Biology subjects

Mather, M. W.

Publications and source records attributed to Mather, M. W..

11 recordsLinked to original sources

bioRxiv2025

ATAD3 megadalton complex in Plasmodium falciparum is essential for mitochondrial and cellular viability

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 ortholog 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. This study is the first characterization, to our knowledge, of ATAD3A in unicellular organisms. ATAD3A has been previously studied in multicellular eukaryotes and has been implicated in several childhood mitochondrial diseases. Our findings here expand our knowledge on apicomplexan mitochondrial biology and our arsenal of potential antimalarial drug targets. Author SummaryEach 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.

microbiology↗

bioRxiv2025

Dual Function of Mitochondrial Complex III in Plasmodium falciparum

Complex III of the malaria parasite mitochondrial electron transport chain (mtETC) has been validated as an attractive target for currently used antimalarials. We previously showed that the main function of mtETC in blood stage Plasmodium falciparum is to regenerate ubiquinone, which serves as an obligatory co-substrate of dihydroorotate dehydrogenase (DHOD), an essential mitochondrial enzyme for pyrimidine biosynthesis. P. falciparum can be rendered resistant to all mtETC inhibitors by provision of a bypass mediated by cytosolic yeast DHOD, a fumarate-reducing enzyme. Malaria parasite mitochondrial DNA (mtDNA) encodes only 3 proteins, each a component of mtETC. However, attempts to eliminate mtDNA in transgenic parasites expressing yDHOD have been unsuccessful, suggesting the possibility that essential function(s) other than the canonical redox reactions of the mtETC also require mtDNA maintenance. Here we have tested the hypothesis that Complex III serves the dual functions of processing imported mitochondrial proteins, as well as ubiquinone regeneration. We have generated transgenic lines that conditionally express mitochondrial processing peptidase a (MPP), which is also a component of Complex III. Using these parasites, we have determined that MPP is essential even when the need for mitochondrial electron transport is bypassed. MPP knockdown also resulted in hypersensitivity of the parasites to proguanil, a drug that synergizes with mtETC inhibitors such as atovaquone. Pulldown with MPP followed by proteomics revealed the association of multiple mitochondrially targeted proteins, in addition to all components of Complex III. These results are consistent with the suggestion that Complex III in P. falciparum serves both mtETC and protein processing functions in mitochondrial physiology.

microbiology↗

bioRxiv2025

Endogenous structure of antimalarial target PfATP4 reveals new class of apicomplexan P-type ATPase modulators

The Plasmodium falciparum sodium efflux pump PfATP4 is a leading antimalarial target, but suffers from a lack of high-resolution structural information needed to identify functionally important features in conserved regions and guide rational design of next generation inhibitors. Here, we determine a 3.7[A] cryoEM structure of PfATP4 purified from CRISPR-engineered P. falciparum parasites, revealing a previously unknown, apicomplexan-specific binding partner, PfABP, which forms a conserved, likely modulatory interaction with PfATP4. The discovery of PfABP presents a new avenue for designing novel PfATP4 inhibitors.

microbiology↗

bioRxiv2024

Mutational Analysis of an Antimalarial Drug Target PfATP4

Among new antimalarials discovered over the past decade are multiple chemical scaffolds that target Plasmodium falciparum P-type ATPase (PfATP4). This essential protein is a Na+ pump responsible for the maintenance of Na+ homeostasis. PfATP4 belongs to the type 2D subfamily of P-type ATPases, for which no structures have been determined. To gain better insight into the structure/function relationship of this validated drug target, we generated a homology model of PfATP4 based on SERCA, a P2A-type ATPase, and refined the model using molecular dynamics in its explicit membrane environment. This model predicted several residues in PfATP4 critical for its function, as well as those that impart resistance to various PfATP4 inhibitors. To validate our model, we developed a genetic system involving merodiploid states of PfATP4 in which the endogenous gene was conditionally expressed, and the second allele was mutated to assess its effect on the parasite. Our model predicted residues involved in Na+ coordination as well as the phosphorylation cycle of PfATP4. Phenotypic characterization of these mutants involved assessment of parasite growth, localization of mutated PfATP4, response to treatment with known PfATP4 inhibitors, and evaluation of the downstream consequences of Na+ influx. Our results were consistent with modeled predictions of the essentiality of the critical residues. Additionally, our approach confirmed the phenotypic consequences of resistance-associated mutations as well as a potential structural basis for the fitness cost associated with some mutations. Taken together, our approach provides a means to explore the structure/function relationship of essential genes in haploid organisms. Significance StatementPlasmodium falciparum ATP4 (PfATP4) is a Na+ efflux pump and represents an important target for antimalarial drugs with nanomolar potency. However, the structure of PfATP4 is unknown, prompting the development of new methodologies to investigate the structure/function relationship. Here, we introduce a dynamic homology modeling approach to (a) identify key residues essential for PfATP4 function and (b) provide a structural basis to understand resistance-associated mutations. To validate these predictions, we developed a genetic system to manipulate the PfATP4 gene to assess the phenotypic consequences of such changes. Our results support the utility of combining homology modeling and genetics to gain functional insights into an antimalarial drug target.

microbiology↗

bioRxiv2022

Genetic disruption of isocitrate dehydrogenase arrests the full development of sexual stage parasites in Plasmodium falciparum

The malaria parasites encode all eight mitochondrial enzymes that can constitute the classical tricarboxylic acid (TCA) cycle. We previously reported that genetic ablation of six out of eight TCA enzymes had no significant impact on the parasite survival and growth in asexual blood stage; aconitase knockout ({Delta}ACO) parasites were, however, arrested as late-stage gametocytes. Herein, we describe a defective gametocyte development phenotype resulting from the knockout of another TCA enzyme, isocitrate dehydrogenase (IDH). Similar to the{Delta} ACO gametocytes,{Delta} IDH gametocytes stall at stage III/IV and cannot form mature gametocytes or undergo exflagellation. Both ACO and IDH KO gametocytes exhibit fragmented mitochondrial morphology. Knockout of IDH also exerts a fitness cost, leading to a decrease in growth rate in erythrocytic stages. We examined two possible causes for the phenotype in the{Delta} IDH gametocytes: 1. Accumulation of reactive oxygen species (ROS) within the parasite mitochondrion due to diminished production of mitochondrial NADPH during the 10-14 days of gametocyte development; or 2. Cytotoxicity of built-up citrate upstream of IDH. We found that antioxidant agents such as N-acetylcysteine and MitoQ did not rescue the phenotype of{Delta} ACO or{Delta} IDH gametocytes. Large amounts of citrate in the medium also had no significant effect on the gametocyte development or gamete formation of wildtype parasites. Our results demonstrate the importance of IDH in gametocytogenesis, providing evidence that IDH as well as aconitase could be further explored as targets for gametocidal agents and malaria transmission blockers.

microbiology↗

bioRxiv2022

Mitochondrially targeted proximity biotinylation and proteomic analysis in Plasmodium falciparum

Despite ongoing efforts to control malaria infection, progress in lowering the number of deaths and infections appears to have stalled. The continued high incidence of malaria infection and mortality is in part due to emergence of parasites resistant to frontline antimalarials. This highlights the need for continued identification of novel protein drug targets. Mitochondrial functions in Plasmodium falciparum, the deadliest species of human malaria parasite, are targets of validated antimalarials including atovaquone and proguanil (Malarone). Thus, there has been great interest in identifying other essential mitochondrial proteins as candidates for novel drug targets. Garnering an increased understanding of the proteomic landscape inside the P. falciparum mitochondrion will also allow us to learn about the basic biology housed within this unique organelle. We employed a proximity biotinylation technique and mass spectrometry to identify novel P. falciparum proteins putatively targeted to the mitochondrion. We fused the leader sequence of a mitochondrially targeted chaperone, Hsp60, to the promiscuous biotin ligase TurboID. Through these experiments, we generated a list of 122 "putative mitochondrial" proteins. To verify whether these proteins were indeed mitochondrial, we chose five candidate proteins of interest for localization studies using ectopic expression and tagging of each full-length protein. This allowed us to localize four candidate proteins of unknown function to the mitochondrion, three of which have previously been assessed to be essential. We suggest that phenotypic characterization of these and other proteins from this list of 122 could be fruitful in understanding the basic mitochondrial biology of these parasites and aid antimalarial drug discovery efforts.

microbiology↗

bioRxiv2022

Transcriptional changes in Plasmodium falciparum upon conditional knock down of mitochondrial ribosomal proteins RSM22 and L23

The mitochondrion of malaria parasites is an attractive antimalarial drug target, which require mitoribosomes to translate genes encoded in the mitochondrial (mt) DNA. Plasmodium mitoribosomes are composed of highly fragmented ribosomal RNA (rRNA) encoded in the mtDNA. All mitoribosomal proteins (MRPs) and other assembly factors are encoded in the nuclear genome. Here, we have studied one small subunit (SSU) MRP, RSM22 (Pf3D7_1027200) and one large subunit (LSU) MRP, L23 (Pf3D7_1239100) in Plasmodium falciparum. We show that both proteins localize to the mitochondrion and are essential for parasite survival. However, the parasites survive conditional knock down (KD) of PfRSM22 for two days longer than PfMRPL23 KD. RNA sequencing revealed transcriptomic changes of the nuclear and mitochondrial genomes upon KD of these MRPs. In the early phase following KD, while most mt rRNAs and transcripts of putative MRPs were downregulated in the absence of PfRSM22, several mt rRNAs and MRPs were upregulated after KD of PfMRPL23. At the late time points of KD, loss of PfRSM22 and PfMRPL23 caused defects in many essential metabolic pathways, leading to parasite death. There was a significant overlap among the mitochondrial related transcripts downregulated in the late phase of PfRSM22 and PfMRPL23 KDs. We have also identified a list of mitochondrial proteins of unknown function that are likely Plasmodium MRPs based on their structural similarity to known MRPs as well as their expression profiles in KD parasites.

microbiology↗

bioRxiv2020

Atypical molecular basis for drug resistance to mitochondrial function inhibitors in Plasmodium falciparum

The continued emergence of drug-resistant Plasmodium falciparum parasites hinders global attempts to eradicate malaria, emphasizing the need to identify new antimalarial drugs. Attractive targets for chemotherapeutic intervention are the cytochrome (cyt) bc1 complex, which is an essential component of the mitochondrial electron transport chain (mtETC) necessary for ubiquinone recycling and mitochondrially localized dihydroorotate dehydrogenase (DHODH) critical for de novo pyrimidine synthesis. Despite the essentiality of this complex, resistance to a novel acridone class of compounds targeting cyt bc1 was readily attained, resulting in a parasite strain (SB1-A6) that was pan-resistant to both mtETC and DHODH inhibitors. Here we describe the molecular mechanism behind the resistance of the SB1-A6 parasite line which lacks the common cyt bc1 point mutations characteristic of resistance to mtETC inhibitors. Using Illumina whole-genome sequencing, we have identified both a copy number variation ([~]2x) and a single-nucleotide polymorphism (C276F) associated with pfdhodh in SB1-A6. We have characterized the role of both genetic lesions by mimicking the copy number variation via episomal expression of pfdhodh and introducing the identified SNP using CRISPR/Cas9 and assessed their contributions to drug resistance. Although both of these genetic polymorphisms have been previously identified as contributing to both DSM-1 (1) and atovaquone resistance (2, 3), SB1-A6 represents a unique genotype in which both alterations are present in a single line, suggesting that the combination contributes to the pan-resistant phenotype. This novel mechanism of resistance to mtETC inhibition has critical implications for the development of future drugs targeting the bc1 complex or de novo pyrimidine synthesis that could help guide future anti-malarial combination therapies and reduce the rapid development of drug resistance in the field.

microbiology↗

bioRxiv2020

Genetic ablation of the mitochondrial ribosome in Plasmodium falciparum sensitizes the human malaria parasite to antimalarial drugs targeting mitochondrial functions

The mitochondrion of malaria parasites contains clinically validated drug targets. Within Plasmodium spp., the mitochondrial DNA (mtDNA) is only 6 kb long, being the smallest mitochondrial genome among all eukaryotes. The mtDNA encodes only three proteins of the mitochondrial electron transport chain and [~] 27 small, fragmented rRNA genes in length of 22-195 nucleotides. The rRNA fragments are thought to form a mitochondrial ribosome (mitoribosome), together with ribosomal proteins imported from the cytosol. The mitoribosome of Plasmodium falciparum has been shown to be essential for maintenance of the mitochondrial membrane potential and parasite viability. However, the role of mitoribosomes in sustaining the metabolic status of the parasite mitochondrion remains unknown. Here, among the 14 annotated mitoribosomal proteins of the small subunit of P. falciparum, we verified the localization and tested the essentiality of three candidates (PfmtRPS12, PfmtRPS17, PfmtRPS18), employing a CRISPR/Cas9 mediated conditional knockdown tool. Using immuno-electron microscopy, we provided evidence that the mitoribosome is closely associated with the mitochondrial inner membrane in the parasite. Upon knockdown of the mitoribosome, the parasites became hypersensitive to inhibitors targeting the bc1 complex, dihydroorotate dehydrogenase and F1Fo ATP synthase complex. Furthermore, knockdown of the mitoribosome blocked the pyrimidine biosynthesis pathway and reduced the pool of pyrimidine nucleotides. Together, our data suggest that disruption of the P. falciparum mitoribosome compromises the metabolic capability of the mitochondrion, rendering the parasite hypersensitive to a panel of inhibitors targeting mitochondrial functions.

biochemistry↗

bioRxiv2019

Oligomerization of the antimalarial drug target PfATP4 is essential for parasite survival

Withdrawal StatementThe authors have withdrawn their manuscript owing to further investigations on PfATP4 that have revealed that the main conclusion of the preprint that PfATP4 forms oligomers is incorrect. Initial cryogenic electron microscopy observations showed a large detergent belt that surrounds monomers of PfATP4, which could have affected migration of the molecule on blue-native polyacrylamide gels. This gave an incorrect impression of the molecule to be in a dimeric or oligomeric state. Authors regret this error in interpreting the data. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

biochemistry↗

bioRxiv2018

Mitochondrial type II NADH dehydrogenase of Plasmodium falciparum is dispensable and not the functional target of putative NDH2 quinolone inhibitors

The battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum, the deadliest human malaria parasite. To counter the problem, novel antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase of P. falciparum, PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P. falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasites susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc1 complex. Our results call into question the validity of PfNDH2 as an antimalarial drug target.\n\nImportanceFor a long time, PfNDH2 has been considered an attractive antimalarial drug target. However, the conclusion that PfNDH2 is essential was based on preliminary and incomplete data. Here we generate a PfNDH2 KO (knockout) parasite in the blood stages of Plasmodium falciparum, showing that the gene is not essential. We also show that previously reported PfNDH2-specific inhibitors kill the parasites primarily via targeting the cytochrome bc1 complex, not PfNDH2. Overall, we provide genetic and biochemical data that help to resolve a long-debated issue in the field regarding the potential of PfNDH2 as an antimalarial drug target.

microbiology↗