RESUMEN
The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including transmembrane proteins, across multiple membranes to the RBC. However, the process by which these exported membrane proteins are extracted from the parasite plasma membrane for export remains unknown. To address this question, we fused the exported membrane protein, skeleton binding protein 1 (SBP1), with TurboID, a rapid, efficient and promiscuous biotin ligase (SBP1TbID). Using time-resolved proximity biotinylation and label-free quantitative proteomics, we identified two groups of SBP1TbID interactors - early interactors (pre-export) and late interactors (post-export). Notably, two promising membrane-associated proteins were identified as pre-export interactors, one of which possesses a predicted translocon domain, that could facilitate the export of membrane proteins. Further investigation using conditional mutants of these candidate proteins showed that these proteins were essential for asexual growth and localize to the host-parasite interface during early stages of the intraerythrocytic cycle. These data suggest that they might play a role in ushering membrane proteins from the parasite plasma membrane for export to the host RBC.
Asunto(s)
Malaria , Plasmodium falciparum , Animales , Humanos , Biotinilación , Eritrocitos/metabolismo , Malaria/parasitología , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Porinas/metabolismo , Transporte de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Diarrhoeal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite Cryptosporidium to be a leading cause of paediatric diarrhoea, with particularly grave impact on infants and immunocompromised individuals. There is neither a vaccine nor an effective treatment. Here we establish a drug discovery process built on scalable phenotypic assays and mouse models that take advantage of transgenic parasites. Screening a library of compounds with anti-parasitic activity, we identify pyrazolopyridines as inhibitors of Cryptosporidium parvum and Cryptosporidium hominis. Oral treatment with the pyrazolopyridine KDU731 results in a potent reduction in intestinal infection of immunocompromised mice. Treatment also leads to rapid resolution of diarrhoea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection. Our results suggest that the Cryptosporidium lipid kinase PI(4)K (phosphatidylinositol-4-OH kinase) is a target for pyrazolopyridines and that KDU731 warrants further preclinical evaluation as a drug candidate for the treatment of cryptosporidiosis.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/antagonistas & inhibidores , Criptosporidiosis/tratamiento farmacológico , Criptosporidiosis/parasitología , Cryptosporidium/efectos de los fármacos , Cryptosporidium/enzimología , Pirazoles/farmacología , Piridinas/farmacología , Animales , Animales Recién Nacidos , Bovinos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Huésped Inmunocomprometido , Interferón gamma/deficiencia , Interferón gamma/genética , Masculino , Ratones , Ratones Noqueados , Pirazoles/química , Pirazoles/farmacocinética , Piridinas/química , Piridinas/farmacocinética , Ratas , Ratas WistarRESUMEN
The human malaria parasite, Plasmodium falciparum, contains an essential plastid called the apicoplast. Most apicoplast proteins are encoded by the nuclear genome and it is unclear how the plastid proteome is regulated. Here, we study an apicoplast-localized caseinolytic-protease (Clp) system and how it regulates organelle proteostasis. Using null and conditional mutants, we demonstrate that the P. falciparum Clp protease (PfClpP) has robust enzymatic activity that is essential for apicoplast biogenesis. We developed a CRISPR/Cas9-based system to express catalytically dead PfClpP, which showed that PfClpP oligomerizes as a zymogen and is matured via transautocatalysis. The expression of both wild-type and mutant Clp chaperone (PfClpC) variants revealed a functional chaperone-protease interaction. Conditional mutants of the substrate-adaptor (PfClpS) demonstrated its essential function in plastid biogenesis. A combination of multiple affinity purification screens identified the Clp complex composition as well as putative Clp substrates. This comprehensive study reveals the molecular composition and interactions influencing the proteolytic function of the apicoplast Clp system and demonstrates its central role in the biogenesis of the plastid in malaria parasites.
Asunto(s)
Apicoplastos/enzimología , Endopeptidasa Clp/metabolismo , Plasmodium falciparum/enzimología , Proteínas Protozoarias/metabolismo , Animales , Apicoplastos/genética , Endopeptidasa Clp/genética , Humanos , Malaria/parasitología , Biogénesis de Organelos , Plasmodium falciparum/genética , Proteolisis , Proteínas Protozoarias/genéticaRESUMEN
Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum/enzimología , Inhibidores Enzimáticos/farmacología , Lisina-ARNt Ligasa/antagonistas & inhibidores , Malaria Falciparum , Plasmodium falciparum/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Criptosporidiosis/tratamiento farmacológico , Criptosporidiosis/enzimología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/química , Humanos , Lisina-ARNt Ligasa/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Ratones SCID , Proteínas Protozoarias/metabolismoRESUMEN
Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus. Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality. Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools. Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivo selection for aminoglycoside resistance. We derived reporter parasites suitable for in vitro and in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.
Asunto(s)
Criptosporidiosis/parasitología , Cryptosporidium parvum/genética , Diarrea/parasitología , Ingeniería Genética/métodos , Aminoglicósidos/farmacología , Animales , Antimaláricos/farmacología , Sistemas CRISPR-Cas , Línea Celular , Criptosporidiosis/complicaciones , Cryptosporidium parvum/enzimología , Cryptosporidium parvum/crecimiento & desarrollo , Diarrea/complicaciones , Evaluación Preclínica de Medicamentos , Resistencia a Medicamentos , Femenino , Eliminación de Gen , Técnicas de Inactivación de Genes , Genes Reporteros , Humanos , Intestinos/parasitología , Ratones , Modelos Animales , Esporozoítos , Timidina Quinasa/deficiencia , Timidina Quinasa/genética , Transfección/métodos , Trimetoprim/farmacologíaRESUMEN
Bumped kinase inhibitors (BKIs) of Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) are leading candidates for treatment of cryptosporidiosis-associated diarrhea. Potential cardiotoxicity related to anti-human ether-à-go-go potassium channel (hERG) activity of the first-generation anti-Cryptosporidium BKIs triggered further testing for efficacy. A luminescence assay adapted for high-throughput screening was used to measure inhibitory activities of BKIs against C. parvum in vitro. Furthermore, neonatal and interferon γ knockout mouse models of C. parvum infection identified BKIs with in vivo activity. Additional iterative experiments for optimum dosing and selecting BKIs with minimum levels of hERG activity and frequencies of other safety liabilities included those that investigated mammalian cell cytotoxicity, C. parvum proliferation inhibition in vitro, anti-human Src inhibition, hERG activity, in vivo pharmacokinetic data, and efficacy in other mouse models. Findings of this study suggest that fecal concentrations greater than parasite inhibitory concentrations correlate best with effective therapy in the mouse model of cryptosporidiosis, but a more refined model for efficacy is needed.
Asunto(s)
Antiprotozoarios/administración & dosificación , Criptosporidiosis/tratamiento farmacológico , Cryptosporidium parvum/efectos de los fármacos , Inhibidores de Proteínas Quinasas/administración & dosificación , Administración Oral , Animales , Diarrea/tratamiento farmacológico , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Noqueados , Ratones SCIDRESUMEN
Apicomplexa are unicellular parasites causing important human and animal diseases, including malaria and toxoplasmosis. Most of these pathogens possess a relict but essential plastid, the apicoplast. The apicoplast was acquired by secondary endosymbiosis between a red alga and a flagellated eukaryotic protist. As a result the apicoplast is surrounded by four membranes. This complex structure necessitates a system of transport signals and translocons allowing nuclear encoded proteins to find their way to specific apicoplast sub-compartments. Previous studies identified translocons traversing two of the four apicoplast membranes. Here we provide functional support for the role of an apicomplexan Toc75 homolog in apicoplast protein transport. We identify two apicomplexan genes encoding Toc75 and Sam50, both members of the Omp85 protein family. We localize the respective proteins to the apicoplast and the mitochondrion of Toxoplasma and Plasmodium. We show that the Toxoplasma Toc75 is essential for parasite growth and that its depletion results in a rapid defect in the import of apicoplast stromal proteins while the import of proteins of the outer compartments is affected only as the secondary consequence of organelle loss. These observations along with the homology to Toc75 suggest a potential role in transport through the second innermost membrane.
Asunto(s)
Apicoplastos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Apicomplexa/genética , Apicomplexa/metabolismo , Apicoplastos/genética , Eritrocitos/parasitología , Fibroblastos/parasitología , Proteínas Fluorescentes Verdes , Humanos , Proteínas de la Membrana/genética , Microscopía Fluorescente , Mitocondrias/metabolismo , Mutagénesis Sitio-Dirigida , Fenilalanina/genética , Filogenia , Transporte de Proteínas , Proteínas Protozoarias/genética , Toxoplasma/genéticaRESUMEN
Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.
Asunto(s)
Proteínas de Cloroplastos/metabolismo , Degradación Asociada con el Retículo Endoplásmico/fisiología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Ubiquitinación/fisiología , Línea Celular , Proteínas de Cloroplastos/genética , Humanos , Plasmodium falciparum/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Transporte de Proteínas/fisiología , Proteínas Protozoarias/genética , Toxoplasma/genéticaRESUMEN
Malaria remains a significant infectious disease that causes millions of clinical cases and >800,000 deaths per year. The Malaria Box is a collection of 400 commercially available chemical entities that have antimalarial activity. The collection contains 200 drug-like compounds, based on their oral absorption and the presence of known toxicophores, and 200 probe-like compounds, which are intended to represent a broad structural diversity. These compounds have confirmed activities against the asexual intraerythrocytic stages of Plasmodium falciparum and low cytotoxicities, but their mechanisms of action and their activities in other stages of the parasite's life cycle remain to be determined. The apicoplast is considered to be a promising source of malaria-specific targets, and its main function during intraerythrocytic stages is to provide the isoprenoid precursor isopentenyl diphosphate, which can be used for phenotype-based screens to identify compounds targeting this organelle. We screened 400 compounds from the Malaria Box using apicoplast-targeting phenotypic assays to identify their potential mechanisms of action. We identified one compound that specifically targeted the apicoplast. Further analyses indicated that the molecular target of this compound may differ from those of the current antiapicoplast drugs, such as fosmidomycin. Moreover, in our efforts to elucidate the mechanisms of action of compounds from the Malaria Box, we evaluated their activities against other stages of the life cycle of the parasite. Gametocytes are the transmission stage of the malaria parasite and are recognized as a priority target in efforts to eradicate malaria. We identified 12 compounds that were active against gametocytes with 50% inhibitory concentration values of <1 µM.
Asunto(s)
Antimaláricos/farmacología , Apicoplastos/efectos de los fármacos , Carbolinas/farmacología , Hemiterpenos/antagonistas & inhibidores , Estadios del Ciclo de Vida/efectos de los fármacos , Compuestos Organofosforados/antagonistas & inhibidores , Ácidos Pipecólicos/farmacología , Plasmodium falciparum/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Antimaláricos/química , Apicoplastos/metabolismo , Carbolinas/química , Descubrimiento de Drogas , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Fosfomicina/análogos & derivados , Fosfomicina/farmacología , Hemiterpenos/biosíntesis , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Estadios del Ciclo de Vida/fisiología , Oligopéptidos/farmacología , Ácidos Pipecólicos/química , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Members of the eukaryotic phylum Apicomplexa are the cause of important human diseases including malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular parasites produce new invasive stages through a complex budding process. The budding cycle is remarkably flexible and can produce varied numbers of progeny to adapt to different host-cell niches. How this complex process is coordinated remains poorly understood. Using Toxoplasma gondii as a genetic model, we show that a key element to this coordination is the centrocone, a unique elaboration of the nuclear envelope that houses the mitotic spindle. Exploiting transgenic parasite lines expressing epitope-tagged centromeric H3 variant CenH3, we identify the centromeres of T. gondii chromosomes by hybridization of chromatin immunoprecipitations to genome-wide microarrays (ChIP-chip). We demonstrate that centromere attachment to the centrocone persists throughout the parasite cell cycle and that centromeres localize to a single apical region within the nucleus. Centromere sequestration provides a mechanism for the organization of the Toxoplasma nucleus and the maintenance of genome integrity.
Asunto(s)
Ciclo Celular , Centrómero/metabolismo , Toxoplasma/citología , Toxoplasma/metabolismo , Ciclo Celular/efectos de los fármacos , Centrómero/efectos de los fármacos , Inmunoprecipitación de Cromatina , Rotura Cromosómica/efectos de los fármacos , Etopósido/farmacología , Técnica del Anticuerpo Fluorescente , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilación/efectos de los fármacos , Mitosis/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reproducibilidad de los Resultados , Toxoplasma/efectos de los fármacosRESUMEN
Most plastids proteins are post-translationally imported into organelles through multisubunit translocons. The TIC and TOC complexes perform this role in the two membranes of the plant chloroplast and in the inner two membranes of the apicoplasts of the apicomplexan parasites, Toxoplasma gondii and Plasmodium falciparum. Tic22 is a ubiquitous intermembrane translocon component that interacts with translocating proteins. Here, we demonstrate that T. gondii Tic22 is an apicoplast-localized protein, essential for parasite survival and protein import into the apicoplast stroma. The structure of Tic22 from P. falciparum reveals a fold conserved from cyanobacteria to plants, which displays a non-polar groove on each side of the molecule. We show that these grooves allow Tic22 to act as a chaperone. General chaperones are common components of protein translocation systems where they maintain cargo proteins in an unfolded conformation during transit. Such a chaperone had not been identified in the intermembrane space of plastids and we propose that Tic22 fulfills this role.
Asunto(s)
Proteínas Portadoras/metabolismo , Chaperonas Moleculares/metabolismo , Plasmodium falciparum/metabolismo , Pliegue de Proteína , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Plasmodium falciparum/química , Plasmodium falciparum/genética , Transporte de Proteínas/fisiología , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Toxoplasma/química , Toxoplasma/genéticaRESUMEN
Apicomplexan parasites are responsible for high impact human diseases such as malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular pathogens are dependent on both de novo lipid biosynthesis as well as the uptake of host lipids for biogenesis of parasite membranes. Genome annotations and biochemical studies indicate that apicomplexan parasites can synthesize fatty acids via a number of different biosynthetic pathways that are differentially compartmentalized. However, the relative contribution of each of these biosynthetic pathways to total fatty acid composition of intracellular parasite stages remains poorly defined. Here, we use a combination of genetic, biochemical, and metabolomic approaches to delineate the contribution of fatty acid biosynthetic pathways in Toxoplasma gondii. Metabolic labeling studies with [(13)C]glucose showed that intracellular tachyzoites synthesized a range of long and very long chain fatty acids (C14:0-26:1). Genetic disruption of the apicoplast-localized type II fatty-acid synthase resulted in greatly reduced synthesis of saturated fatty acids up to 18 carbons long. Ablation of type II fatty-acid synthase activity resulted in reduced intracellular growth that was partially restored by addition of long chain fatty acids. In contrast, synthesis of very long chain fatty acids was primarily dependent on a fatty acid elongation system comprising three elongases, two reductases, and a dehydratase that were localized to the endoplasmic reticulum. The function of these enzymes was confirmed by heterologous expression in yeast. This elongase pathway appears to have a unique role in generating very long unsaturated fatty acids (C26:1) that cannot be salvaged from the host.
Asunto(s)
Retículo Endoplásmico/enzimología , Acido Graso Sintasa Tipo II/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Proteínas Protozoarias/metabolismo , Toxoplasma/enzimología , Retículo Endoplásmico/genética , Acido Graso Sintasa Tipo II/genética , Ácidos Grasos Insaturados/genética , Humanos , Marcaje Isotópico , Metabolómica/métodos , Proteínas Protozoarias/genética , Toxoplasma/genéticaRESUMEN
Autophagy is a cellular process that is highly conserved among eukaryotes and permits the degradation of cellular material. Autophagy is involved in multiple survival-promoting processes. It not only facilitates the maintenance of cell homeostasis by degrading long-lived proteins and damaged organelles, but it also plays a role in cell differentiation and cell development. Equally important is its function for survival in stress-related conditions such as recycling of proteins and organelles during nutrient starvation. Protozoan parasites have complex life cycles and face dramatically changing environmental conditions; whether autophagy represents a critical coping mechanism throughout these changes remains poorly documented. To investigate this in Toxoplasma gondii, we have used TgAtg8 as an autophagosome marker and showed that autophagy and the associated cellular machinery are present and functional in the parasite. In extracellular T. gondii tachyzoites, autophagosomes were induced in response to amino acid starvation, but they could also be observed in culture during the normal intracellular development of the parasites. Moreover, we generated a conditional T. gondii mutant lacking the orthologue of Atg3, a key autophagy protein. TgAtg3-depleted parasites were unable to regulate the conjugation of TgAtg8 to the autophagosomal membrane. The mutant parasites also exhibited a pronounced fragmentation of their mitochondrion and a drastic growth phenotype. Overall, our results show that TgAtg3-dependent autophagy might be regulating mitochondrial homeostasis during cell division and is essential for the normal development of T. gondii tachyzoites.
Asunto(s)
División Celular/fisiología , Mitocondrias/metabolismo , Mutación , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Células Cultivadas , Fibroblastos/parasitología , Humanos , Masculino , Mitocondrias/genética , Mitocondrias/ultraestructura , Proteínas Protozoarias/genética , Toxoplasma/genética , Toxoplasma/ultraestructura , Toxoplasmosis/genética , Toxoplasmosis/metabolismo , Toxoplasmosis/patología , Enzimas Ubiquitina-Conjugadoras/genéticaRESUMEN
Apicomplexa are obligate intracellular pathogens that have fine-tuned their proliferative strategies to match a large variety of host cells. A critical aspect of this adaptation is a flexible cell cycle that remains poorly understood at the mechanistic level. Here we describe a forward genetic dissection of the apicomplexan cell cycle using the Toxoplasma model. By high-throughput screening, we have isolated 165 temperature sensitive parasite growth mutants. Phenotypic analysis of these mutants suggests regulated progression through the parasite cell cycle with defined phases and checkpoints. These analyses also highlight the critical importance of the peculiar intranuclear spindle as the physical hub of cell cycle regulation. To link these phenotypes to parasite genes, we have developed a robust complementation system based on a genomic cosmid library. Using this approach, we have so far complemented 22 temperature sensitive mutants and identified 18 candidate loci, eight of which were independently confirmed using a set of sequenced and arrayed cosmids. For three of these loci we have identified the mutant allele. The genes identified include regulators of spindle formation, nuclear trafficking, and protein degradation. The genetic approach described here should be widely applicable to numerous essential aspects of parasite biology.
Asunto(s)
División Celular/genética , Genes Protozoarios , Prueba de Complementación Genética , Toxoplasma/citología , Toxoplasma/genética , Animales , Núcleo Celular/efectos de los fármacos , Células Cultivadas , Cósmidos/genética , Análisis Mutacional de ADN , ADN Complementario/genética , ADN Complementario/metabolismo , ADN Protozoario/genética , ADN Protozoario/metabolismo , Fibroblastos , Biblioteca Genómica , Humanos , Modelos Genéticos , Mutación , Fenotipo , Recombinación Genética , Toxoplasma/patogenicidad , Toxoplasmosis , TransfecciónRESUMEN
The endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be required for biogenesis and secretion of egress-related organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the Plasmodium falciparumendoplasmic reticulum-resident calcium-binding protein (PfERC), a member of the CREC family. Knockdown of the PfERC gene showed that this gene is essential for asexual growth of P. falciparum Analysis of the intraerythrocytic life cycle revealed that PfERC is essential for parasite egress but is not required for protein trafficking or calcium storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease SUB1. Using double mutant parasites, we showed that PfERC is required for the proteolytic maturation of the essential aspartic protease plasmepsin X, which is required for SUB1 cleavage. Further, we showed that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish that the ER-resident CREC family protein PfERC is a key early regulator of the egress proteolytic cascade of malaria parasites.IMPORTANCE The divergent eukaryotic parasites that cause malaria grow and divide within a vacuole inside a host cell, which they have to break open once they finish cell division. The egress of daughter parasites requires the activation of a proteolytic cascade, and a subtilisin-like protease initiates a proteolytic cascade to break down the membranes blocking egress. It is assumed that the parasite endoplasmic reticulum plays a role in this process, but the proteins in this organelle required for egress remain unknown. We have identified an early ER-resident regulator essential for the maturation of the recently discovered aspartic protease in the egress proteolytic cascade, plasmepsin X, which is required for maturation of the subtilisin-like protease. Conditional loss of PfERC results in the formation of immature and inactive egress proteases that are unable to breakdown the vacuolar membrane barring release of daughter parasites.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Plasmodium falciparum/metabolismo , Proteolisis , Proteínas Protozoarias/metabolismo , Proteínas de Unión al Calcio/genética , Técnicas de Inactivación de Genes , Humanos , Malaria/parasitología , Malaria Falciparum/parasitología , Péptido Hidrolasas/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Vacuolas/metabolismoRESUMEN
Cryptosporidium is a leading cause of diarrheal disease and an important contributor to early childhood mortality, malnutrition, and growth faltering. Older children in high endemicity regions appear resistant to infection, while previously unexposed adults remain susceptible. Experimental studies in humans and animals support the development of disease resistance, but we do not understand the mechanisms that underlie protective immunity to Cryptosporidium. Here, we derive an in vivo model of Cryptosporidium infection in immunocompetent C57BL/6 mice by isolating parasites from naturally infected wild mice. Similar to human cryptosporidiosis, this infection causes intestinal pathology, and interferon-γ controls early infection while T cells are critical for clearance. Importantly, mice that controlled a live infection were resistant to secondary challenge and vaccination with attenuated parasites provided protection equal to live infection. Both parasite and host are genetically tractable and this in vivo model will facilitate mechanistic investigation and rational vaccine design.
Asunto(s)
Inmunidad Adaptativa , Criptosporidiosis/inmunología , Diarrea/inmunología , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Inmunidad Innata , Animales , Criptosporidiosis/patología , Cryptosporidium/crecimiento & desarrollo , Cryptosporidium/inmunología , Diarrea/patología , Resistencia a la Enfermedad , Ratones , Ratones Endogámicos C57BLRESUMEN
The apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease and an important contributor to overall global child mortality. We currently lack effective treatment and immune prophylaxis. Recent advances now permit genetic modification of this important pathogen. We expect this to produce rapid advances in fundamental as well as translational research on cryptosporidiosis. Here we outline genetic engineering for Cryptosporidium in sufficient detail to establish transfection in any laboratory that requires access to this key technology. This chapter details the conceptual design consideration, as well as the experimental steps required to transfect, select, and isolate transgenic parasites. We also provide detail on key in vitro and in vivo assays to detect, validate, and quantify genetically modified Cryptosporidium parasites. © 2017 by John Wiley & Sons, Inc.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Criptosporidiosis/parasitología , Cryptosporidium parvum/crecimiento & desarrollo , Cryptosporidium parvum/genética , Técnicas Genéticas , Microscopía Fluorescente/métodos , Transfección/métodos , Animales , Cryptosporidium parvum/metabolismo , HumanosRESUMEN
UNLABELLED: Apicomplexa are obligate intracellular parasites that cause important diseases in humans and animals. Manipulating the pathogen genome is the most direct way to understand the functions of specific genes in parasite development and pathogenesis. In Toxoplasma gondii, nonhomologous recombination is typically highly favored over homologous recombination, a process required for precise gene targeting. Several approaches, including the use of targeting vectors that feature large flanks to drive site-specific recombination, have been developed to overcome this problem. We have generated a new large-insert repository of T. gondii genomic DNA that is arrayed and sequenced and covers 95% of all of the parasite's genes. Clones from this fosmid library are maintained at single copy, which provides a high level of stability and enhances our ability to modify the organism dramatically. We establish a robust recombineering pipeline and show that our fosmid clones can be easily converted into gene knockout constructs in a 4-day protocol that does not require plate-based cloning but can be performed in multiwell plates. We validated this approach to understand gene function in T. gondii and produced a conditional null mutant for a nucleolar protein belonging to the NOL1/NOP2/SUN family, and we show that this gene is essential for parasite growth. We also demonstrate a powerful complementation strategy in the context of chemical mutagenesis and whole-genome sequencing. This repository is an important new resource that will accelerate both forward and reverse genetic analysis of this important pathogen. IMPORTANCE: Toxoplasma gondii is an important genetic model to understand intracellular parasitism. We show here that large-insert genomic clones are effective tools that enhance homologous recombination and allow us to engineer conditional mutants to understand gene function. We have generated, arrayed, and sequenced a fosmid library of T. gondii genomic DNA in a copy control vector that provides excellent coverage of the genome. The fosmids are maintained in a single-copy state that dramatically improves their stability and allows modification by means of a simple and highly scalable protocol. We show here that modified and unmodified fosmid clones are powerful tools for forward and reverse genetics.
Asunto(s)
Marcación de Gen/métodos , Genética Microbiana/métodos , Genoma de Protozoos , Biología Molecular/métodos , Toxoplasma/genética , Biblioteca de Genes , Vectores Genéticos , Recombinación GenéticaRESUMEN
Apicomplexa are important pathogens that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. Apicomplexan parasites contain a relict chloroplast, the apicoplast. The apicoplast is indispensable and an attractive drug target. The apicoplast is home to a 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway for the synthesis of isoprenoid precursors. This pathway is believed to be the most conserved function of the apicoplast, and fosmidomycin, a specific inhibitor of the pathway, is an effective antimalarial. Surprisingly, fosmidomycin has no effect on most other apicomplexans. Using Toxoplasma gondii, we establish that the pathway is essential in parasites that are highly fosmidomycin resistant. We define the molecular basis of resistance and susceptibility, experimentally testing various host and parasite contributions in T. gondii and Plasmodium. We demonstrate that in T. gondii the parasite plasma membrane is a critical barrier to drug uptake. In strong support of this hypothesis, we engineer de novo drug-sensitive T. gondii parasites by heterologous expression of a bacterial transporter protein. Mice infected with these transgenic parasites can now be cured from a lethal challenge with fosmidomycin. We propose that the varied extent of metabolite exchange between host and parasite is a crucial determinator of drug susceptibility and a predictor of future resistance.
Asunto(s)
Fosfomicina/análogos & derivados , Terpenos/metabolismo , Toxoplasma/efectos de los fármacos , Toxoplasma/metabolismo , Isomerasas Aldosa-Cetosa/genética , Isomerasas Aldosa-Cetosa/metabolismo , Animales , Animales Modificados Genéticamente , Antiprotozoarios/farmacología , Permeabilidad de la Membrana Celular , Resistencia a Medicamentos/fisiología , Fosfomicina/farmacología , Interacciones Huésped-Parásitos/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Redes y Vías Metabólicas , Ratones , Modelos Biológicos , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Orgánulos/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Pentosafosfatos/metabolismo , Plasmodium berghei/efectos de los fármacos , Plasmodium berghei/metabolismo , Plasmodium berghei/patogenicidad , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Toxoplasma/genética , Toxoplasma/patogenicidadRESUMEN
Apicomplexa are unicellular eukaryotic pathogens that carry a vestigial algal endosymbiont, the apicoplast. The physiological function of the apicoplast and its integration into parasite metabolism remain poorly understood and at times controversial. We establish that the Toxoplasma apicoplast membrane-localized phosphate translocator (TgAPT) is an essential metabolic link between the endosymbiont and the parasite cytoplasm. TgAPT is required for fatty acid synthesis in the apicoplast, but this may not be its most critical function. Further analyses demonstrate that TgAPT also functions to supply the apicoplast with carbon skeletons for additional pathways and, indirectly, with energy and reduction power. Genetic ablation of the transporter results in rapid death of parasites. The dramatic consequences of loss of its activity suggest that targeting TgAPT could be a viable strategy to identify antiparasitic compounds.