RESUMO
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.
Assuntos
Antimaláricos , Malária Falciparum , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Variações do Número de Cópias de DNA/genética , Resistência a Medicamentos/genética , Humanos , Malária Falciparum/tratamento farmacológico , Mitocôndrias , Plasmodium falciparum/genéticaRESUMO
The phylum Apicomplexa contains a group of protozoa causing diseases in humans and livestock. Plasmodium spp., the causative agent of malaria, contains a mitochondrion that is very divergent from that of their hosts. The malarial mitochondrion is a clinically validated target for the antimalarial drug atovaquone, which specifically blocks the electron transfer activity of the bc1 complex of the mitochondrial electron transport chain (mtETC). Most mtETC proteins are nuclear-encoded and imported from the cytosol, but three key protein subunits are encoded in the Plasmodium mitochondrial genome: cyt b, COXI, and COXIII. They are translated inside the mitochondrion by mitochondrial ribosomes (mitoribosomes). Here, we characterize the function of one large mitoribosomal protein in Plasmodium falciparum, PfmRPL13. We found that PfmRPL13 localizes to the parasite mitochondrion and is refractory to genetic knockout. Ablation of PfmRPL13 using a conditional knockdown system (TetR-DOZI-aptamer) caused a series of adverse events in the parasite, including mtETC deficiency, loss of mitochondrial membrane potential (Δψm), and death. The PfmRPL13 knockdown parasite also became hypersensitive to proguanil, a drug proposed to target an alternative process for maintaining Δψm Surprisingly, transmission EM revealed that PfmRPL13 disruption also resulted in an unusually elongated and branched mitochondrion. The growth arrest of the knockdown parasite could be rescued with a second copy of PfmRPL13, but not by supplementation with decylubiquinone or addition of a yeast dihydroorotate dehydrogenase gene. In summary, we provide first and direct evidence that mitoribosomes are essential for malaria parasites to maintain the structural and functional integrity of the mitochondrion.
Assuntos
Antimaláricos/farmacologia , Malária/tratamento farmacológico , Mitocôndrias/química , Mitocôndrias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Ribossômicas/metabolismo , Transporte de Elétrons , Genoma Mitocondrial , Humanos , Malária/metabolismo , Malária/parasitologia , Potencial da Membrana Mitocondrial , Mitocôndrias/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/isolamento & purificação , Proteínas Ribossômicas/genéticaRESUMO
Among the several new antimalarials discovered over the past decade are at least three clinical candidate drugs, each with a distinct chemical structure, that disrupt Na+ homeostasis resulting in a rapid increase in intracellular Na+ concentration ([Na+]i) within the erythrocytic stages of Plasmodium falciparum. At present, events triggered by Na+ influx that result in parasite demise are not well-understood. Here we report effects of two such drugs, a pyrazoleamide and a spiroindolone, on intraerythrocytic P. falciparum. Within minutes following the exposure to these drugs, the trophozoite stage parasite, which normally contains little cholesterol, was made permeant by cholesterol-dependent detergents, suggesting it acquired a substantial amount of the lipid. Consistently, the merozoite surface protein 1 and 2 (MSP1 and MSP2), glycosylphosphotidylinositol (GPI)-anchored proteins normally uniformly distributed in the parasite plasma membrane, coalesced into clusters. These alterations were not observed following drug treatment of P. falciparum parasites adapted to grow in a low [Na+] growth medium. Both cholesterol acquisition and MSP1 coalescence were reversible upon the removal of the drugs, implicating an active process of cholesterol exclusion from trophozoites that we hypothesize is inhibited by high [Na+]i. Electron microscopy of drug-treated trophozoites revealed substantial morphological changes normally seen at the later schizont stage including the appearance of partial inner membrane complexes, dense organelles that resemble "rhoptries" and apparent nuclear division. Together these results suggest that [Na+]i disruptor drugs by altering levels of cholesterol in the parasite, dysregulate trophozoite to schizont development and cause parasite demise.
Assuntos
Antimaláricos/farmacologia , Colesterol/metabolismo , Eritrócitos/parasitologia , Malária Falciparum/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Sódio/metabolismo , Western Blotting , Citometria de Fluxo , Imunofluorescência , Humanos , Microscopia Eletrônica de Transmissão , Plasmodium falciparum/metabolismoRESUMO
Caged Garcinia xanthones (CGXs) constitute a family of natural products that are produced by tropical/subtropical trees of the genus Garcinia CGXs have a unique chemical architecture, defined by the presence of a caged scaffold at the C ring of a xanthone moiety, and exhibit a broad range of biological activities. Here we show that synthetic CGXs exhibit antimalarial activity against Plasmodium falciparum, the causative parasite of human malaria, at the intraerythrocytic stages. Their activity can be substantially improved by attaching a triphenylphosphonium group at the A ring of the caged xanthone. Specifically, CR135 and CR142 were found to be highly effective antimalarial inhibitors, with 50% effective concentrations as low as â¼10 nM. CGXs affect malaria parasites at multiple intraerythrocytic stages, with mature stages (trophozoites and schizonts) being more vulnerable than immature rings. Within hours of CGX treatment, malaria parasites display distinct morphological changes, significant reduction of parasitemia (the percentage of infected red blood cells), and aberrant mitochondrial fragmentation. CGXs do not, however, target the mitochondrial electron transport chain, the target of the drug atovaquone and several preclinical candidates. CGXs are cytotoxic to human HEK293 cells at the low micromolar level, which results in a therapeutic window of around 150-fold for the lead compounds. In summary, we show that CGXs are potent antimalarial compounds with structures distinct from those of previously reported antimalarial inhibitors. Our results highlight the potential to further develop Garcinia natural product derivatives as novel antimalarial agents.
Assuntos
Antimaláricos/farmacologia , Garcinia/química , Xantonas/farmacologia , Antimaláricos/química , Antimaláricos/uso terapêutico , Células HEK293 , Humanos , Mitocôndrias/efeitos dos fármacos , Estrutura Molecular , Parasitemia/tratamento farmacológico , Parasitemia/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Esquizontes/efeitos dos fármacos , Relação Estrutura-Atividade , Trofozoítos/efeitos dos fármacos , Xantonas/química , Xantonas/uso terapêuticoRESUMO
Antimalarial combination therapies play a crucial role in preventing the emergence of drug-resistant Plasmodium parasites. Although artemisinin-based combination therapies (ACTs) comprise the majority of these formulations, inhibitors of the mitochondrial cytochrome bc1 complex (cyt bc1) are among the few compounds that are effective for both acute antimalarial treatment and prophylaxis. There are two known sites for inhibition within cyt bc1: atovaquone (ATV) blocks the quinol oxidase (Qo) site of cyt bc1, while some members of the endochin-like quinolone (ELQ) family, including preclinical candidate ELQ-300, inhibit the quinone reductase (Qi) site and retain full potency against ATV-resistant Plasmodium falciparum strains with Qo site mutations. Here, we provide the first in vivo comparison of ATV, ELQ-300, and combination therapy consisting of ATV plus ELQ-300 (ATV:ELQ-300), using P. yoelii murine models of malaria. In our monotherapy assessments, we found that ATV functioned as a single-dose curative compound in suppressive tests whereas ELQ-300 demonstrated a unique cumulative dosing effect that successfully blocked recrudescence even in a high-parasitemia acute infection model. ATV:ELQ-300 therapy was highly synergistic, and the combination was curative with a single combined dose of 1 mg/kg of body weight. Compared to the ATV:proguanil (Malarone) formulation, ATV:ELQ-300 was more efficacious in multiday, acute infection models and was equally effective at blocking the emergence of ATV-resistant parasites. Ultimately, our data suggest that dual-site inhibition of cyt bc1 is a valuable strategy for antimalarial combination therapy and that Qi site inhibitors such as ELQ-300 represent valuable partner drugs for the clinically successful Qo site inhibitor ATV.
Assuntos
Antimaláricos/farmacologia , Atovaquona/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Malária Falciparum/tratamento farmacológico , Quinolonas/farmacologia , Animais , Combinação de Medicamentos , Quimioterapia Combinada/métodos , Feminino , Camundongos , Parasitemia/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Proguanil/farmacologiaRESUMO
A central hub of carbon metabolism is the tricarboxylic acid cycle, which serves to connect the processes of glycolysis, gluconeogenesis, respiration, amino acid synthesis and other biosynthetic pathways. The protozoan intracellular malaria parasites (Plasmodium spp.), however, have long been suspected of possessing a significantly streamlined carbon metabolic network in which tricarboxylic acid metabolism plays a minor role. Blood-stage Plasmodium parasites rely almost entirely on glucose fermentation for energy and consume minimal amounts of oxygen, yet the parasite genome encodes all of the enzymes necessary for a complete tricarboxylic acid cycle. Here, by tracing (13)C-labelled compounds using mass spectrometry we show that tricarboxylic acid metabolism in the human malaria parasite Plasmodium falciparum is largely disconnected from glycolysis and is organized along a fundamentally different architecture from the canonical textbook pathway. We find that this pathway is not cyclic, but rather is a branched structure in which the major carbon sources are the amino acids glutamate and glutamine. As a consequence of this branched architecture, several reactions must run in the reverse of the standard direction, thereby generating two-carbon units in the form of acetyl-coenzyme A. We further show that glutamine-derived acetyl-coenzyme A is used for histone acetylation, whereas glucose-derived acetyl-coenzyme A is used to acetylate amino sugars. Thus, the parasite has evolved two independent production mechanisms for acetyl-coenzyme A with different biological functions. These results significantly clarify our understanding of the Plasmodium metabolic network and highlight the ability of altered variants of central carbon metabolism to arise in response to unique environments.
Assuntos
Ciclo do Ácido Cítrico/fisiologia , Plasmodium falciparum/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Amino Açúcares/metabolismo , Animais , Carbono/metabolismo , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Glucose/metabolismo , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Glutamina/química , Glutamina/metabolismo , Glicólise , Histonas/metabolismo , Malatos/metabolismo , Plasmodium falciparum/citologia , Plasmodium falciparum/fisiologiaRESUMO
Heme is an essential cofactor for aerobic organisms. Its redox chemistry is central to a variety of biological functions mediated by hemoproteins. In blood stages, malaria parasites consume most of the hemoglobin inside the infected erythrocytes, forming nontoxic hemozoin crystals from large quantities of heme released during digestion. At the same time, the parasites possess a heme de novo biosynthetic pathway. This pathway in the human malaria parasite Plasmodium falciparum has been considered essential and is proposed as a potential drug target. However, we successfully disrupted the first and last genes of the pathway, individually and in combination. These knock-out parasite lines, lacking 5-aminolevulinic acid synthase and/or ferrochelatase (FC), grew normally in blood-stage culture and exhibited no changes in sensitivity to heme-related antimalarial drugs. We developed a sensitive LC-MS/MS assay to monitor stable isotope incorporation into heme from its precursor 5-[(13)C4]aminolevulinic acid, and this assay confirmed that de novo heme synthesis was ablated in FC knock-out parasites. Disrupting the FC gene also caused no defects in gametocyte generation or maturation but resulted in a greater than 70% reduction in male gamete formation and completely prevented oocyst formation in female Anopheles stephensi mosquitoes. Our data demonstrate that the heme biosynthesis pathway is not essential for asexual blood-stage growth of P. falciparum parasites but is required for mosquito transmission. Drug inhibition of pathway activity is therefore unlikely to provide successful antimalarial therapy. These data also suggest the existence of a parasite mechanism for scavenging host heme to meet metabolic needs.
Assuntos
Anopheles/parasitologia , Eritrócitos/parasitologia , Heme/biossíntese , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , 5-Aminolevulinato Sintetase/deficiência , 5-Aminolevulinato Sintetase/genética , Animais , Feminino , Ferroquelatase/genética , Técnicas de Inativação de Genes , Heme/metabolismo , Humanos , Masculino , Plasmodium falciparum/genética , Plasmodium falciparum/fisiologia , Espectrometria de Massas em TandemRESUMO
The cytochrome bc1 complex (cyt bc1) is the third component of the mitochondrial electron transport chain and is the target of several potent antimalarial compounds, including the naphthoquinone atovaquone (ATV) and the 4(1H)-quinolone ELQ-300. Mechanistically, cyt bc1 facilitates the transfer of electrons from ubiquinol to cytochrome c and contains both oxidative (Qo) and reductive (Qi) catalytic sites that are amenable to small-molecule inhibition. Although many antimalarial compounds, including ATV, effectively target the Qo site, it has been challenging to design selective Qi site inhibitors with the ability to circumvent clinical ATV resistance, and little is known about how chemical structure contributes to site selectivity within cyt bc1. Here, we used the proposed Qi site inhibitor ELQ-300 to generate a drug-resistant Plasmodium falciparum clone containing an I22L mutation at the Qi region of cyt b. Using this D1 clone and the Y268S Qo mutant strain, P. falciparum Tm90-C2B, we created a structure-activity map of Qi versus Qo site selectivity for a series of endochin-like 4(1H)-quinolones (ELQs). We found that Qi site inhibition was associated with compounds containing 6-position halogens or aryl 3-position side chains, while Qo site inhibition was favored by 5,7-dihalogen groups or 7-position substituents. In addition to identifying ELQ-300 as a preferential Qi site inhibitor, our data suggest that the 4(1H)-quinolone scaffold is compatible with binding to either site of cyt bc1 and that minor chemical changes can influence Qo or Qi site inhibition by the ELQs.
Assuntos
Antimaláricos/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Quinolonas/farmacologia , Animais , Citocromos b/genética , Citocromos b/metabolismo , Resistência a Medicamentos , Complexo III da Cadeia de Transporte de Elétrons/genética , Modelos Moleculares , Mutação/genética , Plasmodium falciparum/genética , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Mitocôndrias/metabolismo , Plasmodium falciparum/citologia , Plasmodium falciparum/metabolismo , Animais , Animais Geneticamente Modificados , Atovaquona/farmacologia , Di-Hidro-Orotato Desidrogenase , Transporte de Elétrons/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Proguanil/farmacologia , Saccharomyces cerevisiae/enzimologia , Ubiquinona/metabolismoRESUMO
The rotary nanomotor ATP synthase is a central player in the bioenergetics of most organisms. Yet the role of ATP synthase in malaria parasites has remained unclear, as blood stages of Plasmodium falciparum appear to derive ATP largely through glycolysis. Also, genes for essential subunits of the F(O) sector of the complex could not be detected in the parasite genomes. Here, we have used molecular genetic and immunological tools to investigate the localization, complex formation, and functional significance of predicted ATP synthase subunits in P. falciparum. We generated transgenic P. falciparum lines expressing seven epitope-tagged canonical ATP synthase subunits, revealing localization of all but one of the subunits to the mitochondrion. Blue native gel electrophoresis of P. falciparum mitochondrial membranes suggested the molecular mass of the ATP synthase complex to be greater than 1 million daltons. This size is consistent with the complex being assembled as a dimer in a manner similar to the complexes observed in other eukaryotic organisms. This observation also suggests the presence of previously unknown subunits in addition to the canonical subunits in P. falciparum ATP synthase complex. Our attempts to disrupt genes encoding ß and γ subunits were unsuccessful, suggesting an essential role played by the ATP synthase complex in blood stages of P. falciparum. These studies suggest that, despite some unconventional features and its minimal contribution to ATP synthesis, P. falciparum ATP synthase is localized to the parasite mitochondrion, assembled as a large dimeric complex, and is likely essential for parasite survival.
Assuntos
Merozoítos/enzimologia , Mitocôndrias/enzimologia , Complexos Multienzimáticos/metabolismo , Plasmodium falciparum/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Protozoários/metabolismo , Glicólise/fisiologia , Mitocôndrias/genética , Complexos Multienzimáticos/genética , Plasmodium falciparum/genética , ATPases Translocadoras de Prótons/genética , Proteínas de Protozoários/genéticaRESUMO
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.
Assuntos
Mitocôndrias/metabolismo , Plasmodium falciparum/fisiologia , Antimaláricos/farmacologia , Atovaquona/farmacologia , Células Cultivadas , Di-Hidro-Orotato Desidrogenase , Resistência a Medicamentos , Transporte de Elétrons , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Mitocôndrias/enzimologia , Organismos Geneticamente Modificados , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Parasitemia/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/farmacologiaRESUMO
Cholesterol is the most abundant lipid in the erythrocyte. During its blood-stage development, the malaria parasite establishes an active cholesterol gradient across the various membrane systems within the infected erythrocyte. Interestingly, some antimalarial compounds have recently been shown to disrupt cholesterol homeostasis in the intraerythrocytic stages of Plasmodium falciparum. These studies point to the importance of cholesterol for parasite growth. Previously, reduction of cholesterol from the erythrocyte membrane by treatment with methyl-ß-cyclodextrin (MßCD) was shown to inhibit parasite invasion and growth. In addition, MßCD treatment of trophozoite-stage P. falciparum was shown to result in parasite expulsion from the host cell. We have revisited these phenomena by using live video microscopy, ultrastructural analysis, and response to antimalarial compounds. By using time-lapse video microscopy of fluorescently tagged parasites, we show that MßCD treatment for just 30 min causes dramatic expulsion of the trophozoite-stage parasites. This forceful expulsion occurs within 10 s. Remarkably, the plasma membrane of the host cell from which the parasite has been expelled does not appear to be compromised. The parasitophorous vacuolar membrane (PVM) continued to surround the extruded parasite, but the PVM appeared damaged. Treatment with antimalarial compounds targeting PfATP4 or PfNCR1 prevented MßCD-mediated extrusion of the parasites, pointing to a potential role of cholesterol dynamics underlying the expulsion phenomena. We also confirmed the essential role of erythrocyte plasma membrane cholesterol for invasion and growth of P. falciparum. This defect can be partially complemented by cholesterol and desmosterol but not with epicholesterol, revealing stereospecificity underlying cholesterol function. Overall, our studies advance previous observations and reveal unusual cell biological features underlying cholesterol depletion of the infected erythrocyte plasma membrane. IMPORTANCE Malaria remains a major challenge in much of the world. Symptoms of malaria are caused by the growth of parasites belonging to Plasmodium spp. inside the red blood cells (RBCs), leading to their destruction. The parasite depends upon its host for much of its nutritional needs. Cholesterol is a major lipid in the RBC plasma membrane, which is the only source of this lipid for malaria parasites. We have previously shown that certain new antimalarial compounds disrupt cholesterol homeostasis in P. falciparum. Here, we use live time-lapse video microscopy to show dramatic expulsion of the parasite from the host RBC when the cholesterol content of the RBC is reduced. Remarkably, this expulsion is inhibited by the antimalarials that disrupt lipid homeostasis. We also show stereospecificity of cholesterol in supporting parasite growth inside RBC. Overall, these results point to a critical role of cholesterol in the physiology of malaria parasites.
Assuntos
Colesterol/metabolismo , Membrana Eritrocítica/metabolismo , Malária Falciparum/metabolismo , Plasmodium falciparum/metabolismo , Antimaláricos/farmacologia , Membrana Eritrocítica/genética , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
The mitochondrion of malaria parasites is an attractive antimalarial drug target, which require mitoribosomes to translate genes encoded in the mitochondrial (mt) DNA. Plasmodium mitoribosomes are composed of highly fragmented ribosomal RNA (rRNA) encoded in the mtDNA. All mitoribosomal proteins (MRPs) and other assembly factors are encoded in the nuclear genome. Here, we have studied one putative assembly factor, RSM22 (Pf3D7_1027200) and one large subunit (LSU) MRP, L23 (Pf3D7_1239100) in Plasmodium falciparum. We show that both proteins localize to the mitochondrion. Conditional knock down (KD) of PfRSM22 or PfMRPL23 leads to reduced cytochrome bc1 complex activity and increased sensitivity to bc1 inhibitors such as atovaquone and ELQ-300. Using RNA sequencing as a tool, we reveal the transcriptomic changes of nuclear and mitochondrial genomes upon KD of these two proteins. In the early phase of KD, while most mt rRNAs and transcripts of putative MRPs were downregulated in the absence of PfRSM22, many mt rRNAs and several MRPs were upregulated after KD of PfMRPL23. The contrast effects in the early phase of KD likely suggests non-redundant roles of PfRSM22 and PfMRPL23 in the assembly of P. falciparum mitoribosomes. At the late time points of KD, loss of PfRSM22 and PfMRPL23 caused defects in many essential metabolic pathways and transcripts related to essential mitochondrial functions, leading to parasite death. In addition, we enlist mitochondrial proteins of unknown function that are likely novel Plasmodium MRPs based on their structural similarity to known MRPs as well as their expression profiles in KD parasites.
Assuntos
Antimaláricos , Malária Falciparum , Plasmodium , Antimaláricos/uso terapêutico , Atovaquona/farmacologia , DNA Mitocondrial/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Malária Falciparum/tratamento farmacológico , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Plasmodium/genética , Plasmodium falciparum , RNA Ribossômico/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transcrição GênicaRESUMO
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.
Assuntos
Antimaláricos/farmacologia , Atovaquona/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Eritrócitos/parasitologia , Mitocôndrias/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Hemólise/efeitos dos fármacos , HumanosRESUMO
Malaria is endemic in most developing countries, with nearly 500 million cases estimated to occur each year. The need to design a new generation of antimalarial drugs that can combat the most drug-resistant forms of the malarial parasite is well recognized. In this study, we wanted to develop inhibitors of key proteins that form the invasion machinery of the malarial parasite. A critical feature of host-cell invasion by apicomplexan parasites is the interaction between the carboxy terminal tail of myosin A (MyoA) and the myosin tail interacting protein (MTIP). Using the cocrystal structure of the Plasmodium knowlesi MTIP and the MyoA tail peptide as input to the hybrid structure-based virtual screening approach, we identified a series of small molecules as having the potential to inhibit MTIP-MyoA interactions. Of the initial 15 compounds tested, a pyrazole-urea compound inhibited P. falciparum growth with an EC(50) value of 145 nM. We screened an additional 51 compounds belonging to the same chemical class and identified 8 compounds with EC(50) values less than 400 nM. Interestingly, the compounds appeared to act at several stages of the parasite's life cycle to block growth and development. The pyrazole-urea compounds identified in this study could be effective antimalarial agents because they competitively inhibit a key protein-protein interaction between MTIP and MyoA responsible for the gliding motility and the invasive features of the malarial parasite.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Modelos Moleculares , Miosina não Muscular Tipo IIA/antagonistas & inibidores , Miosina não Muscular Tipo IIA/química , Miosina não Muscular Tipo IIA/metabolismo , Testes de Sensibilidade Parasitária , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Ureia/química , Ureia/farmacologiaRESUMO
Lipid homeostasis is essential to the maintenance of life. We previously reported that disruptions of the parasite Na+ homeostasis via inhibition of PfATP4 resulted in elevated cholesterol within the parasite plasma membrane as assessed by saponin sensitivity. A large number of compounds have been shown to target the parasite Na+ homeostasis. We screened 800 compounds from the Malaria and Pathogen Boxes to identify chemotypes that disrupted the parasite plasma membrane lipid homeostasis. Here, we show that the compounds disrupting parasite Na+ homeostasis also induced saponin sensitivity, an indication of parasite lipid homeostasis disruption. Remarkably, 13 compounds were identified that altered the plasma membrane lipid composition independently of the Na+ homeostasis disruption. Further studies suggest that these compounds target the Plasmodium falciparum Niemann-Pick type C1-related (PfNCR1) protein, which is hypothesized to be involved in maintaining plasma membrane lipid composition. PfNCR1, like PfATP4, appears to be targeted by multiple chemotypes with the potential for drug discovery.
Assuntos
Antimaláricos/farmacologia , Membrana Celular/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Eritrócitos/parasitologia , Homeostase/efeitos dos fármacos , Humanos , Lipídeos/química , Malária Falciparum/parasitologia , Plasmodium falciparum/química , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sódio/metabolismoRESUMO
The battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum, the deadliest human malaria parasite. To counter the problem, novel antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase (NDH2) of P. falciparum, PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P. falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasite's susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc1 complex. Our results suggest that PfNDH2 is not likely a good antimalarial drug target.
Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos/genética , NADH Desidrogenase/genética , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Animais , Antimaláricos/uso terapêutico , Sistemas CRISPR-Cas , Células Cultivadas , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Eritrócitos/parasitologia , Técnicas de Inativação de Genes , Humanos , Malária Falciparum/sangue , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , NADH Desidrogenase/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Quinolonas/farmacologia , Quinolonas/uso terapêuticoRESUMO
Coenzyme Q (CoQ, ubiquinone) is a central electron carrier in mitochondrial respiration. CoQ is synthesized through multiple steps involving a number of different enzymes. The prevailing view that the CoQ used in respiration exists as a free pool that diffuses throughout the mitochondrial inner membrane bilayer has recently been challenged. In the yeast Saccharomyces cerevisiae, deletion of the gene encoding Coq10p results in respiration deficiency without inhibiting the synthesis of CoQ, suggesting that the Coq10 protein is critical for the delivery of CoQ to the site(s) of respiration. The precise mechanism by which this is achieved remains unknown at present. We have identified a Plasmodium orthologue of Coq10 (PfCoq10), which is predominantly expressed in trophozoite-stage parasites, and localizes to the parasite mitochondrion. Expression of PfCoq10 in the S. cerevisiae coq10 deletion strain restored the capability of the yeast to grow on respiratory substrates, suggesting a remarkable functional conservation of this protein over a vast evolutionary distance, and despite a relatively low level of amino acid sequence identity. As the antimalarial drug atovaquone acts as a competitive inhibitor of CoQ, we assessed whether over-expression of PfCoq10 altered the atovaquone sensitivity in parasites and in yeast mitochondria, but found no alteration of its activity.
Assuntos
Proteínas de Transporte/genética , Malária Falciparum/genética , Plasmodium falciparum/genética , Ubiquinona/análogos & derivados , Atovaquona/administração & dosagem , Proteínas de Transporte/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Plasmodium falciparum/patogenicidade , Respiração/efeitos dos fármacos , Respiração/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Ubiquinona/biossíntese , Ubiquinona/deficiência , Ubiquinona/genéticaRESUMO
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.