RESUMEN
Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ± 1.2, a coefficient of variation of 6.8% ± 2.9%, and a Z'-factor of 0.75 ± 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as â¼1 µM, and several were found to have low-micromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development.
Asunto(s)
Antimaláricos/farmacología , Inhibidores Enzimáticos/farmacología , Hexoquinasa/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/biosíntesis , Antimaláricos/química , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/química , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Expresión Génica , Genes Reporteros , Glucólisis/efectos de los fármacos , Células HEK293 , Células HeLa , Hexoquinasa/genética , Hexoquinasa/metabolismo , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Relación Señal-Ruido , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world's population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite-host cell interactions, forming the basis of the parasite's cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.
Asunto(s)
Interacciones Huésped-Parásitos , Parásitos/fisiología , Infecciones por Protozoos/parasitología , Animales , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/parasitología , Humanos , Leishmania/inmunología , Leishmania/patogenicidad , Leishmania/fisiología , Leishmaniasis/inmunología , Leishmaniasis/parasitología , Malaria/inmunología , Malaria/parasitología , Parásitos/patogenicidad , Fagocitos/inmunología , Fagocitos/parasitología , Plasmodium/inmunología , Plasmodium/patogenicidad , Plasmodium/fisiología , Infecciones por Protozoos/inmunología , Toxoplasma/inmunología , Toxoplasma/patogenicidad , Toxoplasma/fisiología , Trypanosoma cruzi/inmunología , Trypanosoma cruzi/patogenicidad , Trypanosoma cruzi/fisiologíaRESUMEN
Parasites in the genus Plasmodium cause disease throughout the tropic and subtropical regions of the world. P. falciparum, one of the deadliest species of the parasite, relies on glycolysis for the generation of ATP while it inhabits the mammalian red blood cell. The first step in glycolysis is catalyzed by hexokinase (HK). While the 55.3-kDa P. falciparum HK (PfHK) shares several biochemical characteristics with mammalian HKs, including being inhibited by its products, it has limited amino acid identity (~26%) to the human HKs, suggesting that enzyme-specific therapeutics could be generated. To that end, interrogation of a selected small-molecule library of HK inhibitors has identified a class of PfHK inhibitors, isobenzothiazolinones, some of which have 50% inhibitory concentrations (IC50s) of <1 µM. Inhibition was reversible by dilution but not by treatment with a reducing agent, suggesting that the basis for enzyme inactivation was not covalent association with the inhibitor. Lastly, six of these compounds and the related molecule ebselen inhibited P. falciparum growth in vitro (50% effective concentration [EC50] of ≥ 0.6 and <6.8 µM). These findings suggest that the chemotypes identified here could represent leads for future development of therapeutics against P. falciparum.
Asunto(s)
Antimaláricos/farmacología , Benzotiazoles/farmacología , Hexoquinasa/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Secuencia de Aminoácidos , Azoles/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Glucólisis , Concentración 50 Inhibidora , Isoindoles , Datos de Secuencia Molecular , Compuestos de Organoselenio/farmacología , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Semisynthetic 8,8-dialkyldihydroberberines (8,8-DDBs) were found to possess mid- to low-nanomolar potency against Plasmodium falciparum blood-stage parasites, Leishmania donovani intracellular amastigotes, and Trypanosoma brucei brucei bloodstream forms. For example, 8,8-diethyldihydroberberine chloride (5b) exhibited in vitro IC50 values of 77, 100, and 5.3 nM against these three parasites, respectively. In turn, two 8,8-dialkylcanadines, obtained by reduction of the corresponding 8,8-DDBs, were much less potent against these parasites in vitro. While the natural product berberine is a weak DNA binder, the 8,8-DDBs displayed no affinity for DNA, as assessed by changes in the melting temperature of poly(dA·dT) DNA. Selected 8,8-DDBs showed efficacy in mouse models of visceral leishmaniasis and African trypanosomiasis, with 8,8-dimethyldihydroberberine chloride (5a) reducing liver parasitemia by 46% in L. donovani-infected BALB/c mice when given at an intraperitoneal dose of 10 mg/kg/day for five days. The 8,8-DDBs may thus serve as leads for discovering new antimalarial, antileishmanial, and antitrypanosomal drug candidates.
Asunto(s)
Antimaláricos/farmacología , Antiprotozoarios/farmacología , Alcaloides de Berberina/farmacología , Animales , Antimaláricos/química , Antiprotozoarios/química , Alcaloides de Berberina/síntesis química , Alcaloides de Berberina/química , Cristalografía por Rayos X , Femenino , Concentración 50 Inhibidora , Leishmania donovani/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/efectos de los fármacos , Trypanosoma/efectos de los fármacosRESUMEN
Dihydroquinoline derivative OSU-40 (1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate) is selectively potent against Trypanosma brucei rhodesiense in vitro (50% inhibitory concentration [IC(50)], 14 nM; selectivity index, 1,700) and has been proposed to cause the formation of reactive oxygen species (ROS) in African trypanosomes (J. Fotie et al., J. Med. Chem. 53:966-982, 2010). In the present study, we sought to provide further support for the hypothesis that OSU-40 kills trypanosomes through oxidative stress. Inducible RNA interference (RNAi) was applied to downregulate key enzymes in parasite antioxidant defense, including T. brucei trypanothione synthetase (TbTryS) and superoxide dismutase B (TbSODB). Both TbTryS RNAi-induced and TbSODB RNAi-induced cells showed impaired growth and increased sensitivity toward OSU-40 by 2.4-fold and 3.4-fold, respectively. Decreased expression of key parasite antioxidant enzymes was thus associated with increased sensitivity to OSU-40, consistent with the hypothesis that OSU-40 acts through oxidative stress. Finally, the dose-dependent formation of free radicals was observed after incubation of T. brucei with OSU-40 utilizing electron spin resonance (ESR) spectroscopy. These data support the notion that the mode of antitrypanosomal action for this class of compounds is to induce oxidative stress.
Asunto(s)
Acetatos/farmacología , Amida Sintasas/antagonistas & inhibidores , Proteínas Protozoarias/antagonistas & inhibidores , Compuestos de Quinolinio/farmacología , Superóxido Dismutasa/antagonistas & inhibidores , Tripanocidas/farmacología , Trypanosoma brucei rhodesiense/efectos de los fármacos , Amida Sintasas/metabolismo , Células Cultivadas , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Concentración 50 Inhibidora , Estrés Oxidativo/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Trypanosoma brucei rhodesiense/enzimología , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitologíaRESUMEN
Treatment of diseases such as African sleeping sickness and leishmaniasis often depends on relatively expensive or toxic drugs, and resistance to current chemotherapeutics is an issue in treating these diseases and malaria. In this study, a new semi-synthetic berberine analogue, 5,6-didehydro-8,8-diethyl-13-oxodihydroberberine chloride (1), showed nanomolar level potency against in vitro models of leishmaniasis, malaria, and trypanosomiasis as well as activity in an in vivo visceral leishmaniasis model. Since the synthetic starting material, berberine hemisulfate, is inexpensive, 8,8-dialkyl-substituted analogues of berberine may lead to a new class of affordable antiprotozoal compounds.
Asunto(s)
Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Berberina/farmacología , Parásitos/efectos de los fármacos , Infecciones por Protozoos/tratamiento farmacológico , Animales , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Berberina/síntesis química , Berberina/química , Berberina/uso terapéutico , Chlorocebus aethiops , Modelos Animales de Enfermedad , Concentración 50 Inhibidora , Leishmania/efectos de los fármacos , Leishmaniasis/tratamiento farmacológico , Malaria/tratamiento farmacológico , Ratones , Modelos Moleculares , Plasmodium falciparum/efectos de los fármacos , Trypanosoma brucei brucei/efectos de los fármacos , Tripanosomiasis/tratamiento farmacológico , Células VeroRESUMEN
Malaria is a devastating disease caused by a protozoan parasite. It affects over 300 million individuals and results in over 400â 000 deaths annually, most of whom are young children under the age of five. Hexokinase, the first enzyme in glucose metabolism, plays an important role in the infection process and represents a promising target for therapeutic intervention. Here, cryo-EM structures of two conformational states of Plasmodium vivax hexokinase (PvHK) are reported at resolutions of â¼3â Å. It is shown that unlike other known hexokinase structures, PvHK displays a unique tetrameric organization (â¼220â kDa) that can exist in either open or closed quaternary conformational states. Despite the resemblance of the active site of PvHK to its mammalian counterparts, this tetrameric organization is distinct from that of human hexokinases, providing a foundation for the structure-guided design of parasite-selective antimalarial drugs.
RESUMEN
Autophagy is a highly regulated, biological process that provides energy during periods of stress and starvation. This conserved process also acts as a defense mechanism and clears microbes from the host cell. Autophagy is impaired in Cystic Fibrosis (CF) patients and CF mice, as their cells exhibit low expression levels of essential autophagy molecules. The genetic disorder in CF is due to mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene that encodes for a chloride channel. CF patients are particularly prone to infection by pathogens that are otherwise cleared by autophagy in healthy immune cells including Burkholderia cenocepacia (B. cenocepacia). The objective of this study is to determine the mechanism underlying weak autophagic activity in CF macrophages and find therapeutic targets to correct it. Using reduced representation bisulfite sequencing (RRBS) to determine DNA methylation profile, we found that the promoter regions of Atg12 in CF macrophages are significantly more methylated than in the wild-type (WT) immune cells, accompanied by low protein expression. The natural product epigallocatechin-3-gallate (EGCG) significantly reduced the methylation of Atg12 promoter improving its expression. Accordingly, EGCG restricted B. cenocepacia replication within CF mice and their derived macrophages by improving autophagy and preventing dissemination. In addition, EGCG improved the function of CFTR protein. Altogether, utilizing RRBS for the first time in the CF field revealed a previously unrecognized mechanism for reduced autophagic activity in CF. Our data also offers a mechanism by which EGCG exerts its positive effects in CF.
Asunto(s)
Autofagia , Fibrosis Quística/fisiopatología , Macrófagos/fisiología , Animales , Catequina/análogos & derivados , Catequina/fisiología , Células Cultivadas , Ratones , Ratones Endogámicos C57BLRESUMEN
Analysis of the Plasmodium falciparum genome reveals a limited number of putative autophagy genes, specifically the four genes involved in ATG8 lipidation, an essential step in formation of autophagosomes. In yeast, Atg8 lipidation requires the E1-type ligase Atg7, an E2-type ligase Atg3, and a cysteine protease Atg4. These four putative P. falciparum ATG (PfATG) genes are transcribed during the parasite's erythrocytic stages. PfAtg7 has relatively low identity and similarity to yeast Atg7 (14.7% and 32.2%, respectively), due primarily to long insertions typical of P. falciparum. Excluding the insertions the identity and similarity are higher (38.0% and 70.8%, respectively). This and the fact that key residues are conserved, including the catalytic cysteine and ATP binding domain, we hypothesize that PfAtg7 is the activating enzyme of PfAtg8. To assess the role of PfAtg7 we have generated two transgenic parasite lines. In one, the PfATG7 locus was modified to introduce a C-terminal hemagglutinin tag. Western blotting reveals two distinct protein species, one migrating near the predicted 150 kDa and one at approximately 65 kDa. The second transgenic line introduces an inducible degradation domain into the PfATG7 locus, allowing us to rapidly attenuate PfAtg7 protein levels. Corresponding species are also observed in this parasite line at approximately 200 kDa and 100 kDa. Upon PfATG7 attenuation parasites exhibit a slow growth phenotype indicating the essentiality of this putative enzyme for normal growth.
Asunto(s)
Autofagia , Eritrocitos/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Biología Computacional , Secuencia Conservada , Regulación de la Expresión Génica , Metabolismo de los Lípidos , Datos de Secuencia Molecular , Plasmodium falciparum/citología , Plasmodium falciparum/fisiología , Estabilidad Proteica , Proteínas Protozoarias/químicaRESUMEN
Multidrug resistant gram-negative bacteria are an increasing therapeutic challenge. The beta-lactamases are a group of enzymes that confer resistance to the beta-lactam antibiotics. The carbapenems have been in wide use to treat beta-lactamase producing, multidrug resistant gram-negative bacterial infections. However, the emergence of carbapenemases, enzymes capable of hydrolyzing the carbapenems, has limited our therapeutic options. In the recent years, there has been some development in the discovery of new agents such as boronic acid derivatives, ME1071 and Ca-EDTA that may enhance the activity of existing antibiotics, CTC-96 which reverses antibiotic resistance and polymyxin derivatives with decreased renal toxicity. While global efforts towards new drug development should continue, appropriate use of currently available antibiotics is equally important. In this review, we will discuss the general characteristics of carbapenemases, recent patents with drugs under development and current treatment options.
Asunto(s)
Antibacterianos/farmacocinética , Carbapenémicos/farmacocinética , Bacterias Gramnegativas/enzimología , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Carbapenémicos/farmacología , Carbapenémicos/uso terapéutico , Descubrimiento de Drogas/métodos , Farmacorresistencia Bacteriana Múltiple , Bacterias Gramnegativas/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Hidrólisis , beta-Lactamasas/metabolismoRESUMEN
Intraerythrocytic malaria parasites use host hemoglobin as a major nutrient source. Aspartic proteases (plasmepsins) and cysteine proteases (falcipains) function in the early steps of the hemoglobin degradation pathway. There is extensive functional redundancy within and between these protease families. Plasmepsins are synthesized as integral membrane proenzymes that are activated by cleavage from the membrane. This cleavage is mediated by a maturase activity whose identity has been elusive. We have used a combination of cell biology, chemical biology, and enzymology approaches to analyze this processing event. These studies reveal that plasmepsin processing occurs primarily via the falcipains; however, if falcipain activity is blocked, autoprocessing can take place, serving as an alternate activation system. These results establish a further level of redundancy between the protease families involved in Plasmodium hemoglobin degradation.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Cisteína Endopeptidasas/metabolismo , Plasmodium falciparum/citología , Plasmodium falciparum/enzimología , Vacuolas/enzimología , Secuencia de Aminoácidos , Animales , Activación Enzimática , Cinética , Leucina/análogos & derivados , Leucina/farmacología , Espectrometría de Masas , Pepstatinas/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Plasmodium falciparum/efectos de los fármacosRESUMEN
The mitochondrial genome of trypanosomes, termed kinetoplast DNA (kDNA), contains thousands of minicircles and dozens of maxicircles topologically interlocked in a network. To identify proteins involved in network replication, we screened an inducible RNA interference-based genomic library for cells that lose kinetoplast DNA. In one cloned cell line with inducible kinetoplast DNA loss, we found that the RNA interference vector had aberrantly integrated into the genome resulting in overexpression of genes down-stream of the integration site (Motyka, S. A., Zhao, Z., Gull, K., and Englund, P. T. (2004) Mol. Biochem. Parasitol. 134, 163-167). We now report that the relevant overexpressed gene encodes a mitochondrial cytochrome b(5) reductase-like protein. This overexpression caused kDNA loss by oxidation/inactivation of the universal minicircle sequence-binding protein, which normally binds the minicircle replication origin and triggers replication. The rapid loss of maxicircles suggests that the universal minicircle sequence-binding protein might also control maxicircle replication. Several lines of evidence indicate that the cytochrome b(5) reductase-like protein controls the oxidization status of the universal minicircle sequence-binding protein via tryparedoxin, a mitochondrial redox protein. For example, overexpression of mitochondrial tryparedoxin peroxidase, which utilizes tryparedoxin, also caused oxidation of the universal minicircle sequence-binding protein and kDNA loss. Furthermore, the growth defect caused by overexpression of cytochrome b(5) reductase-like protein could be partially rescued by simultaneously overexpressing tryparedoxin.
Asunto(s)
ADN de Cinetoplasto/metabolismo , Proteínas Protozoarias/biosíntesis , Trypanosoma brucei brucei/genética , Animales , Citocromo-B(5) Reductasa/biosíntesis , Citocromo-B(5) Reductasa/genética , Replicación del ADN , Regulación de la Expresión Génica , Oxidación-Reducción , Proteínas Protozoarias/genética , Tiorredoxinas/biosíntesis , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
Plasmepsins (PMs) are thought to have an important function in hemoglobin degradation in the malarial parasite Plasmodium falciparum and have generated interest as antimalarial drug targets. Four paralogous plasmepsins reside in the food vacuole of P. falciparum. Targeted gene disruption by double crossover homologous recombination has been employed to study food vacuole plasmepsin function in cultured parasites. Parasite clones with deletions in each of the individual PM I, PM II, and HAP genes as well as clones with a double PM IV/PM I disruption have been generated. All of these clones lack the corresponding PMs, are viable, and appear morphologically normal. PM II and PM IV/I disruptions have longer doubling times than the 3D7 parental line in rich RPMI medium. This appears to be because of a decreased level of productive progeny rather than an increased cell cycle time. In amino acid-limited medium, all four knockouts exhibit slower growth than the parental strain. Compared with 3D7, knock-out clone sensitivity to aspartic and cysteine protease inhibitors is changed minimally. These results suggest substantial functional redundancy and have important implications for the design of antimalarial drugs. The slow growth phenotype may explain why P. falciparum has maintained four plasmepsin genes with overlapping functions.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Alimentos , Leucina/análogos & derivados , Vacuolas/metabolismo , Aminoácidos/deficiencia , Animales , Ácido Aspártico Endopeptidasas/deficiencia , Ácido Aspártico Endopeptidasas/genética , Ciclo Celular , Medios de Cultivo/química , Medios de Cultivo/farmacología , Genes Protozoarios/genética , Leucina/farmacología , Parásitos/citología , Parásitos/efectos de los fármacos , Parásitos/genética , Parásitos/crecimiento & desarrollo , Pepstatinas/farmacología , Plasmodium falciparum/citología , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismoRESUMEN
RNA interference (RNAi) is a powerful tool for identifying gene function in Trypanosoma brucei. We generated an RNAi library, the first of its kind in any organism, by ligation of genomic fragments into the vector pZJMbeta. After transfection at approximately 5-fold genome coverage, trypanosomes were induced to express double-stranded RNA and screened for reduced con canavalin A (conA) binding. Since this lectin binds the surface glycoprotein EP-procyclin, we predicted that cells would lose affinity to conA if RNAi silenced genes affecting EP-procyclin expression or modification. We found a cell line in which RNAi switches expression from glycosylated EP-procyclins to the unglycosylated GPEET-procyclin. This switch results from silencing a hexokinase gene. The relationship between procyclin expression and glycolysis was supported by silencing other genes in the glycolytic pathway, and confirmed by observation of a similar upregulation of GPEET- procyclin when parental cells were grown in medium depleted of glucose. These data suggest that T.brucei 'senses' changes in glucose level and modulates procyclin expression accordingly.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Silenciador del Gen , Glucólisis/fisiología , Hexoquinasa/genética , Glicoproteínas de Membrana/genética , Procesamiento Proteico-Postraduccional/genética , Proteínas Protozoarias/genética , ARN Protozoario/genética , ARN no Traducido/genética , Trypanosoma brucei brucei/genética , Animales , Línea Celular , Sistema Libre de Células , Concanavalina A/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Biblioteca de Genes , Genes Protozoarios , Glucosa/farmacología , Glucólisis/genética , Glicosilación , Glicosilfosfatidilinositoles/metabolismo , Hexoquinasa/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Piruvato Quinasa/biosíntesis , Piruvato Quinasa/genética , ARN Bicatenario/biosíntesis , ARN Interferente Pequeño , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transfección , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/metabolismoRESUMEN
Trypanosome mitochondrial DNA is a network containing thousands of interlocked minicircles. Silencing of a mitochondrial topoisomerase II by RNA interference (RNAi) causes progressive network shrinking, allowing assessment of the minimal network size compatible with viability. We cloned surviving cells after short-term RNAi and found, as expected, that the number of surviving clones decreased with the duration of RNAi. Unexpectedly, a clonal cell line contained heterogeneously sized networks, some being very small. Several experiments showed that cells survived network shrinkage by asymmetrical division of replicated networks, sacrificing daughters with the small progeny network. Therefore, the average network size gradually increased. During the network shrinkage and early stages of recovery, there were changes in the minicircle repertoire.
Asunto(s)
ADN de Cinetoplasto/genética , Trypanosomatina/genética , Animales , ADN-Topoisomerasas de Tipo II/metabolismo , ADN de Cinetoplasto/metabolismo , ADN de Cinetoplasto/ultraestructura , Citometría de Flujo , Colorantes Fluorescentes/metabolismo , Silenciador del Gen , Mitocondrias/genética , Mitocondrias/metabolismo , Conformación de Ácido Nucleico , ARN Bicatenario/metabolismo , Factores de Tiempo , Inhibidores de Topoisomerasa II , Trypanosomatina/citología , Trypanosomatina/metabolismoRESUMEN
We used an RNA interference (RNAi) library in a forward genetic selection to study the mechanism of toxicity of tubercidin (7-deazaadenosine) to procyclic Trypanosoma brucei. Following transfection of cells with an RNAi-based genomic library, we used 5 microm tubercidin to select a drug-resistant cell line. Surprisingly, we found in these resistant cells that the hexose transporters had been silenced. We subsequently found that silencing of hexokinase, a glycolytic enzyme, also yielded tubercidin-resistant parasites. These observations suggested that glycolysis could be a target of tubercidin action and that RNAi silencing of glycolytic enzymes was gradual enough to allow the parasites to adapt to alternative sources of energy. Indeed, adaptation of procyclic trypanosomes to a glucose-independent metabolism by reduction of glucose in the culture medium caused tubercidin resistance. High pressure liquid chromatography analysis of glycolytic intermediates from parasites treated with tubercidin showed a dose-dependent increase in concentration of 1,3-bisphosphoglycerate, a substrate of phosphoglycerate kinase. Furthermore, tubercidin triphosphate inhibited recombinant T. brucei phosphoglycerate kinase activity in vitro with an IC50 of 7.5 microm. We conclude that 5 microm tubercidin kills trypanosomes by targeting glycolysis, especially by inhibition of phosphoglycerate kinase.
Asunto(s)
Antiprotozoarios/farmacología , Glucólisis/efectos de los fármacos , Interferencia de ARN , Trypanosoma brucei brucei/efectos de los fármacos , Tubercidina/farmacología , Adaptación Fisiológica/genética , Animales , Ácidos Difosfoglicéricos/análisis , Resistencia a Medicamentos , Hexoquinasa/antagonistas & inhibidores , Hexoquinasa/genética , Fosfoglicerato Quinasa/antagonistas & inhibidores , Fosfoglicerato Quinasa/genética , ARN Interferente Pequeño , Trypanosoma brucei brucei/metabolismoRESUMEN
Understanding mitochondrial transcription is a requisite first step toward understanding the regulation of mitochondrial gene expression in kinetoplastids. Here we report the identification and functional characterization of a mitochondrial RNA polymerase (mtRNAP) from Trypanosoma brucei, the first trans-acting factor involved in kinetoplast mitochondrial transcription to be identified. Using sequences conserved among the catalytic domains of the single-subunit mtRNAPs, we were able to obtain a full-length sequence for a candidate mtRNAP from T. brucei. Sequence comparison indicates that it shares homology in its catalytic domain with other single-subunit mtRNAPs, including functionally conserved residues that are identical in all single-subunit RNAPs. We used RNA interference to functionally knock out the gene product to determine whether the candidate gene represents an mtRNAP. As predicted for a mitochondrial specific RNA polymerase, reduction of the gene product resulted in a specific decrease of mitochondrial versus nuclear transcripts. Additionally, similar to the mtRNAP of other organisms, the mtRNAP characterized here is involved in replication of the mitochondrial genome. Thus, based on sequence comparison and functional studies, we have cloned an mtRNAP from trypanosomes.