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 dormant 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 when actively replicating parasites recrudesced. 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.

Clinically relevant atovaquone-resistant human malaria parasites fail to transmit by mosquito.

Long-acting injectable medications, such as atovaquone, offer the prospect of a "chemical vaccine" for malaria, combining drug efficacy with vaccine durability. However, selection and transmission of drug-resistant parasites is of concern. Laboratory studies have indicated that atovaquone resistance disadvantages parasites in mosquitoes, but lack of data on clinically relevant Plasmodium falciparum has hampered integration of these variable findings into drug development decisions. Here we generate atovaquone-resistant parasites that differ from wild type parent by only a Y268S mutation in cytochrome b, a modification associated with atovaquone treatment failure in humans. Relative to wild type, Y268S parasites evidence multiple defects, most marked in their development in mosquitoes, whether from Southeast Asia (Anopheles stephensi) or Africa (An. gambiae). Growth of asexual Y268S P. falciparum in human red cells is impaired, but parasite loss in the mosquito is progressive, from reduced gametocyte exflagellation, to smaller number and size of oocysts, and finally to absence of sporozoites. The Y268S mutant fails to transmit from mosquitoes to mice engrafted with human liver cells and erythrocytes. The severe-to-lethal fitness cost of clinically relevant atovaquone resistance to P. falciparum in the mosquito substantially lessens the likelihood of its transmission in the field.

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. Complementation of MnmA and SufS activity with a bacterial MnmA and its cognate cysteine desulfurase strongly suggests 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.

A Chemical Proteomic Strategy Reveals Inhibitors of Lipoate Salvage in Bacteria and Parasites.

The development of novel anti-infectives requires unprecedented strategies targeting pathways which are solely present in pathogens but absent in humans. Following this principle, we developed inhibitors of lipoic acid (LA) salvage, a crucial pathway for the survival of LA auxotrophic bacteria and parasites but non-essential in human cells. An LA-based probe was selectively transferred onto substrate proteins via lipoate protein ligase (LPL) in intact cells, and their binding sites were determined by mass spectrometry. Probe labeling served as a proxy of LPL activity, enabling in situ screenings for cell-permeable LPL inhibitors. Profiling a focused compound library revealed two substrate analogs (LAMe and C3) as inhibitors, which were further validated by binding studies and co-crystallography. Importantly, LAMe exhibited low toxicity in human cells and achieved killing of Plasmodium falciparum in erythrocytes with an EC50 value of 15 µM, making it the most effective LPL inhibitor reported to date.

The mitochondrion of Plasmodium falciparum is required for cellular acetyl-CoA metabolism and 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 the mitochondrion is required for cellular acetyl-CoA biosynthesis and identify a synthetic lethal relationship between the two main ketoacid dehydrogenase enzymes. 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 required for cellular acetyl-CoA metabolism and protein acetylation essential for parasite survival.

Metabolic responses in blood-stage malaria parasites associated with increased and decreased sensitivity to PfATP4 inhibitors.

BACKGROUND: Spiroindolone and pyrazoleamide antimalarial compounds target Plasmodium falciparum P-type ATPase (PfATP4) and induce disruption of intracellular Na+ homeostasis. Recently, a PfATP4 mutation was discovered that confers resistance to a pyrazoleamide while increasing sensitivity to a spiroindolone. Transcriptomic and metabolic adaptations that underlie this seemingly contradictory response of P. falciparum to sublethal concentrations of each compound were examined to understand the different cellular accommodation to PfATP4 disruptions. METHODS: A genetically engineered P. falciparum Dd2 strain (Dd2A211V) carrying an Ala211Val (A211V) mutation in PfATP4 was used to identify metabolic adaptations associated with the mutation that results in decreased sensitivity to PA21A092 (a pyrazoleamide) and increased sensitivity to KAE609 (a spiroindolone). First, sublethal doses of PA21A092 and KAE609 causing substantial reduction (30-70%) in Dd2A211V parasite replication were identified. Then, at this sublethal dose of PA21A092 (or KAE609), metabolomic and transcriptomic data were collected during the first intraerythrocytic developmental cycle. Finally, the time-resolved data were integrated with a whole-genome metabolic network model of P. falciparum to characterize antimalarial-induced physiological adaptations. RESULTS: Sublethal treatment with PA21A092 caused significant (p < 0.001) alterations in the abundances of 91 Plasmodium gene transcripts, whereas only 21 transcripts were significantly altered due to sublethal treatment with KAE609. In the metabolomic data, a substantial alteration (≥ fourfold) in the abundances of carbohydrate metabolites in the presence of either compound was found. The estimated rates of macromolecule syntheses between the two antimalarial-treated conditions were also comparable, except for the rate of lipid synthesis. A closer examination of parasite metabolism in the presence of either compound indicated statistically significant differences in enzymatic activities associated with synthesis of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol. CONCLUSION: The results of this study suggest that malaria parasites activate protein kinases via phospholipid-dependent signalling in response to the ionic perturbation induced by the Na+ homeostasis disruptor PA21A092. Therefore, targeted disruption of phospholipid signalling in PA21A092-resistant parasites could be a means to block the emergence of resistance to PA21A092.

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.

New insights into apicoplast metabolism in blood-stage malaria parasites.

The apicoplast of Plasmodium falciparum is the only source of essential isoprenoid precursors and Coenzyme A (CoA) in the parasite. Isoprenoid precursor synthesis relies on the iron-sulfur cluster (FeS) cofactors produced within the apicoplast, rendering FeS synthesis an essential function of this organelle. Recent reports provide important insights into the roles of FeS cofactors and the use of isoprenoid precursors and CoA both inside and outside the apicoplast. Here, we review the recent insights into the roles of these metabolites in blood-stage malaria parasites and discuss new questions that have been raised in light of these discoveries.

Screening the Pathogen Box for Inhibition of Plasmodium falciparum Sporozoite Motility Reveals a Critical Role for Kinases in Transmission Stages.

As the malaria parasite becomes resistant to every drug that we develop, the identification and development of novel drug candidates are essential. Many studies have screened compounds designed to target the clinically important blood stages. However, if we are to shrink the malaria map, new drugs that block the transmission of the parasite are needed. Sporozoites are the infective stage of the malaria parasite, transmitted to the mammalian host as mosquitoes probe for blood. Sporozoite motility is critical to their ability to exit the inoculation site and establish infection, and drug-like compounds targeting motility are effective at blocking infection in the rodent malaria model. In this study, we established a moderate-throughput motility assay for sporozoites of the human malaria parasite Plasmodium falciparum, enabling us to screen the 400 drug-like compounds from the pathogen box provided by the Medicines for Malaria Venture for their activity. Compounds exhibiting inhibitory effects on P. falciparum sporozoite motility were further assessed for transmission-blocking activity and asexual-stage growth. Five compounds had a significant inhibitory effect on P. falciparum sporozoite motility in the nanomolar range. Using membrane feeding assays, we demonstrate that four of these compounds had inhibitory activity against the transmission of P. falciparum to the mosquito. Interestingly, of the four compounds with inhibitory activity against both transmission stages, three are known kinase inhibitors. Together with a previous study that found that several of these compounds could inhibit asexual blood-stage parasite growth, our findings provide new antimalarial drug candidates that have multistage activity.

The ferredoxin redox system - an essential electron distributing hub in the apicoplast of Apicomplexa.

The apicoplast, a relict plastid found in most species of the phylum Apicomplexa, harbors the ferredoxin redox system which supplies electrons to enzymes of various metabolic pathways in this organelle. Recent reports in Toxoplasma gondii and Plasmodium falciparum have shown that the iron-sulfur cluster (FeS)-containing ferredoxin is essential in tachyzoite and blood-stage parasites, respectively. Here we review ferredoxin's crucial contribution to isoprenoid and lipoate biosynthesis as well as tRNA modification in the apicoplast, highlighting similarities and differences between the two species. We also discuss ferredoxin's potential role in the initial reductive steps required for FeS synthesis as well as recent evidence that offers an explanation for how NADPH required by the redox system might be generated in Plasmodium spp.

Metabolic changes accompanying the loss of fumarate hydratase and malate-quinone oxidoreductase in the asexual blood stage of Plasmodium falciparum.

In the glucose-rich milieu of red blood cells, asexually replicating malarial parasites mainly rely on glycolysis for ATP production, with limited carbon flux through the mitochondrial tricarboxylic acid (TCA) cycle. By contrast, gametocytes and mosquito-stage parasites exhibit an increased dependence on the TCA cycle and oxidative phosphorylation for more economical energy generation. Prior genetic studies supported these stage-specific metabolic preferences by revealing that six of eight TCA cycle enzymes are completely dispensable during the asexual blood stages of Plasmodium falciparum, with only fumarate hydratase (FH) and malate-quinone oxidoreductase (MQO) being refractory to deletion. Several hypotheses have been put forth to explain the possible essentiality of FH and MQO, including their participation in a malate shuttle between the mitochondrial matrix and the cytosol. However, using newer genetic techniques like CRISPR and dimerizable Cre, we were able to generate deletion strains of FH and MQO in P. falciparum. We employed metabolomic analyses to characterize a double knockout mutant of FH and MQO (ΔFM) and identified changes in purine salvage and urea cycle metabolism that may help to limit fumarate accumulation. Correspondingly, we found that the ΔFM mutant was more sensitive to exogenous fumarate, which is known to cause toxicity by modifying and inactivating proteins and metabolites. Overall, our data indicate that P. falciparum is able to adequately compensate for the loss of FH and MQO, rendering them unsuitable targets for drug development.

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. PPS knockdown is lethal to parasites, rescued by IPP and long- (C50) but not short-chain (≤C20) prenyl alcohols, 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.

Metabolic adjustments of blood-stage Plasmodium falciparum in response to sublethal pyrazoleamide exposure.

Due to the recurring loss of antimalarial drugs to resistance, there is a need for novel targets, drugs, and combination therapies to ensure the availability of current and future countermeasures. Pyrazoleamides belong to a novel class of antimalarial drugs that disrupt sodium ion homeostasis, although the exact consequences of this disruption in Plasmodium falciparum remain under investigation. In vitro experiments demonstrated that parasites carrying mutations in the metabolic enzyme PfATP4 develop resistance to pyrazoleamide compounds. However, the underlying mechanisms that allow mutant parasites to evade pyrazoleamide treatment are unclear. Here, we first performed experiments to identify the sublethal dose of a pyrazoleamide compound (PA21A092) that caused a significant reduction in growth over one intraerythrocytic developmental cycle (IDC). At this drug concentration, we collected transcriptomic and metabolomic data at multiple time points during the IDC to quantify gene- and metabolite-level alterations in the treated parasites. To probe the effects of pyrazoleamide treatment on parasite metabolism, we coupled the time-resolved omics data with a metabolic network model of P. falciparum. We found that the drug-treated parasites adjusted carbohydrate metabolism to enhance synthesis of myoinositol-a precursor for phosphatidylinositol biosynthesis. This metabolic adaptation caused a decrease in metabolite flux through the pentose phosphate pathway, causing a decreased rate of RNA synthesis and an increase in oxidative stress. Our model analyses suggest that downstream consequences of enhanced myoinositol synthesis may underlie adjustments that could lead to resistance emergence in P. falciparum exposed to a sublethal dose of a pyrazoleamide drug.

Inter-study and time-dependent variability of metabolite abundance in cultured red blood cells.

BACKGROUND: Cultured human red blood cells (RBCs) provide a powerful ex vivo assay platform to study blood-stage malaria infection and propagation. In recent years, high-resolution metabolomic methods have quantified hundreds of metabolites from parasite-infected RBC cultures under a variety of perturbations. In this context, the corresponding control samples of the uninfected culture systems can also be used to examine the effects of these perturbations on RBC metabolism itself and their dependence on blood donors (inter-study variations). METHODS: Time-course datasets from five independent studies were generated and analysed, maintaining uninfected RBCs (uRBC) at 2% haematocrit for 48 h under conditions originally designed for parasite cultures. Using identical experimental protocols, quadruplicate samples were collected at six time points, and global metabolomics were employed on the pellet fraction of the uRBC cultures. In total, ~ 500 metabolites were examined across each dataset to quantify inter-study variability in RBC metabolism, and metabolic network modelling augmented the analyses to characterize the metabolic state and fluxes of the RBCs. RESULTS: To minimize inter-study variations unrelated to RBC metabolism, an internal standard metabolite (phosphatidylethanolamine C18:0/20:4) was identified with minimal variation in abundance over time and across all the samples of each dataset to normalize the data. Although the bulk of the normalized data showed a high degree of inter-study consistency, changes and variations in metabolite levels from individual donors were noted. Thus, a total of 24 metabolites were associated with significant variation in the 48-h culture time window, with the largest variations involving metabolites in glycolysis and synthesis of glutathione. Metabolic network analysis was used to identify the production of superoxide radicals in cultured RBCs as countered by the activity of glutathione oxidoreductase and synthesis of reducing equivalents via the pentose phosphate pathway. Peptide degradation occurred at a rate that is comparable with central carbon fluxes, consistent with active degradation of methaemoglobin, processes also commonly associated with storage lesions in RBCs. CONCLUSIONS: The bulk of the data showed high inter-study consistency. The collected data, quantification of an expected abundance variation of RBC metabolites, and characterization of a subset of highly variable metabolites in the RBCs will help in identifying non-specific changes in metabolic abundances that may obscure accurate metabolomic profiling of Plasmodium falciparum and other blood-borne pathogens.

Dephospho-CoA kinase, a nuclear-encoded apicoplast protein, remains active and essential after Plasmodium falciparum apicoplast disruption.

Malaria parasites contain an essential organelle called the apicoplast that houses metabolic pathways for fatty acid, heme, isoprenoid, and iron-sulfur cluster synthesis. Surprisingly, malaria parasites can survive without the apicoplast as long as the isoprenoid precursor isopentenyl pyrophosphate (IPP) is supplemented in the growth medium, making it appear that isoprenoid synthesis is the only essential function of the organelle in blood-stage parasites. In the work described here, we localized an enzyme responsible for coenzyme A synthesis, DPCK, to the apicoplast, but we were unable to delete DPCK, even in the presence of IPP. However, once the endogenous DPCK was complemented with the E. coli DPCK (EcDPCK), we were successful in deleting it. We were then able to show that DPCK activity is required for parasite survival through knockdown of the complemented EcDPCK. Additionally, we showed that DPCK enzyme activity remains functional and essential within the vesicles present after apicoplast disruption. These results demonstrate that while the apicoplast of blood-stage P. falciparum parasites can be disrupted, the resulting vesicles remain biochemically active and are capable of fulfilling essential functions.

Metabolic Survival Adaptations of Plasmodium falciparum Exposed to Sublethal Doses of Fosmidomycin.

The malaria parasite Plasmodium falciparum contains the apicoplast organelle that synthesizes isoprenoids, which are metabolites necessary for posttranslational modification of Plasmodium proteins. We used fosmidomycin, an antibiotic that inhibits isoprenoid biosynthesis, to identify mechanisms that underlie the development of the parasite's adaptation to the drug at sublethal concentrations. We first determined a concentration of fosmidomycin that reduced parasite growth by ∼50% over one intraerythrocytic developmental cycle (IDC). At this dose, we maintained synchronous parasite cultures for one full IDC and collected metabolomic and transcriptomic data at multiple time points to capture global and stage-specific alterations. We integrated the data with a genome-scale metabolic model of P. falciparum to characterize the metabolic adaptations of the parasite in response to fosmidomycin treatment. Our simulations showed that, in treated parasites, the synthesis of purine-based nucleotides increased, whereas the synthesis of phosphatidylcholine during the trophozoite and schizont stages decreased. Specifically, the increased polyamine synthesis led to increased nucleotide synthesis, while the reduced methyl-group cycling led to reduced phospholipid synthesis and methyltransferase activities. These results indicate that fosmidomycin-treated parasites compensate for the loss of prenylation modifications by directly altering processes that affect nucleotide synthesis and ribosomal biogenesis to control the rate of RNA translation during the IDC. This also suggests that combination therapies with antibiotics that target the compensatory response of the parasite, such as nucleotide synthesis or ribosomal biogenesis, may be more effective than treating the parasite with fosmidomycin alone.

Roles of Ferredoxin-Dependent Proteins in the Apicoplast of Plasmodium falciparum Parasites.

Ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) form a redox system that is hypothesized to play a central role in the maintenance and function of the apicoplast organelle of malaria parasites. The Fd/FNR system provides reducing power to various iron-sulfur cluster (FeS)-dependent proteins in the apicoplast and is believed to help to maintain redox balance in the organelle. While the Fd/FNR system has been pursued as a target for antimalarial drug discovery, Fd, FNR, and the FeS proteins presumably reliant on their reducing power play an unknown role in parasite survival and apicoplast maintenance. To address these questions, we generated genetic deletions of these proteins in a parasite line containing an apicoplast bypass system. Through these deletions, we discovered that Fd, FNR, and certain FeS proteins are essential for parasite survival but found that none are required for apicoplast maintenance. Additionally, we addressed the question of how Fd and its downstream FeS proteins obtain FeS cofactors by deleting the FeS transfer proteins SufA and NfuApi. While individual deletions of these proteins revealed their dispensability, double deletion resulted in synthetic lethality, demonstrating a redundant role in providing FeS clusters to Fd and other essential FeS proteins. Our data support a model in which the reducing power from the Fd/FNR system to certain downstream FeS proteins is essential for the survival of blood-stage malaria parasites but not for organelle maintenance, while other FeS proteins are dispensable for this stage of parasite development. IMPORTANCE Ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) form one of the few known redox systems in the apicoplast of malaria parasites and provide reducing power to iron-sulfur (FeS) cluster proteins within the organelle. While the Fd/FNR system has been explored as a drug target, the essentiality and roles of this system and the identity of its downstream FeS proteins have not been determined. To answer these questions, we generated deletions of these proteins in an apicoplast metabolic bypass line (PfMev) and determined the minimal set of proteins required for parasite survival. Moving upstream of this pathway, we also generated individual and dual deletions of the two FeS transfer proteins that deliver FeS clusters to Fd and downstream FeS proteins. We found that both transfer proteins are dispensable, but double deletion displayed a synthetic lethal phenotype, demonstrating their functional redundancy. These findings provide important insights into apicoplast biochemistry and drug development.

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 multicomponent 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 the 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. falciparumIMPORTANCE Malaria 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 can be lost by recombination. By interspacing the aptamers with unique sequences, we created a stable genetic system that remains effective at controlling target gene expression.

The NTP generating activity of pyruvate kinase II is critical for apicoplast maintenance in Plasmodium falciparum.

The apicoplast of Plasmodium falciparum parasites is believed to rely on the import of three-carbon phosphate compounds for use in organelle anabolic pathways, in addition to the generation of energy and reducing power within the organelle. We generated a series of genetic deletions in an apicoplast metabolic bypass line to determine which genes involved in apicoplast carbon metabolism are required for blood-stage parasite survival and organelle maintenance. We found that pyruvate kinase II (PyrKII) is essential for organelle maintenance, but that production of pyruvate by PyrKII is not responsible for this phenomenon. Enzymatic characterization of PyrKII revealed activity against all NDPs and dNDPs tested, suggesting that it may be capable of generating a broad range of nucleotide triphosphates. Conditional mislocalization of PyrKII resulted in decreased transcript levels within the apicoplast that preceded organelle disruption, suggesting that PyrKII is required for organelle maintenance due to its role in nucleotide triphosphate generation.

A mevalonate bypass system facilitates elucidation of plastid biology in malaria parasites.

Malaria parasites rely on a plastid organelle for survival during the blood stages of infection. However, the entire organelle is dispensable as long as the isoprenoid precursor, isopentenyl pyrophosphate (IPP), is supplemented in the culture medium. We engineered parasites to produce isoprenoid precursors from a mevalonate-dependent pathway, creating a parasite line that replicates normally after the loss of the apicoplast organelle. We show that carbon-labeled mevalonate is specifically incorporated into isoprenoid products, opening new avenues for researching this essential class of metabolites in malaria parasites. We also show that essential apicoplast proteins, such as the enzyme target of the drug fosmidomycin, can be deleted in this mevalonate bypass parasite line, providing a new method to determine the roles of other important apicoplast-resident proteins. Several antibacterial drugs kill malaria parasites by targeting basic processes, such as transcription, in the organelle. We used metabolomic and transcriptomic methods to characterize parasite metabolism after azithromycin treatment triggered loss of the apicoplast and found that parasite metabolism and the production of apicoplast proteins is largely unaltered. These results provide insight into the effects of apicoplast-disrupting drugs, several of which have been used to treat malaria infections in humans. Overall, the mevalonate bypass system provides a way to probe essential aspects of apicoplast biology and study the effects of drugs that target apicoplast processes.