Biology subjects

Prigge, S. T.

Publications and source records attributed to Prigge, S. T..

13 recordsLinked to original sources

bioRxiv2026

Reduced expression of an essential blood-stage Plasmodium phosphatidylserine synthase does not modulate parasite resistance to PfATP4 inhibitors

Plasmodium falciparum ATP4 mutations A211V and G223R allow parasites to survive the lethal effects of antimalarials PA21A092 (PA92) and cipargamin (CIP), respectively. An A211V mutant line (Dd2A211V) treated with PA92 showed enhanced levels of lipid production, which prompted the idea that components of the phospholipid biosynthesis pathway could be involved in the survival mechanism of PfATP4 mutant parasites. As phosphatidylserine synthase (PfPSS) is the only enzyme that produces phosopholipid phosphatidylserine (PS) in P. falciparum parasites, we hypothesized that PfPSS is both essential for parasite survival and that reduced PfPSS expression would cause resistant PfATP4 mutant parasites to become susceptible to PA92 or CIP. We created a CIP-resistant G223R mutant line (Dd2G223R) via CRISPR-Cas9 and integrated a conditional PfPSS knockdown construct into a Dd2A211V ({downarrow}PSS-Dd2A211V) and our Dd2G223R line ({downarrow}PSS-Dd2G223R). We treated these knockdown lines with PA92 or CIP to determine the half-maximal effective concentration (EC50) of each antimalarial with normal or reduced PfPSS levels. While we found that PfPSS is essential for parasite survival, we did not find any significant alterations to the EC50 values of PA92 or CIP based on the reduced levels of PfPSS in our mutant lines. Although PfPSS does not appear to be involved, other components of the phospholipid production pathway could still affect the resistance mechanism of PfATP4 mutations. Identification of novel targets to counteract the mechanism by which PfATP4 mutant parasites resist lethal drug effects is crucial for the successful application of antimalarials in endemic countries where resistance is on the rise.

microbiology↗

bioRxiv2026

Acyl Carrier Protein is Essential for Apicoplast Biogenesis in Malaria Parasites Independent of Fatty Acid Synthesis

Acyl carrier protein (ACP) and its 4-phosphopantetheine prosthetic group canonically function as the soluble scaffold for acyl chain assembly and elongation during type-II fatty acid synthesis (FASII). Plasmodium malaria parasites retain a FASII pathway in the apicoplast organelle that has been the subject of considerable scrutiny and confusion. Although apicoplast FASII is essential for P. falciparum growth within mosquitoes and the human liver, this pathway is dispensable and largely inactive in blood-stage parasites that can scavenge host fatty acids. In contrast to FASII enzymes that can be disrupted without fitness defect, we report that knockout or ligand-dependent knockdown of apicoplast ACP is lethal to blood-stage P. falciparum, indicating an essential FASII-independent function. Loss of ACP impairs the biosynthesis of essential isoprenoid precursors and blocks apicoplast biogenesis. Using proximity biotinylation and biochemical interaction studies, we identified a key role for ACP in binding and stabilizing apicoplast pyruvate kinase II (PKII). This critical enzyme is the only known source of nucleoside triphosphates (NTPs) in this organelle and is required for isoprenoid synthesis and apicoplast biogenesis. Our work reveals that ACP knockdown results in destabilization and loss of PKII, which is sufficient to explain ACP essentiality in this stage. This work unveils essential ACP function at a key biochemical hub controlling broad apicoplast metabolism in malaria parasites that is independent of the canonical ACP role in FASII.

biochemistry↗

bioRxiv2025

Genetic analysis of pyrimidine biosynthetic enzymes in Plasmodium falciparum

The malaria parasite Plasmodium falciparum depends entirely on de novo pyrimidine synthesis, as it is unable to salvage these essential nucleotides. This reliance makes the pyrimidine biosynthesis pathway a compelling target for antimalarial drugs, with several inhibitors targeting its rate-limiting enzyme, dihydroorotate dehydrogenase (PfDHODH), already in clinical development. In this study, we investigated the roles of three other pathway enzymes - aspartate transcarbamoylase (PfATC), carbamoyl phosphate synthetase II (PfCPSII), and dihydroorotase (PfDHO). PfATC features a unique N-terminal extension predicted to serve as an apicoplast trafficking peptide. However, using antibodies against the native protein and an epitope-tagged version, we found no evidence of apicoplast localization. Knockdown of PfATC expression proved lethal and could not be rescued by an apicoplast metabolic bypass. Complementation assays further revealed that truncation of the N-terminal domain impaired parasite growth, suggesting that this region is important for PfATC function or stability in vivo. PfCPSII, which harbors large Plasmodium-specific insertions between its catalytic domains, was likewise found to be essential for parasite proliferation. To assess the role of PfDHO, we engineered parasites to salvage uracil via heterologous expression of a yeast enzyme. Deletion of PfDHO in this parasite line resulted in uracil auxotrophy, confirming the enzymes essential function in pyrimidine synthesis. Together, these findings reveal multiple vulnerable nodes within the pyrimidine biosynthesis pathway. AUTHOR SUMMARYNucleotides are central metabolites that serve as building blocks for DNA and RNA, act as key energy carriers, and function as cofactors or regulators in several metabolic pathways. To satisfy these diverse demands, most organisms rely on both nucleotide salvage and de novo synthesis. The malaria parasite Plasmodium falciparum acquires purine nucleotides from the host but lacks the capacity to salvage pyrimidines, making de novo pyrimidine synthesis essential. Several enzymes in this pathway differ from their human counterparts in sequence, domain architecture, and evolutionary origin, enhancing their potential as selective drug targets. Dihydroorotate dehydrogenase (PfDHODH), the fourth enzyme in the pathway, has already been validated as an antimalarial target. Here, we systematically examined upstream enzymes using molecular genetic approaches. Each proved essential for asexual blood-stage parasite survival, with the Plasmodium-specific N-terminal extension of aspartate carbamoyltransferase (PfATC) required for optimal growth. The introduction of a yeast uracil salvage enzyme rescued parasites depleted of these biosynthetic enzymes, demonstrating that their essential functions are confined to pyrimidine production and that their distinctive structural features do not support additional metabolic roles. In summary, these results delineate additional enzymatic steps in this important metabolic pathway that warrant continued investigation from both biological and translational angles.

microbiology↗

bioRxiv2025

An Essential Adaptor for Apicoplast Fission and Inheritance in Malaria Parasites

Blood-stage Plasmodium falciparum parasites rely on a non-photosynthetic plastid, the apicoplast, for survival, making it an attractive target for antimalarial intervention. Like the mitochondrion, the apicoplast cannot be generated de novo and must be inherited by daughter parasites during cell division. This inheritance relies on coordinated apicoplast positioning and fission, but the molecular mechanisms controlling these processes remain poorly understood. Here, we identify a previously uncharacterized P. falciparum protein (Pf3D7_0613600), which we name PfAnchor, as a key regulator of apicoplast fission. Using Ultrastructure Expansion Microscopy (U-ExM), we show that PfAnchor localizes to the apicoplast throughout the asexual blood-stage. Conditional depletion disrupts apicoplast fission, leading to incomplete cytokinesis and parasite death. Notably, loss of the apicoplasts elongated branched structure via azithromycin treatment rescues these defects, underscoring Anchors specific role in apicoplast fission. Immunoprecipitation identified an interaction with the dynamin-like GTPase PfDyn2, a key mediator of both apicoplast and mitochondrial fission, establishing PfAnchor as the first apicoplast-specific dynamin adaptor protein. Our findings define PfAnchor as an essential factor for apicoplast fission and inheritance in P. falciparum blood-stage parasites, highlighting parasite-specific organelle division as a potential vulnerability for therapeutic intervention.

cell biology↗

bioRxiv2024

tRNA modifying enzymes MnmE and MnmG are essential for Plasmodium falciparum apicoplast maintenance

The circular genome of the Plasmodium falciparum apicoplast contains a complete minimal set of tRNAs, positioning the apicoplast as an ideal model for studying the fundamental factors required for protein translation. Modifications at tRNA wobble base positions, such as xm5s2U, are critical for accurate protein translation. These modifications are ubiquitously found in tRNAs decoding two-family box codons ending in A or G in prokaryotes and in eukaryotic organelles. Here, we investigated the xm5s2U biosynthetic pathway in the apicoplast organelle of P. falciparum. Through comparative genomics, we identified orthologs of enzymes involved in this process: SufS, MnmA, MnmE, and MnmG. While SufS and MnmA were previously shown to catalyze s2U modifications, we now show that MnmE and MnmG are apicoplast-localized and contain features required for xm5s2U biosynthetic activity. Notably, we found that P. falciparum lacks orthologs of MnmC, MnmL, and MnmM, suggesting that the parasites contain a minimal xm5s2U biosynthetic pathway similar to that found in bacteria with reduced genomes. Deletion of either MnmE or MnmG resulted in apicoplast disruption and parasite death, mimicking the phenotype observed in {Delta}mnmA and {Delta}sufS parasites. Our data strongly support the presence and essentiality of xm5s2U modifications in apicoplast tRNAs. This study advances our understanding of the minimal requirements for protein translation in the apicoplast organelle. ImportanceThe apicoplast of Plasmodium falciparum is an ideal model for studying minimal translation machinery since it contains the fewest number of tRNA isotypes required for protein translation. In such reduced systems, tRNA modifications, particularly at the wobble anticodon position, are critical for proper decoding of mRNA codons by tRNAs. In this study, we investigated the enzymes responsible for the wobble modification xm5s2U in the apicoplast. Our findings reveal that only four enzymes are required in the apicoplast to carry out this modification, similar to the situation found in bacteria with highly reduced genomes. Based on their phylogenetic conservation, these four enzymes have been included in all proposed minimal genome concepts to date and it appears that the apicoplast has retained the same core machinery. Our findings provide novel insight into apicoplast biology and into minimal requirements for protein translation.

microbiology↗

bioRxiv2024

MULTIPLE, REDUNDANT CARBOXYLIC ACID TRANSPORTERS SUPPORT MITOCHONDRIAL METABOLISM IN PLASMODIUM FALCIPARUM

The mitochondrion of the deadliest human malaria parasite, Plasmodium falciparum, is an essential source of cellular acetyl-CoA during the asexual blood-stage of the parasite life cycle. Blocking mitochondrial acetyl-CoA synthesis leads to a hypoacetylated proteome and parasite death. We previously determined that mitochondrial acetyl-CoA is primarily synthesized from glucose-derived pyruvate by -ketoacid dehydrogenases. Here, we asked if inhibiting the import of glycolytic pyruvate across the mitochondrial inner membrane would affect acetyl-CoA production and, thus, could be a potential target for antimalarial drug development. We selected the two predicted mitochondrial pyruvate carrier proteins (PfMPC1 and PfMPC2) for genetic knockout and isotopic metabolite tracing via HPLC-MS metabolomic analysis. Surprisingly, we observed that asexual blood-stage parasites could survive the loss of either or both PfMPCs with only minor growth defects, despite a substantial reduction in the amount of glucose-derived isotopic labelling into acetyl-CoA. Furthermore, genetic deletion of two additional mitochondrial carboxylic acid transporters - DTC (di/tricarboxylic acid carrier) and YHM2 (a putative citrate/-ketoglutarate carrier protein) - only mildly affected asexual blood-stage replication, even in the context of PfMPC deficiency. Although we observed no added impact on the incorporation of glucose carbon into acetyl-CoA in these quadruple knockout mutants, we noted a large decrease in glutamine-derived label in tricarboxylic acid cycle metabolites, suggesting that DTC and YHM2 both import glutamine derivatives into the mitochondrion. Altogether, our results expose redundant routes used to fuel the blood-stage malaria parasite mitochondrion with imported carbon from two major sources - glucose and glutamine. SIGNIFICANCEThe mitochondrion of malaria parasites generates key molecules, such as acetyl-CoA, that are required for numerous cellular processes. To support mitochondrial biosynthetic pathways, the parasites must transport carbon sources into this organelle. By studying how the mitochondrion obtains pyruvate, a molecule derived from glucose, we have uncovered redundant carbon transport systems that ensure parasite survival in red blood cells. This metabolic redundancy poses a challenge for drug development, as it enables the parasite to adapt and survive by relying on alternative pathways when one is disrupted.

microbiology↗

bioRxiv2024

tRNA lysidinylation is essential for the minimal translation system found in the apicoplast of Plasmodium falciparum

For decades, researchers have sought to define minimal genomes to elucidate the fundamental principles of life and advance biotechnology. tRNAs, essential components of this machinery, decode mRNA codons into amino acids. The apicoplast of malaria parasites encodes 25 tRNA isotypes in its organellar genome - the lowest number found in known translation systems. Efficient translation in such minimal systems depends heavily on post-transcriptional tRNA modifications, especially at the wobble anticodon position. Lysidine modification at the wobble position (C34) of tRNACAU distinguishes between methionine (AUG) and isoleucine (AUA) codons, altering the amino acid delivered by this tRNA and ensuring accurate protein synthesis. Lysidine is formed by the enzyme tRNA isoleucine lysidine synthetase (TilS) and is nearly ubiquitous in bacteria and essential for cellular viability. We identified a TilS ortholog (PfTilS) located in the apicoplast of Plasmodium falciparum parasites. By complementing PfTilS with a bacterial ortholog, we demonstrated that the lysidinylation activity of PfTilS is critical for parasite survival and apicoplast maintenance, likely due to its impact on apicoplast protein translation. Our findings represent the first characterization of TilS in an endosymbiotic organelle, advancing eukaryotic organelle research and our understanding of minimal translational machinery. Due to the absence of lysidine modifications in humans, this research also exposes a potential vulnerability in malaria parasites that could be targeted by antimalarial strategies. SignificanceIn recent decades, synthetic biologists have sought the minimal cellular components required for life, focusing on simpler systems for easier modeling. The apicoplast organelle of malaria parasites, with only 25 tRNA isotypes, contains the smallest known complete tRNA set, even smaller than in synthetic organisms. This makes it an ideal model for studying minimal translational machinery, where tRNAs depend on post-transcriptional modifications for efficient protein translation. A key modification, lysidine, is crucial for decoding isoleucine and methionine. This study describes a tRNA-isoleucine lysidine synthetase (TilS) enzyme, essential for apicoplast protein translation. These findings have implications for understanding eukaryotic organelles and minimal translation machinery. Additionally, the absence of lysidine in humans suggests a potential target for antimalarial strategies.

microbiology↗

bioRxiv2024

Progressive heterogeneity of enlarged and irregularly shaped apicoplasts in P. falciparum persister blood stages after drug treatment

Morphological modifications and shifts in organelle relationships are hallmarks of dormancy in eukaryotic cells. Communications between altered mitochondria and nuclei are associated with metabolic quiescence of cancer cells that can survive chemotherapy. In plants, changes in the pathways between nuclei, mitochondria, and chloroplasts are associated with cold stress and bud dormancy. Plasmodium falciparum parasites, the deadliest agent of malaria in humans, contain a chloroplast-like organelle (apicoplast) derived from an ancient photosynthetic symbiont. Antimalarial treatments can fail because a small fraction of the blood stage parasites enter dormancy and recrudesce after drug exposure. Altered mitochondrial-nuclear interactions in these persisters have been described for P. falciparum, but interactions of the apicoplast remained to be characterized. In the present study, we examined the apicoplasts of persisters obtained after exposure to dihydroartemisinin (a first-line antimalarial drug) followed by sorbitol treatment, or after exposure to sorbitol treatment alone. As previously observed, the mitochondrion of persisters was consistently enlarged and in close association with the nucleus. In contrast, the apicoplast varied from compact and oblate, like those of active ring stage parasites, to enlarged and irregularly shaped. Enlarged apicoplasts became more prevalent later in dormancy, but regular size apicoplasts subsequently predominated in actively replicating recrudescent parasites. All three organelles, nucleus, mitochondrion, and apicoplast, became closer during dormancy. Understanding their relationships in erythrocytic-stage persisters may lead to new strategies to prevent recrudescences and protect the future of malaria chemotherapy. Significance StatementDormancy of blood-stage malaria parasites (as persister forms) frequently undermines treatment and may facilitate the evolution of drug resistance. Here, we examine changes that occur in dormancy with two P. falciparum organelles relative to the nucleus: the mitochondrion and the plastid-like apicoplast. As previously reported, the mitochondrion of persisters is consistently enlarged, irregularly shaped, and shifted into close apposition with the nucleus. However, apicoplasts exhibit a greater variety of shapes, volumes, and relative positioning during dormancy: some persisters maintain a regular appearing apicoplast, while others show dramatically altered apicoplasts, reminiscent of the chloroplast swelling and degradation that occurs with death from reactive oxygen species in various plant cells. Improved understanding of these processes will support new approaches in antimalarial chemotherapy.

microbiology↗

bioRxiv2023

Transmissibility of clinically relevant atovaquone-resistant Plasmodium falciparum by anopheline mosquitoes

Rising numbers of malaria cases and deaths underscore the need for new interventions. Long-acting injectable medications, such as those now in use for HIV prophylaxis, offer the prospect of a malaria "chemical vaccine", combining the efficacy of a drug (like atovaquone) with the durability of a biological vaccine. Of concern, however, is the possible selection and transmission of drug-resistant parasites. We addressed this question by generating clinically relevant, highly atovaquone-resistant, Plasmodium falciparum mutants competent to infect mosquitoes. Isogenic paired strains, that differ only by a single Y268S mutation in cytochrome b, were evaluated in parallel in southeast Asian (Anopheles stephensi) or African (Anopheles gambiae) mosquitoes, and thence in humanized mice. Fitness costs of the mutation were evident along the lifecycle, in asexual parasite growth in vitro and in a progressive loss of parasites in the mosquito. In numerous independent experiments, microscopic exam of salivary glands from hundreds of mosquitoes failed to detect even one Y268S sporozoite, a defect not rescued by coinfection with wild type parasites. Furthermore, despite uniformly successful transmission of wild type parasites from An. stephensi to FRG NOD huHep mice bearing human hepatocytes and erythrocytes, multiple attempts with Y268S-fed mosquitoes failed: there was no evidence of parasites in mouse tissues by microscopy, in vitro culture, or PCR. These studies confirm a severe-to-lethal fitness cost of clinically relevant atovaquone-resistant P. falciparum in the mosquito, and they significantly lessen the likelihood of their transmission in the field. SignificanceNew tools are needed to protect individuals from malaria and to control malaria in the field. Atovaquone plus proguanil is a commonly used and well-tolerated medicine to prevent malaria. No drug resistance has been reported from its prophylactic use, but tablets must be taken daily. Giving atovaquone as a single injection may provide much longer-lasting protection, against both falciparum and vivax malaria, but there is concern this may create drug resistance. In this study we showed that clinically relevant atovaquone-resistant malaria parasites survive poorly, if at all, in mosquitoes, and that mosquitoes do not transmit drug-resistant parasites to humanized mice. These findings lessen the likelihood that an atovaquone "chemical vaccine" would lead to the spread of atovaquone resistance.

microbiology↗

bioRxiv2022

The Plasmodium falciparum apicoplast cysteine desulfurase provides sulfur for both iron sulfur cluster assembly and tRNA modification

Iron sulfur clusters (FeS) are ancient and ubiquitous protein cofactors that play fundamental roles in many aspects of cell biology. These cofactors cannot be scavenged or trafficked within a cell and thus must be synthesized in any subcellular compartment where they are required. We examined the FeS synthesis proteins found in the relict plastid organelle, called the apicoplast, of the human malaria parasite Plasmodium falciparum. Using a chemical bypass method, we deleted four of the FeS pathway proteins involved in sulfur acquisition and cluster assembly and demonstrated that they are all essential for parasite survival. However, the effect that these deletions had on the apicoplast organelle differed. Deletion of the cysteine desulfurase SufS led to disruption of the apicoplast organelle and loss of the organellar genome, whereas the other deletions did not affect organelle maintenance. Ultimately, we discovered that the requirement of SufS for organelle maintenance is not driven by its role in FeS biosynthesis, but rather, by its function in generating sulfur for use by MnmA, a tRNA modifying enzyme that we localized to the apicoplast. By complementing the activity of the parasite MnmA and SufS with a bacterial MnmA and its cognate cysteine desulfurase, we showed that the parasite SufS provides sulfur for both FeS biosynthesis and tRNA modification in the apicoplast. The dual role of parasite SufS is likely to be found in other plastid-containing organisms and highlights the central role of this enzyme in plastid biology.

microbiology↗

bioRxiv2022

The mitochondrion of Plasmodium falciparum generates essential acetyl-CoA for protein acetylation

Coenzyme A (CoA) biosynthesis is an excellent target for antimalarial intervention. While most studies have focused on the use of CoA to produce acetyl-CoA in the apicoplast and the cytosol of malaria parasites, mitochondrial acetyl-CoA production is less well understood. In the current study, we performed metabolite labeling experiments to measure endogenous metabolites in Plasmodium falciparum lines with genetic deletions affecting mitochondrial dehydrogenase activity. Our results show that mitochondrial acetyl-CoA biosynthesis is essential for parasite growth and identify a catalytic redundancy between the two main ketoacid dehydrogenase enzymes, both of which are able to produce acetyl-CoA. The activity of these enzymes is dependent on the lipoate attachment enzyme LipL2, which is essential for parasite survival solely based on its role in supporting acetyl-CoA metabolism. We also find that acetyl-CoA produced in the mitochondrion is essential for the acetylation of histones and other proteins outside of the mitochondrion. Taken together, our results demonstrate that the mitochondrion is an essential de novo source of acetyl-CoA and is required for P. falciparum protein acetylation critical to parasite survival.

microbiology↗

bioRxiv2021

Critical Role for Isoprenoids in Apicoplast Biogenesis by Malaria Parasites

Isopentenyl pyrophosphate (IPP) is an essential metabolic output of the apicoplast organelle in Plasmodium falciparum malaria parasites and is required for prenylation-dependent vesicular trafficking and other cellular processes. We have elucidated a critical and previously uncharacterized role for IPP in apicoplast biogenesis. Inhibiting IPP synthesis blocks apicoplast elongation and inheritance by daughter merozoites, and apicoplast biogenesis is rescued by exogenous IPP and polyprenols. Knockout of the only known isoprenoid-dependent apicoplast pathway, tRNA prenylation by MiaA, has no effect on blood-stage parasites and thus cannot explain apicoplast reliance on IPP. However, we have localized an annotated polyprenyl synthase (PPS) to the apicoplast lumen. PPS knockdown is lethal to parasites, rescued by IPP, and blocks apicoplast biogenesis, thus explaining apicoplast dependence on isoprenoid synthesis. We hypothesize that PPS synthesizes long-chain polyprenols critical for apicoplast membrane fluidity and biogenesis. This work critically expands the paradigm for isoprenoid utilization in malaria parasites and identifies a novel essential branch of apicoplast metabolism suitable for therapeutic targeting.

microbiology↗

bioRxiv2020

A redesigned TetR-aptamer system to control gene expression in Plasmodium falciparum

One of the most powerful approaches to understanding gene function involves turning genes on and off at will and measuring the impact at the cellular or organismal level. This particularly applies to the cohort of essential genes where traditional gene knockouts are inviable. In Plasmodium falciparum, conditional control of gene expression has been achieved by using multi-component systems in which individual modules interact with each other to regulate DNA recombination, transcription or posttranscriptional processes. The recently devised TetR-DOZI aptamer system relies on the ligand-regulatable interaction of a protein module with synthetic RNA aptamers to control the translation of a target gene. This technique has been successfully employed to study essential genes in P. falciparum and involves the insertion of several aptamer copies into their 3 untranslated regions (UTRs) which provide control over mRNA fate. However, aptamer repeats are prone to recombination and one or more copies can be lost from the system, resulting in a loss of control over target gene expression. We rectified this issue by redesigning the aptamer array to minimize recombination while preserving the control elements. As proof of concept, we compared the original and modified arrays for their ability to knock down the levels of a putative essential apicoplast protein (PF3D7_0815700) and demonstrated that the modified array is highly stable and efficient. This redesign will enhance the utility of a tool that is quickly becoming a favored strategy for genetic studies in P. falciparum. ImportanceMalaria elimination efforts have been repeatedly hindered by the evolution and spread of multidrug-resistant strains of Plasmodium falciparum. The absence of a commercially available vaccine emphasizes the need for a better understanding of Plasmodium biology in order to further translational research. This has been partly facilitated by targeted gene deletion strategies for the functional analysis of parasite genes. However, genes that are essential for parasite replication in erythrocytes are refractory to such methods, and require conditional knockdown or knockout approaches to dissect their function. One such approach is the TetR-DOZI system that employs multiple synthetic aptamers in the untranslated regions of target genes to control their expression in a tetracycline-dependent manner. Maintaining modified parasites with intact aptamer copies has been challenging since these repeats are frequently lost by recombination. By interspacing the aptamers with unique sequences, we created a stable genetic system that remains effective at controlling target gene expression.

molecular biology↗