opin Struct Biol. 2021;66:119–28. pmid:33246198
- 44. White SR, Lauring B. AAA+ ATPases: achieving diversity of function with conserved machinery. Traffic. 2007;8(12):1657–67. pmid:17897320
- 45. Peralta S, Goffart S, Williams SL, Diaz F, Garcia S, Nissanka N, et al. ATAD3 controls mitochondrial cristae structure in mouse muscle, influencing mtDNA replication and cholesterol levels. J Cell Sci. 2018;131(13):jcs217075. pmid:29898916
- 46. He J, Cooper HM, Reyes A, Di Re M, Sembongi H, Litwin TR, et al. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis. Nucleic Acids Res. 2012;40(13):6109–21. pmid:22453275
- 47. Goller T, Seibold UK, Kremmer E, Voos W, Kolanus W. Atad3 function is essential for early post-implantation development in the mouse. PLoS One. 2013;8(1):e54799. pmid:23372768
- 48. Hoffmann M, Bellance N, Rossignol R, Koopman WJH, Willems PHGM, Mayatepek E, et al. C. elegans ATAD-3 is essential for mitochondrial activity and development. PLoS One. 2009;4(10):e7644. pmid:19888333
- 49. Skopkova M, Stufkova H, Rambani V, Stranecky V, Brennerova K, Kolnikova M, et al. ATAD3A-related pontocerebellar hypoplasia: new patients and insights into phenotypic variability. Orphanet J Rare Dis. 2023;18(1):92. pmid:37095554
- 50. Harel T, Yoon WH, Garone C, Gu S, Coban-Akdemir Z, Eldomery MK, et al. Recurrent de novo and biallelic variation of ATAD3A, encoding a mitochondrial membrane protein, results in distinct neurological syndromes. Am J Human Genetics. 2016;99(4):831–45.
- 51. Khan YA, White KI, Brunger AT. The AAA+ superfamily: a review of the structural and mechanistic principles of these molecular machines. Crit Rev Biochem Mol Biol. 2022;57(2):156–87. pmid:34632886
- 52. Suplick K, Morrisey J, Vaidya AB. Complex transcription from the extrachromosomal DNA encoding mitochondrial functions of Plasmodium yoelii. Mol Cell Biol. 1990;10(12):6381–8. pmid:1701017
- 53.
Dass S. Investigation of Plasmodium falciparum mitochondrial ribosomes. Drexel University; 2022.
- 54. Painter HJ, Morrisey JM, Vaidya AB. Mitochondrial electron transport inhibition and viability of intraerythrocytic Plasmodium falciparum. Antimicrob Agents Chemother. 2010;54(12):5281–7. pmid:20855748
- 55. Gerhold JM, Cansiz-Arda Ş, Lõhmus M, Engberg O, Reyes A, van Rennes H, et al. Human mitochondrial DNA-protein complexes attach to a cholesterol-rich membrane structure. Sci Rep. 2015;5:15292. pmid:26478270
- 56. Peralta S, Goffart S, Williams SL, Diaz F, Garcia S, Nissanka N, et al. ATAD3 controls mitochondrial cristae structure in mouse muscle, influencing mtDNA replication and cholesterol levels. J Cell Sci. 2018;131(13):jcs217075. pmid:29898916
- 57. Vale RD. AAA proteins. Lords of the ring. J Cell Biol. 2000;150(1):F13-9. pmid:10893253
- 58. van Dooren GG, Stimmler LM, McFadden GI. Metabolic maps and functions of the Plasmodium mitochondrion. FEMS Microbiol Rev. 2006;30(4):596–630. pmid:16774588
- 59. Tanveer A, Allen SM, Jackson KE, Charan M, Ralph SA, Habib S. An FtsH protease is recruited to the mitochondrion of Plasmodium falciparum. PLoS One. 2013;8(9):e74408. pmid:24058559
- 60. Boltryk SD, Passecker A, Alder A, Carrington E, van de Vegte-Bolmer M, van Gemert G-J, et al. CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research. Nat Commun. 2021;12(1):4806. pmid:34376675
- 61. Chen S. fastp 1.0: An ultra-fast all-round tool for FASTQ data quality control and preprocessing. Imeta. 2025;4(5):e70078. pmid:41112039
- 62. Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. ArXiv. 2013.
- 63. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25(16):2078–9. pmid:19505943
- 64. Institute B. Picard tools. 2023. https://example.com/picard-tools
- 65. Prjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A. Using SPAdes de novo assembler. Curr Protoc Bioinformatics. 2020;70(1):e102.
- 66. Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, et al. Twelve years of SAMtools and BCFtools. Gigascience. 2021;10(2):giab008. pmid:33590861
- 67. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20(9):1297–303. pmid:20644199
- 68. Ginsburg H, Nissani E, Krugliak M. Alkalinization of the food vacuole of malaria parasites by quinoline drugs and alkylamines is not correlated with their antimalarial activity. Biochem Pharmacol. 1989;38(16):2645–54. pmid:2669763
- 69. Haynes JD, Moch JK. Automated synchronization of Plasmodium falciparum parasites by culture in a temperature-cycling incubator. Methods Mol Med. 2002;72:489–97. pmid:12125145
- 70. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30(4):772–80. pmid:23329690
- 71. Chang J-M, Di Tommaso P, Notredame C. TCS: a new multiple sequence alignment reliability measure to estimate alignment accuracy and improve phylogenetic tree reconstruction. Mol Biol Evol. 2014;31(6):1625–37. pmid:24694831
- 72. Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59(3):307–21. pmid:20525638
- 73. Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25(9):1189–91.
- 74. Alvarez-Jarreta J, Amos B, Aurrecoechea C, Bah S, Barba M, Barreto A, et al. VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center in 2023. Nucleic Acids Res. 2024;52(D1):D808–16. pmid:37953350
- 75. Zhang M, Wang C, Otto TD, Oberstaller J, Liao X, Adapa SR, et al. Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis. Science. 2018;360(6388):eaap7847. pmid:29724925
- 76. Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024;630(8016):493–500. pmid:38718835
- 77. Zhang C, Pyle AM. A unified approach to sequential and non-sequential structure alignment of proteins, RNAs, and DNAs. iScience. 2022;25(10):105218. pmid:36248743
- 78. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004;25(13):1605–12. pmid:15264254