RESUMEN
Cripowellins from Crinum erubescens are known pesticidal and have potent antiplasmodial activity. To gain mechanistic insights to this class of natural products, studies to determine the timing of action of cripowellins within the asexual intraerythrocytic cycle of Plasmodium falciparum were performed and led to the observation that this class of natural products induced reversible cytostasis in the ring stage within the first 24 h of treatment. The transcriptional program necessary for P. falciparum to progress through the asexual intraerythrocytic life cycle is well characterized. Whole transcriptome abundance analysis showed that cripowellin B "pauses" the transcriptional program necessary to progress through the intraerythrocytic life cycle coinciding with the lack of morphological progression of drug treated parasites. In addition, cripowellin B-treated parasites re-enter transcriptional progression after treatment was removed. This study highlights the use of cripowellins as chemical probes to reveal new aspects of cell cycle progression of the asexual ring stage of P. falciparum which could be leveraged for the generation of future antimalarial therapeutics.
Asunto(s)
Alcaloides de Amaryllidaceae , Antimaláricos , Malaria Falciparum , Animales , Plasmodium falciparum , Antimaláricos/farmacología , Antimaláricos/metabolismo , Alcaloides de Amaryllidaceae/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Estadios del Ciclo de Vida , EritrocitosRESUMEN
Differentiation from asexual blood stages to mature sexual gametocytes is required for the transmission of malaria parasites. Here, we report that the ApiAP2 transcription factor, PfAP2-G2 (PF3D7_1408200) plays a critical role in the maturation of Plasmodium falciparum gametocytes. PfAP2-G2 binds to the promoters of a wide array of genes that are expressed at many stages of the parasite life cycle. Interestingly, we also find binding of PfAP2-G2 within the gene body of almost 3,000 genes, which strongly correlates with the location of H3K36me3 and several other histone modifications as well as Heterochromatin Protein 1 (HP1), suggesting that occupancy of PfAP2-G2 in gene bodies may serve as an alternative regulatory mechanism. Disruption of pfap2-g2 does not impact asexual development, but the majority of sexual parasites are unable to mature beyond stage III gametocytes. The absence of pfap2-g2 leads to overexpression of 28% of the genes bound by PfAP2-G2 and none of the PfAP2-G2 bound genes are downregulated, suggesting that it is a repressor. We also find that PfAP2-G2 interacts with chromatin remodeling proteins, a microrchidia (MORC) protein, and another ApiAP2 protein (PF3D7_1139300). Overall our data demonstrate that PfAP2-G2 establishes an essential gametocyte maturation program in association with other chromatin-related proteins.
Asunto(s)
Células Germinativas/crecimiento & desarrollo , Malaria Falciparum/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Gametogénesis , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/metabolismo , Humanos , Estadios del Ciclo de Vida , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Factores de Transcripción/genéticaRESUMEN
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) required 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 panresistant 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 (â¼2×) 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 single nucleotide polymorphism (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 and atovaquone resistance, SB1-A6 represents a unique genotype in which both alterations are present in a single line, suggesting that the combination contributes to the panresistant 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 antimalarial combination therapies and reduce the rapid development of drug resistance in the field.
Asunto(s)
Antimaláricos , Malaria Falciparum , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Variaciones en el Número de Copia de ADN/genética , Resistencia a Medicamentos/genética , Humanos , Malaria Falciparum/tratamiento farmacológico , Mitocondrias , Plasmodium falciparum/genéticaRESUMEN
To capture the transcriptional dynamics within proliferating cells, methods to differentiate nascent transcription from preexisting mRNAs are desired. One approach is to label newly synthesized mRNA transcripts in vivo through the incorporation of modified pyrimidines. However, the human malaria parasite, Plasmodium falciparum, is incapable of pyrimidine salvage for mRNA biogenesis. To capture cellular mRNA dynamics during Plasmodium development, we engineered parasites that can salvage pyrimidines through the expression of a single bifunctional yeast fusion gene, cytosine deaminase/uracil phosphoribosyltransferase (FCU). We show that expression of FCU allows for the direct incorporation of thiol-modified pyrimidines into nascent mRNAs. Using developmental stage-specific promoters to express FCU-GFP enables the biosynthetic capture and in-depth analysis of mRNA dynamics from subpopulations of cells undergoing differentiation. We demonstrate the utility of this method by examining the transcriptional dynamics of the sexual gametocyte stage transition, a process that is essential to malaria transmission between hosts. Using the pfs16 gametocyte-specific promoter to express FCU-GFP in 3D7 parasites, we found that sexual stage commitment is governed by transcriptional reprogramming and stabilization of a subset of essential gametocyte transcripts. We also measured mRNA dynamics in F12 gametocyte-deficient parasites and demonstrate that the transcriptional program required for sexual commitment and maturation is initiated but likely aborted due to the absence of the PfAP2-G transcriptional regulator and a lack of gametocyte-specific mRNA stabilization. Biosynthetic labeling of Plasmodium mRNAs is incredibly versatile, can be used to measure transcriptional dynamics at any stage of parasite development, and will allow for future applications to comprehensively measure RNA-protein interactions in the malaria parasite.
Asunto(s)
Estadios del Ciclo de Vida/genética , Plasmodium falciparum/genética , ARN Mensajero/biosíntesis , ARN Protozoario/biosíntesis , Transcripción Genética , Transgenes , Biotina/química , Células Cultivadas , Citosina Desaminasa/genética , Citosina Desaminasa/metabolismo , Eritrocitos/parasitología , Expresión Génica , Humanos , Organismos Modificados Genéticamente , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Pirimidinas/metabolismo , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Protozoario/genética , ARN Protozoario/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Coloración y Etiquetado/métodos , Estreptavidina/químicaRESUMEN
BACKGROUND: Malaria pathogenesis relies on sexual gametocyte forms of the malaria parasite to be transmitted between the infected human and the mosquito host but the molecular mechanisms controlling gametocytogenesis remains poorly understood. Here we provide a high-resolution transcriptome of Plasmodium falciparum as it commits to and develops through gametocytogenesis. RESULTS: The gametocyte-associated transcriptome is significantly different from that of the asexual parasites, with dynamic gene expression shifts characterizing early, intermediate and late-stage gametocyte development and results in differential timing for sex-specific transcripts. The transcriptional dynamics suggest strict transcriptional control during gametocytogenesis in P. falciparum, which we propose is mediated by putative regulators including epigenetic mechanisms (driving active repression of proliferation-associated processes) and a cascade-like expression of ApiAP2 transcription factors. CONCLUSIONS: The gametocyte transcriptome serves as the blueprint for sexual differentiation and will be a rich resource for future functional studies on this critical stage of Plasmodium development, as the intraerythrocytic transcriptome has been for our understanding of the asexual cycle.
Asunto(s)
Gametogénesis/genética , Regulación de la Expresión Génica , Plasmodium falciparum/genética , Transcripción Genética , Células Germinativas/metabolismo , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Reproducción/genética , Reproducción Asexuada/genética , Diferenciación Sexual/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The threat of widespread drug resistance to frontline antimalarials has renewed the urgency for identifying inexpensive chemotherapeutic compounds that are effective against Plasmodium falciparum, the parasite species responsible for the greatest number of malaria-related deaths worldwide. To aid in the fight against malaria, a recent extensive screening campaign has generated thousands of lead compounds with low micromolar activity against blood stage parasites. A subset of these leads has been compiled by the Medicines for Malaria Venture (MMV) into a collection of structurally diverse compounds known as the MMV Malaria Box. Currently, little is known regarding the activity of these Malaria Box compounds on parasite metabolism during intraerythrocytic development, and a majority of the targets for these drugs have yet to be defined. Here we interrogated the in vitro metabolic effects of 189 drugs (including 169 of the drug-like compounds from the Malaria Box) using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). The resulting metabolic fingerprints provide information on the parasite biochemical pathways affected by pharmacologic intervention and offer a critical blueprint for selecting and advancing lead compounds as next-generation antimalarial drugs. Our results reveal several major classes of metabolic disruption, which allow us to predict the mode of action (MoA) for many of the Malaria Box compounds. We anticipate that future combination therapies will be greatly informed by these results, allowing for the selection of appropriate drug combinations that simultaneously target multiple metabolic pathways, with the aim of eliminating malaria and forestalling the expansion of drug-resistant parasites in the field.
Asunto(s)
Antimaláricos/farmacología , Quimioterapia Combinada/métodos , Estadios del Ciclo de Vida/efectos de los fármacos , Redes y Vías Metabólicas/efectos de los fármacos , Terapia Molecular Dirigida/métodos , Plasmodium falciparum/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Antimaláricos/química , Atovacuona/farmacología , Células Cultivadas , Cromatografía Líquida de Alta Presión , Bases de Datos de Compuestos Químicos , Resistencia a Medicamentos/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Humanos , Estadios del Ciclo de Vida/fisiología , Metabolómica/métodos , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Espectrometría de Masas en TándemRESUMEN
The malaria parasite Plasmodium falciparum depends on glucose to meet its energy requirements during blood-stage development. Although glycolysis is one of the best understood pathways in the parasite, it is unclear if glucose metabolism appreciably contributes to the acetyl-CoA pools required for tricarboxylic acid metabolism (TCA) cycle and fatty acid biosynthesis. P. falciparum possesses a pyruvate dehydrogenase (PDH) complex that is localized to the apicoplast, a specialized quadruple membrane organelle, suggesting that separate acetyl-CoA pools are likely. Herein, we analyze PDH-deficient parasites using rapid stable-isotope labeling and show that PDH does not appreciably contribute to acetyl-CoA synthesis, tricarboxylic acid metabolism, or fatty acid synthesis in blood stage parasites. Rather, we find that acetyl-CoA demands are supplied through a "PDH-like" enzyme and provide evidence that the branched-chain keto acid dehydrogenase (BCKDH) complex is performing this function. We also show that acetyl-CoA synthetase can be a significant contributor to acetyl-CoA biosynthesis. Interestingly, the PDH-like pathway contributes glucose-derived acetyl-CoA to the TCA cycle in a stage-independent process, whereas anapleurotic carbon enters the TCA cycle via a stage-dependent phosphoenolpyruvate carboxylase/phosphoenolpyruvate carboxykinase process that decreases as the parasite matures. Although PDH-deficient parasites have no blood-stage growth defect, they are unable to progress beyond the oocyst phase of the parasite mosquito stage.
Asunto(s)
Acetilcoenzima A/biosíntesis , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/metabolismo , Acetato CoA Ligasa/metabolismo , Animales , Anopheles/parasitología , Ciclo del Ácido Cítrico , Ácidos Grasos/metabolismo , Cinética , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Fosfoenolpiruvato Carboxilasa/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
The origin of all mitochondria can be traced to the symbiotic arrangement that resulted in the emergence of eukaryotes in a world that was exclusively populated by prokaryotes. This arrangement, however, has been in continuous genetic flux: the varying degrees of gene loss and transfer from the mitochondrial genome in different eukaryotic lineages seem to signify an ongoing 'conflict' between the host and the symbiont. Eukaryotic parasites belonging to the phylum Apicomplexa provide an excellent example to support this view. These organisms contain the smallest mitochondrial genomes known, with an organization that differs among various genera; one genus, Cryptosporidium, seems to have lost the entire mitochondrial genome. Here we show that erythrocytic stages of the human malaria parasite Plasmodium falciparum seem to maintain an active mitochondrial electron transport chain to serve just one metabolic function: regeneration of ubiquinone required as the electron acceptor for dihydroorotate dehydrogenase, an essential enzyme for pyrimidine biosynthesis. Transgenic P. falciparum parasites expressing Saccharomyces cerevisiae dihydroorotate dehydrogenase, which does not require ubiquinone as an electron acceptor, were completely resistant to inhibitors of mitochondrial electron transport. Maintenance of mitochondrial membrane potential, however, was essential in these parasites, as indicated by their hypersensitivity to proguanil, a drug that collapsed the membrane potential in the presence of electron transport inhibitors. Thus, acquisition of just one enzyme can render mitochondrial electron transport nonessential in erythrocytic stages of P. falciparum.
Asunto(s)
Mitocondrias/metabolismo , Plasmodium falciparum/citología , Plasmodium falciparum/metabolismo , Animales , Animales Modificados Genéticamente , Atovacuona/farmacología , Dihidroorotato Deshidrogenasa , Transporte de Electrón/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Proguanil/farmacología , Saccharomyces cerevisiae/enzimología , Ubiquinona/metabolismoRESUMEN
BACKGROUND: DNA microarrays have been a valuable tool in malaria research for over a decade but remain in limited use in part due their relatively high cost, poor availability, and technical difficulty. With the aim of alleviating some of these factors next-generation DNA microarrays for genome-wide transcriptome analysis for both Plasmodium falciparum and Plasmodium berghei using the Agilent 8 x 15 K platform were designed. METHODS: Probe design was adapted from previously published methods and based on the most current transcript predictions available at the time for P. falciparum or P. berghei. Array performance and transcriptome analysis was determined using dye-coupled, aminoallyl-labelled cDNA and streamlined methods for hybridization, washing, and array analysis were developed. RESULTS: The new array design marks a notable improvement in the number of transcripts covered and average number of probes per transcript. Array performance was excellent across a wide range of transcript abundance, with low inter-array and inter-probe variability for relative abundance measurements and it recapitulated previously observed transcriptional patterns. Additionally, improvements in sensitivity permitted a 20-fold reduction in necessary starting RNA amounts, further reducing experimental costs and widening the range of application. CONCLUSIONS: DNA microarrays utilizing the Agilent 8 x 15 K platform for genome-wide transcript analysis in P. falciparum and P. berghei mark an improvement in coverage and sensitivity, increased availability to the research community, and simplification of the experimental methods.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Parasitología/métodos , Plasmodium berghei/genética , Plasmodium falciparum/genética , Plasmodium berghei/patogenicidad , Plasmodium berghei/fisiología , Plasmodium falciparum/patogenicidad , Plasmodium falciparum/fisiologíaRESUMEN
Previous studies demonstrated that Plasmodium falciparum strain D10 became highly resistant to the mitochondrial electron transport chain (mtETC) inhibitor atovaquone when the mtETC was decoupled from the pyrimidine biosynthesis pathway by expressing the fumarate-dependent (ubiquinone-independent) yeast dihydroorotate dehydrogenase (yDHODH) in parasites. To investigate the requirement for decoupled mtETC activity in P. falciparum with different genetic backgrounds, we integrated a single copy of the yDHODH gene into the genomes of D10attB, 3D7attB, Dd2attB, and HB3attB strains of the parasite. The yDHODH gene was equally expressed in all of the transgenic lines. All four yDHODH transgenic lines showed strong resistance to atovaquone in standard short-term growth inhibition assays. During longer term growth with atovaquone, D10attB-yDHODH and 3D7attB-yDHODH parasites remained fully resistant, but Dd2attB-yDHODH and HB3attB-yDHODH parasites lost their tolerance to the drug after 3 to 4 days of exposure. No differences were found, however, in growth responses among all of these strains to the Plasmodium-specific DHODH inhibitor DSM1 in either short- or long-term exposures. Thus, DSM1 works well as a selective agent in all parasite lines transfected with the yDHODH gene, whereas atovaquone works for some lines. We found that the ubiquinone analog decylubiquinone substantially reversed the atovaquone inhibition of Dd2attB-yDHODH and HB3attB-yDHODH transgenic parasites during extended growth. Thus, we conclude that there are strain-specific differences in the requirement for mtETC activity among P. falciparum strains, suggesting that, in erythrocytic stages of the parasite, ubiquinone-dependent dehydrogenase activities other than those of DHODH are dispensable in some strains but are essential in others.
Asunto(s)
Mitocondrias/metabolismo , Plasmodium falciparum/fisiología , Antimaláricos/farmacología , Atovacuona/farmacología , Células Cultivadas , Dihidroorotato Deshidrogenasa , Resistencia a Medicamentos , Transporte de Electrón , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Mitocondrias/enzimología , Organismos Modificados Genéticamente , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Parasitemia/parasitología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/farmacologíaRESUMEN
Although mitochondrial electron transport is a validated target of the antimalarial drug atovaquone, the molecular details underlying parasite demise are unclear. We have shown that a critical function of mitochondrial electron transport in blood-stage Plasmodium falciparum is to support pyrimidine biosynthesis. Here, we explore the effects of atovaquone, alone and in combination with proguanil, on P. falciparum viability. Our results suggest that the effects of inhibition depend upon the erythrocytic stage of the parasites and the duration of exposure. Ring- and schizont-stage parasites are most resilient to drug treatment and can survive for 48 h, with a fraction remaining viable even after 96 h. Survival of parasites does not appear to require nutrient uptake. Thus, intraerythrocytic parasites with inhibited mitochondrial electron transport and collapsed mitochondrial membrane potential do not undergo apoptosis but enter an apparent static state. These results have significant implications for desirable properties of antimalarials under development that target mitochondrial functions.
Asunto(s)
Antimaláricos/farmacología , Atovacuona/farmacología , Transporte de Electrón/efectos de los fármacos , Eritrocitos/parasitología , Mitocondrias/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Apoptosis/efectos de los fármacos , Hemólisis/efectos de los fármacos , HumanosRESUMEN
In the malaria parasite Plasmodium falciparum, the switch from asexual multiplication to sexual differentiation into gametocytes is essential for transmission to mosquitos. The transcription factor PfAP2-G is a key determinant of sexual commitment that orchestrates this crucial cell fate decision. Here we identify the direct targets of PfAP2-G and demonstrate that it dynamically binds hundreds of sites across the genome. We find that PfAP2-G is a transcriptional activator of early gametocyte genes, and identify differences in PfAP2-G occupancy between gametocytes derived via next-cycle and same-cycle conversion. Our data implicate PfAP2-G not only as a transcriptional activator of gametocyte genes, but also as a potential regulator of genes important for red blood cell invasion. We also find that regulation by PfAP2-G requires interaction with a second transcription factor, PfAP2-I. These results clarify the functional role of PfAP2-G during sexual commitment and early gametocytogenesis.
Asunto(s)
Malaria/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Sistemas CRISPR-Cas , Eritrocitos/parasitología , Regulación de la Expresión Génica , Genes Protozoarios/genética , Malaria/transmisión , Malaria Falciparum/parasitología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/sangre , Factores de Transcripción/metabolismoRESUMEN
Genome-wide analysis of transcription in the malaria parasite Plasmodium falciparum has revealed robust variation in steady-state mRNA abundance throughout the 48-h intraerythrocytic developmental cycle (IDC), suggesting that this process is highly dynamic and tightly regulated. Here, we utilize rapid 4-thiouracil (4-TU) incorporation via pyrimidine salvage to specifically label, capture, and quantify newly-synthesized RNA transcripts at every hour throughout the IDC. This high-resolution global analysis of the transcriptome captures the timing and rate of transcription for each newly synthesized mRNA in vivo, revealing active transcription throughout all IDC stages. Using a statistical model to predict the mRNA dynamics contributing to the total mRNA abundance at each timepoint, we find varying degrees of transcription and stabilization for each mRNA corresponding to developmental transitions. Finally, our results provide new insight into co-regulation of mRNAs throughout the IDC through regulatory DNA sequence motifs, thereby expanding our understanding of P. falciparum mRNA dynamics.
Asunto(s)
Genes Protozoarios/genética , Genoma de Protozoos/genética , Plasmodium falciparum/genética , Transcripción Genética , Eritrocitos/parasitología , Perfilación de la Expresión Génica , Ontología de Genes , Humanos , Malaria Falciparum/parasitología , Plasmodium falciparum/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismoRESUMEN
Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target-inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite.
Asunto(s)
Antimaláricos/farmacología , Resistencia a Medicamentos/genética , Genoma de Protozoos , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Activación Metabólica , Alelos , Variaciones en el Número de Copia de ADN , Evolución Molecular Dirigida , Resistencia a Múltiples Medicamentos/genética , Genes Protozoarios , Metabolómica , Mutación , Plasmodium falciparum/crecimiento & desarrollo , Selección Genética , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Gene expression DNA microarrays have been vital for characterizing whole-genome transcriptional profiles. Nevertheless, their effectiveness relies heavily on the accuracy of genome sequences, the annotation of gene structures, and the sequence-dependent performance of individual probes. Currently available gene expression arrays for the malaria parasite Plasmodium falciparum rely on an average of 2 probes per gene, usually positioned near the 3' end of genes; consequently, existing designs are prone to measurement bias and cannot capture complexities such as the occurrence of transcript isoforms arising from alternative splicing or alternative start/ stop sites. Here, we describe two novel gene expression arrays with exon-focused probes designed with an average of 12 and 20 probes spanning each gene. This high probe density minimizes signal noise inherent in probe-to-probe sequence-dependent hybridization intensity. We demonstrate that these exon arrays accurately profile genome-wide expression, comparing favorably to currently available arrays and RNA-seq profiling, and can detect alternatively spliced transcript isoforms as well as non-coding RNAs (ncRNAs). Of the 964 candidate alternate splicing events from published RNA-seq studies, 162 are confirmed using the exon array. Furthermore, the exon array predicted 330 previously unidentified alternate splicing events. Gene expression microarrays continue to offer a cost-effective alternative to RNA-seq for the simultaneous monitoring of gene expression and alternative splicing events. Microarrays may even be preferred in some cases due to their affordability and the rapid turn-around of results when hundreds of samples are required for fine-scale systems biology investigations, including the monitoring of the networks of gene co-expression in the emergence of drug resistance.
Asunto(s)
Expresión Génica , Plasmodium/genética , ARN Mensajero/genética , Empalme Alternativo , Animales , Exones , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
New antimalarial drugs are urgently needed to control drug-resistant forms of the malaria parasite Plasmodium falciparum. Mitochondrial electron transport is the target of both existing and new antimalarials. Herein, we describe 11 genetic knockout (KO) lines that delete six of the eight mitochondrial tricarboxylic acid (TCA) cycle enzymes. Although all TCA KOs grew normally in asexual blood stages, these metabolic deficiencies halted life-cycle progression in later stages. Specifically, aconitase KO parasites arrested as late gametocytes, whereas α-ketoglutarate-dehydrogenase-deficient parasites failed to develop oocysts in the mosquitoes. Mass spectrometry analysis of (13)C-isotope-labeled TCA mutant parasites showed that P. falciparum has significant flexibility in TCA metabolism. This flexibility manifested itself through changes in pathway fluxes and through altered exchange of substrates between cytosolic and mitochondrial pools. Our findings suggest that mitochondrial metabolic plasticity is essential for parasite development.
Asunto(s)
Enzimas/genética , Malaria Falciparum/genética , Mitocondrias/metabolismo , Plasmodium falciparum/genética , Ácidos Tricarboxílicos/metabolismo , Animales , Antimaláricos/química , Antimaláricos/aislamiento & purificación , Antimaláricos/metabolismo , Ciclo del Ácido Cítrico/genética , Enzimas/metabolismo , Eritrocitos/metabolismo , Técnicas de Inactivación de Genes , Humanos , Estadios del Ciclo de Vida , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Malaria Falciparum/parasitología , Mitocondrias/patología , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/patogenicidadRESUMEN
Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.