RESUMEN
We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of "pseudoschizonts," which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.
Asunto(s)
Eritrocitos/parasitología , Ácido Mirístico/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Eritrocitos/efectos de los fármacos , Lipoilación/efectos de los fármacos , Merozoítos/efectos de los fármacos , Merozoítos/metabolismo , Parásitos/efectos de los fármacos , Parásitos/crecimiento & desarrollo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Plasmodium falciparum/ultraestructura , Solubilidad , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Cystoisospora suis is a common diarrheal pathogen of piglets and typically controlled by metaphylactic toltrazuril application. Recently, toltrazuril resistance has been reported in the field; however, both evaluation of toltrazuril efficacy against field isolates and the anticoccidial drug development for pigs is hampered by costs and labor of animal experimentation. Therefore an in vitro merozoite development assay was developed to evaluate the efficacy of compounds against C. suis in vitro. Monolayers of IPEC-1 cells were infected with sporozoites derived from oocysts of defined C. suis laboratory strains and the optimal infection dose as well as concentration, time point and duration of treatment were evaluated by quantitative real-time PCR. Cell cultures were treated with bumped kinase inhibitor (BKI) 1369 at different time points to evaluate the possibility to delineate effects on different developmental stages in vitro during invasion and early infection, and to determine different inhibitory concentrations (IC50, IC95). BKI 1369 had an IC50 of 35 nM and an IC95 of 350 nM. Dose- and duration-dependent efficacy was seen when developing stages were treated with BKI 1369 after infection (days 0-1, 2-3 and 2-5) but not when sporozoites were pre-incubated with BKI 1369 before infection. Efficacies of further BKIs were also evaluated at 200 nM. BKI 1318, 1708, 1748 and 1862 had an efficacy comparable to that of BKI 1369 (which is also effective in vivo). BKI 1862 showed a more pronounced loss of efficacy in lower concentrations than BKI 1369, signifying pharmacokinetic differences of similar compounds detectable in vitro. In addition, the effects of toltrazuril and its metabolites, toltrazuril sulfoxide and toltrazuril sulfone, on a toltrazuril sensitive and a resistant strain of C. suis were evaluated. Inhibition of merozoite growth in vitro by toltrazuril and its metabolites was dose-dependent only for toltrazuril. Clear differences were noted for the effect on a toltrazuril-sensitive vs. a resistant strain, indicating that this in vitro assay has the capacity to delineate susceptible from resistant strains in vitro. It could also be used to evaluate and compare the efficacy of novel compounds against C. suis and support the determination of the optimal time point of treatment in vivo.
Asunto(s)
Coccidiosis/veterinaria , Coccidiostáticos/farmacología , Sarcocystidae/efectos de los fármacos , Enfermedades de los Porcinos/parasitología , Triazinas/farmacología , Animales , Línea Celular , Coccidiosis/tratamiento farmacológico , Coccidiosis/parasitología , Coccidiostáticos/metabolismo , Coccidiostáticos/uso terapéutico , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/veterinaria , Resistencia a Medicamentos , Concentración 50 Inhibidora , Merozoítos/efectos de los fármacos , Merozoítos/crecimiento & desarrollo , Proyectos Piloto , Piperidinas/farmacología , Pirimidinas/farmacología , Quinolinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Sarcocystidae/crecimiento & desarrollo , Sulfonas/química , Sulfóxidos/química , Porcinos , Enfermedades de los Porcinos/tratamiento farmacológico , Triazinas/metabolismo , Triazinas/uso terapéuticoRESUMEN
Chicken coccidiosis, caused by an obligate intracellular protozoan parasite of the genus Eimeria, is a major parasitic disease in the intensively reared poultry industry. Due to the widespread use of anticoccidial drugs, resistance has become an inevitable problem. In our previous study, Eimeria tenella citrate synthase (EtCS) was found to be up-expressed in two drug-resistant strains (diclazuril-resistant and maduramycin-resistant strains) compared to drug-sensitive strain by RNA sequence. In this study, we cloned and expressed EtCS and obtain its polyclonal antibodies. Quantitative real-time polymerase chain (qPCR) reactions and Western blots were used to analyze the transcription and translation levels of EtCS in sensitive and three drug-resistant strains. Compared with the sensitive strain, the transcription of EtCS was both significantly upregulated in diclazuril-resistant and maduramycin-resistant strains, but was not significantly different in salinomycin-resistant strain. No significant difference was seen in translation level in the three drug-resistant strains. Indirect immunofluorescence indicated that EtCS was mainly located in the cytoplasm of sporozoites except for posterior refractile bodies and in the cytoplasm and surface of merozoites. Anti-rEtCS antibody has inhibitory effects on E. tenella sporozoite invasion of DF-1 cells and the inhibition rate is more than 83%. Binding of the protein to chicken macrophage (HD11) cells was confirmed by immunofluorescence assays. When macrophages were treated with rEtCS, secretion of nitric oxide and cell proliferation of the macrophages were substantially reduced. These results showed that EtCS may be related to host cell invasion of E. tenella and involve in the development of E.tenella resistance to some drugs.
Asunto(s)
Pollos/parasitología , Citrato (si)-Sintasa/genética , Citrato (si)-Sintasa/metabolismo , Coccidiosis/veterinaria , Eimeria tenella/enzimología , Enfermedades de las Aves de Corral/parasitología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Secuencia de Bases , Western Blotting , Citrato (si)-Sintasa/inmunología , Citrato (si)-Sintasa/aislamiento & purificación , Clonación Molecular , Coccidiosis/parasitología , Eimeria tenella/genética , Eimeria tenella/fisiología , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Sueros Inmunes/inmunología , Macrófagos/citología , Macrófagos/metabolismo , Merozoítos/efectos de los fármacos , Ratones , Óxido Nítrico/biosíntesis , Nitrilos/farmacología , Piranos/farmacología , Conejos , Reacción en Cadena en Tiempo Real de la Polimerasa , Organismos Libres de Patógenos Específicos , Esporozoítos/enzimología , Esporozoítos/inmunología , Triazinas/farmacologíaRESUMEN
We report a systematic, cellular phenotype-based antimalarial screening of the Medicines for Malaria Venture Pathogen Box collection, which facilitated the identification of specific blockers of late-stage intraerythrocytic development of Plasmodium falciparum First, from standard growth inhibition assays, we identified 173 molecules with antimalarial activity (50% effective concentration [EC50] ≤ 10 µM), which included 62 additional molecules over previously known antimalarial candidates from the Pathogen Box. We identified 90 molecules with EC50 of ≤1 µM, which had significant effect on the ring-trophozoite transition, while 9 molecules inhibited the trophozoite-schizont transition and 21 molecules inhibited the schizont-ring transition (with ≥50% parasites failing to proceed to the next stage) at 1 µM. We therefore rescreened all 173 molecules and validated hits in microscopy to prioritize 12 hits as selective blockers of the schizont-ring transition. Seven of these molecules inhibited the calcium ionophore-induced egress of Toxoplasma gondii, a related apicomplexan parasite, suggesting that the inhibitors may be acting via a conserved mechanism which could be further exploited for target identification studies. We demonstrate that two molecules, MMV020670 and MMV026356, identified as schizont inhibitors in our screens, induce the fragmentation of DNA in merozoites, thereby impairing their ability to egress and invade. Further mechanistic studies would facilitate the therapeutic exploitation of these molecules as broadly active inhibitors targeting late-stage development and egress of apicomplexan parasites relevant to human health.
Asunto(s)
Antimaláricos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Fragmentación del ADN/efectos de los fármacos , Humanos , Merozoítos/efectos de los fármacos , Pruebas de Sensibilidad Parasitaria , Esquizontes/efectos de los fármacos , Trofozoítos/efectos de los fármacosRESUMEN
An effective vaccine is a priority for malaria control and elimination. The leading candidate in the Plasmodium falciparum blood stage is PfRh5. PfRh5 assembles into trimeric complex with PfRipr and PfCyRPA in the parasite, and this complex is essential for erythrocyte invasion. In this study, we show that antibodies specific for PfRh5 and PfCyRPA prevent trimeric complex formation. We identify the EGF-7 domain on PfRipr as a neutralising epitope and demonstrate that antibodies against this region act downstream of complex formation to prevent merozoite invasion. Antibodies against the C-terminal region of PfRipr were more inhibitory than those against either PfRh5 or PfCyRPA alone, and a combination of antibodies against PfCyRPA and PfRipr acted synergistically to reduce invasion. This study supports prioritisation of PfRipr for development as part of a next-generation antimalarial vaccine.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Antígenos de Protozoos/genética , Proteínas Portadoras/genética , Malaria Falciparum/tratamiento farmacológico , Proteínas Protozoarias/genética , Anticuerpos Neutralizantes/inmunología , Proteínas Portadoras/antagonistas & inhibidores , Eritrocitos/efectos de los fármacos , Eritrocitos/inmunología , Humanos , Vacunas contra la Malaria/inmunología , Vacunas contra la Malaria/farmacología , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Merozoítos/efectos de los fármacos , Merozoítos/inmunología , Plasmodium falciparum/inmunología , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/inmunologíaRESUMEN
Ethanamizuril (EZL) is a novel triazine compound with excellent anticoccidial activity. We carried out a preliminary investigation of the effects of EZL on the different life cycle stages of Eimeria tenella. EZL mainly acted on the schizogony stage, with peak activity during the second-generation merozoite stage. We also studied the possible target of EZL by identifying the majorly differentially expressed gene affected by EZL in second-generation merozoites using real-time polymerase chain reaction, and screening for surface antigen proteins (SAGs). The relative expression levels of SAGs were compared by Western blot analysis showing that expression levels of surface antigen family member (SAGfm) and SAG19 were significantly downregulated by EZL. Immunofluorescence analysis indicated that SAGfm and SAG19 were localized on the surface of second-generation merozoites. In addition, fluorescence signals were significantly stronger in second-generation merozoites of infected non-medicated control (INC) group compared with that of the EZL group. Therefore, it was speculated that SAGs might be a potential target of EZL action. The inhibitory effects of anticoccidial drugs on SAG levels in coccidia thus warrant further research.
Asunto(s)
Coccidiosis/tratamiento farmacológico , Eimeria tenella/efectos de los fármacos , Enfermedades de las Aves de Corral/prevención & control , Triazinas/farmacología , Animales , Antígenos de Superficie/metabolismo , Western Blotting , Pollos/parasitología , Coccidiosis/parasitología , Estadios del Ciclo de Vida/efectos de los fármacos , Merozoítos/efectos de los fármacos , Proteínas Protozoarias/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Bloodstream malaria parasites require Ca++ for their development, but the sites and mechanisms of Ca++ utilization are not well understood. We hypothesized that there may be differences in Ca++ uptake or utilization by genetically distinct lines of P. falciparum. These differences, if identified, may provide insights into molecular mechanisms. RESULTS: Dose response studies with the Ca++ chelator EGTA (ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid) revealed stable differences in Ca++ requirement for six geographically divergent parasite lines used in previous genetic crosses, with the largest difference seen between the parents of the HB3 x Dd2 cross. Genetic mapping of Ca++ requirement yielded complex inheritance in 34 progeny clones with a single significant locus on chromosome 7 and possible contributions from other loci. Although encoded by a gene in the significant locus and a proposed Ca++ target, PfCRT (P. falciparum chloroquine resistance transporter), the primary determinant of clinical resistance to the antimalarial drug chloroquine, does not appear to contribute to this quantitative trait. Stage-specific application of extracellular EGTA also excluded determinants associated with merozoite egress and erythrocyte reinvasion. CONCLUSIONS: We have identified differences in Ca++ utilization amongst P. falciparum lines. These differences are under genetic regulation, segregating as a complex trait in genetic cross progeny. Ca++ uptake and utilization throughout the bloodstream asexual cycle of malaria parasites represents an unexplored target for therapeutic intervention.
Asunto(s)
Calcio/metabolismo , Sitios Genéticos , Malaria Falciparum/parasitología , Parásitos/genética , Plasmodium falciparum/genética , Animales , Cruzamientos Genéticos , Ácido Egtácico/farmacología , Femenino , Estudios de Asociación Genética , Haplotipos/genética , Patrón de Herencia/genética , Masculino , Proteínas de Transporte de Membrana/metabolismo , Merozoítos/efectos de los fármacos , Merozoítos/metabolismo , Parásitos/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/metabolismoRESUMEN
BACKGROUND: Despite treatment with effective antimalarial drugs, the mortality rate is still high in severe cases of the disease, highlighting the need to find adjunct therapies that can inhibit the adhesion of Plasmodium falciparum-infected erythrocytes (Pf-iEs). OBJECTIVES: In this context, we evaluated a new heparan sulfate (HS) from Nodipecten nodosus for antimalarial activity and inhibition of P. falciparum cytoadhesion and rosetting. METHODS: Parasite inhibition was measured by SYBR green using a cytometer. HS was assessed in rosetting and cytoadhesion assays under static and flow conditions using Chinese hamster ovary (CHO) and human lymphatic endothelial cell (HLEC) cells expressing intercellular adhesion molecule-1 (ICAM1) and chondroitin sulfate A (CSA), respectively. FINDINGS: This HS inhibited merozoite invasion similar to heparin. Moreover, mollusk HS decreased cytoadherence of P. falciparum to CSA and ICAM-1 on the surface of endothelial cells under static and flow conditions. In addition, this glycan efficiently disrupted rosettes. CONCLUSIONS: These findings support a potential use for mollusk HS as adjunct therapy for severe malaria.
Asunto(s)
Heparitina Sulfato/farmacología , Merozoítos/efectos de los fármacos , Moluscos/química , Plasmodium falciparum/efectos de los fármacos , Animales , Adhesión Celular/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Proteínas Protozoarias/efectos de los fármacos , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Toxoplasmosis is a common parasitic disease caused by Toxoplasma gondii (Nicolle et Manceaux, 1908), an obligate parasite capable of infecting a range of cell types in almost all warm-blooded animals. Upon infecting an intermediate host, the parasites differentiate into tachyzoites which rapidly infect host tissues. Usually, the invading parasites are cleared by the immune system and administered drugs, but some tachyzoites differentiate into bradyzoites forming tissue cysts. These tissue cysts could serve as a source for re-infection and exacerbations. Currently, treatment for toxoplasmosis is limited and, moreover, there are no drugs for treating the cystic stage thus rendering toxoplasmosis a global burden. Recently, we demonstrated that inorganic nanoparticles showed promising activity against the tachyzoite stage T. gondii. In the present study, we evaluated nanoparticles for effect on bradyzoite formation in vitro. Data revealed that the nanoparticles limited bradyzoite burden in vitro. Further, the nanoparticles decreased the bradyzoite-specific BAG-1 promoter activity relative to the untreated control under a bradyzoite-inducing culture condition, even though this reduction in BAG-1 promoter activity waned with increasing concentrations of nanoparticles. In contrast, a parallel experiment under normal cell culture conditions showed that the nanoparticle treatment mildly increased the BAG-1 promoter activity relative to the untreated control. Taken together, the findings are evidence that nanoparticles not only possess anti-tachyzoite potential but they also have anti-bradyzoite potential in vitro.
Asunto(s)
Coccidiostáticos/farmacología , Merozoítos/efectos de los fármacos , Nanopartículas del Metal , Toxoplasma/efectos de los fármacos , Merozoítos/crecimiento & desarrollo , Toxoplasma/crecimiento & desarrolloRESUMEN
The MIC is an essential quantitative measure of the asexual blood-stage effect of an antimalarial drug. In areas of high malaria transmission, and thus frequent individual infection, patients who are treated with slowly eliminated antimalarials become reinfected as drug concentrations decline. In the frequent relapse forms of Plasmodium vivax and in Plasmodium ovale malaria, recurrent infection occurs from relapses which begin to emerge from the liver approximately 2 weeks after the primary illness. An important determinant of the interval from starting treatment of a symptomatic infection to the patency of these recurrent infections is the in vivo concentration-response relationship and thus the in vivo MIC. Using mechanistic knowledge of parasite asexual replication and the pharmacokinetic and pharmacodynamic properties of the antimalarial drugs, a generative statistical model was derived which relates the concentration-response relationship to time of reinfection patency. This model was used to estimate the in vivo MIC of chloroquine in the treatment of Plasmodium vivax malaria.
Asunto(s)
Antimaláricos/farmacocinética , Antimaláricos/uso terapéutico , Adolescente , Adulto , Niño , Preescolar , Cloroquina/farmacocinética , Cloroquina/uso terapéutico , Femenino , Humanos , Lactante , Malaria Vivax/tratamiento farmacológico , Masculino , Merozoítos/efectos de los fármacos , Merozoítos/patogenicidad , Persona de Mediana Edad , Pruebas de Sensibilidad Parasitaria , Plasmodium vivax/efectos de los fármacos , Plasmodium vivax/patogenicidad , Adulto JovenRESUMEN
Erythrocyte invasion by merozoite is a multistep process involving multiple ligand-receptor interactions. The Plasmodium falciparum reticulocyte binding protein homologues (PfRHs) consists of five functional members. The differential expression of PfRHs has been linked to the utilization of different invasion pathways by the merozoites as well as a mechanism of immune evasion. PfRHs are expressed at the apical end of merozoite and form interactions with distinct red blood cell (RBC) surface receptors that are important for successful invasion. Here we show that PfRH2b undergoes processing before and during merozoite invasion. The different processed fragments bind to chymotrypsin sensitive RBC surface receptors. We also show that PfRH2b follows the merozoite tight junction during invasion. Monoclonal antibodies (mAbs) inhibit merozoites invasion by blocking tight junction formation. mAbs binding to PfRH2b block merozoites intracellular Ca2+ signal necessary for EBA175 surface expression. The data suggests that a conserved function of PfRHs, where their interaction with RBC surface receptors facilitated recruitment of EBA175 and other tight junction proteins necessary for merozoite invasion by modulating merozoite intracellular Ca2+ signals.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Eritrocitos/parasitología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Quimotripsina/metabolismo , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Interacciones Huésped-Parásitos , Humanos , Merozoítos/efectos de los fármacos , Merozoítos/metabolismo , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
Despite recent successful control efforts, malaria remains a leading global health burden. Alarmingly, resistance to current antimalarials is increasing and the development of new drug families is needed to maintain malaria control. Current antimalarials target the intraerythrocytic developmental stage of the Plasmodium falciparum life cycle. However, the invasive extracellular parasite form, the merozoite, is also an attractive target for drug development. We have previously demonstrated that heparin-like molecules, including those with low molecular weights and low anticoagulant activities, are potent and specific inhibitors of merozoite invasion and blood-stage replication. Here we tested a large panel of heparin-like molecules and sulfated polysaccharides together with various modified chemical forms for their inhibitory activity against P. falciparum merozoite invasion. We identified chemical modifications that improve inhibitory activity and identified several additional sulfated polysaccharides with strong inhibitory activity. These studies have important implications for the further development of heparin-like molecules as antimalarial drugs and for understanding merozoite invasion.
Asunto(s)
Antimaláricos/farmacología , Heparina/análogos & derivados , Heparina/farmacología , Merozoítos/crecimiento & desarrollo , Plasmodium falciparum/efectos de los fármacos , Polisacáridos/farmacología , Descubrimiento de Drogas/métodos , Resistencia a Medicamentos/genética , Malaria Falciparum/tratamiento farmacológico , Merozoítos/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Polisacáridos/químicaRESUMEN
Nitromezuril is a novel triazine compound for preventing coccidiosis in broiler chickens. A single treatment of chickens inoculated with Eimeria tenella during the endogenous phase were used to evaluate the developmental stages of action of nitromezuril by clinically anticoccidial indices and histopathology. Results showed that a single dose of nitromezuril at 5 mg/kg b.w. during 56 to 80 h post-inoculation can most effectively prevent weight loss and reduce both oocyst shedding and caecal lesions. The anticoccidial index reached the level of middle efficacy. Histological examinations indicated that administration of nitromezuril during 44 to 104 h after infection significantly reduced the merozoite population and the pathological damage to the caecum. Nitromezuril treatment could disturb the process of schizonts division into schizoites and produce abnormal schizonts. Overall, nitromezuril may exert its effects during the entire endogenous stage of the parasites but the schizogony stages were intrinsically more vulnerable. Nitromezuril is a potential novel anticoccidial agent suitable for further development.
Asunto(s)
Pollos , Coccidiosis/veterinaria , Coccidiostáticos/farmacología , Eimeria tenella/efectos de los fármacos , Enfermedades de las Aves de Corral/tratamiento farmacológico , Triazinas/farmacología , Animales , Ciego/parasitología , Ciego/patología , Pollos/parasitología , Coccidiosis/tratamiento farmacológico , Coccidiosis/patología , Merozoítos/efectos de los fármacos , Oocistos/efectos de los fármacos , Enfermedades de las Aves de Corral/parasitología , Enfermedades de las Aves de Corral/patología , Esquizontes/efectos de los fármacosRESUMEN
To explore the primary stage or site of action of acetamizuril (AZL), a novel triazine anticoccidial compound, the ultrastructural development of Eimeria tenella at different endogenous stages was studied in experimentally infected chickens treated with a single oral dose of 15 mg/kg AZL. As a result of drug action, the differentiations of second-generation schizonts and microgamonts were largely inhibited and merozoites became irregular in shape. Meanwhile, the outer membrane blistering and perinuclear space enlargement were obvious in the second-generation schizonts and microgamonts, which were never observed in the classic triazine anticoccidiosis drug diclazuril-treated E. tenella. The chromatin aggregation, anachromasis, and marginalization were visible in merozoites and microgamonts. Furthermore, the abnormal evagination of microgametes finally resulted in the degeneration of microgamonts and the failure of subsequent fertilization. The most marked micromorphological alteration occurring in the macrogamonts was the WFB2. Even if the fertilization occurred, the formation of oocyst wall became malformed and the zygote proceeded to the obvious degeneration. In addition, mitochondria swelling and cytoplasm vacuolization were discerned in respective intracellular stages, while endoplasmic reticulum and Golgi body swelling was less seen. These alterations may be the causes leading to the final death of E. tenella and also provide some information for further exploring the mechanism of action of AZL at the molecular level.
Asunto(s)
Coccidiosis/veterinaria , Coccidiostáticos/farmacología , Eimeria tenella/efectos de los fármacos , Triazinas/farmacología , Animales , Ciego/parasitología , Ciego/ultraestructura , Pollos , Coccidiosis/tratamiento farmacológico , Coccidiosis/parasitología , Eimeria tenella/crecimiento & desarrollo , Eimeria tenella/ultraestructura , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/ultraestructura , Merozoítos/efectos de los fármacos , Merozoítos/ultraestructura , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Nitrilos/farmacología , Oocistos , Distribución Aleatoria , Esquizontes/efectos de los fármacos , Esquizontes/ultraestructuraRESUMEN
Widespread drug resistance calls for the urgent development of new antimalarials that target novel steps in the life cycle of Plasmodium falciparum and Plasmodium vivax. The essential subtilisin-like serine protease SUB1 of Plasmodium merozoites plays a dual role in egress from and invasion into host erythrocytes. It belongs to a new generation of attractive drug targets against which specific potent inhibitors are actively searched. We characterize here the P. vivax SUB1 enzyme and show that it displays a typical auto-processing pattern and apical localization in P. vivax merozoites. To search for small PvSUB1 inhibitors, we took advantage of the similarity of SUB1 with bacterial subtilisins and generated P. vivax SUB1 three-dimensional models. The structure-based virtual screening of a large commercial chemical compounds library identified 306 virtual best hits, of which 37 were experimentally confirmed inhibitors and 5 had Ki values of <50 µM for PvSUB1. Interestingly, they belong to different chemical families. The most promising competitive inhibitor of PvSUB1 (compound 2) was equally active on PfSUB1 and displayed anti-P. falciparum and Plasmodium berghei activity in vitro and in vivo, respectively. Compound 2 inhibited the endogenous PfSUB1 as illustrated by the inhibited maturation of its natural substrate PfSERA5 and inhibited parasite egress and subsequent erythrocyte invasion. These data indicate that the strategy of in silico screening of three-dimensional models to select for virtual inhibitors combined with stringent biological validation successfully identified several inhibitors of the PvSUB1 enzyme. The most promising hit proved to be a potent cross-inhibitor of PlasmodiumSUB1, laying the groundwork for the development of a globally active small compound antimalarial.
Asunto(s)
Plasmodium vivax/enzimología , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Serina Proteasas/química , Secuencia de Aminoácidos , Animales , Antimaláricos/química , Antimaláricos/farmacología , Sitios de Unión/genética , Biocatálisis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Femenino , Cinética , Malaria/parasitología , Malaria/prevención & control , Merozoítos/efectos de los fármacos , Merozoítos/enzimología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Plasmodium berghei/efectos de los fármacos , Plasmodium berghei/enzimología , Plasmodium vivax/efectos de los fármacos , Plasmodium vivax/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Homología de Secuencia de Aminoácido , Serina Proteasas/genética , Serina Proteasas/metabolismo , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Células Sf9 , Especificidad por SustratoRESUMEN
Calcium-dependent protein kinases (CDPKs) play important roles in the life cycle of Plasmodium falciparum and other apicomplexan parasites. CDPKs commonly have an N-terminal kinase domain (KD) and a C-terminal calmodulin-like domain (CamLD) with calcium-binding EF hands. The KD and CamLD are separated by a junction domain (JD). Previous studies on Plasmodium and Toxoplasma CDPKs suggest a role for the JD and CamLD in the regulation of kinase activity. Here, we provide direct evidence for the binding of the CamLD with the P3 region (Leu(356) to Thr(370)) of the JD in the presence of calcium (Ca(2+)). Moreover, site-directed mutagenesis of conserved hydrophobic residues in the JD (F363A/I364A, L356A, and F350A) abrogates functional activity of PfCDPK1, demonstrating the importance of these residues in PfCDPK1 function. Modeling studies suggest that these residues play a role in interaction of the CamLD with the JD. The P3 peptide, which specifically inhibits the functional activity of PfCDPK1, blocks microneme discharge and erythrocyte invasion by P. falciparum merozoites. Purfalcamine, a previously identified specific inhibitor of PfCDPK1, also inhibits microneme discharge and erythrocyte invasion, confirming a role for PfCDPK1 in this process. These studies validate PfCDPK1 as a target for drug development and demonstrate that interfering with its mechanistic regulation may provide a novel approach to design-specific PfCDPK1 inhibitors that limit blood stage parasite growth and clear malaria parasite infections.
Asunto(s)
Merozoítos/enzimología , Orgánulos/enzimología , Plasmodium falciparum/enzimología , Proteínas Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Secuencia de Aminoácidos , Transporte Biológico/efectos de los fármacos , Calcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Ciclohexilaminas/farmacología , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Escherichia coli/genética , Expresión Génica , Humanos , Merozoítos/efectos de los fármacos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Orgánulos/efectos de los fármacos , Orgánulos/metabolismo , Péptidos/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Proteínas Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Malaria parasites grow within erythrocytes, but are also free in host plasma between cycles of asexual replication. As a result, the parasite is exposed to fluctuating levels of Na(+) and K(+) , ions assumed to serve important roles for the human pathogen, Plasmodium falciparum. We examined these assumptions and the parasite's ionic requirements by establishing continuous culture in novel sucrose-based media. With sucrose as the primary osmoticant and K(+) and Cl(-) as the main extracellular ions, we obtained parasite growth and propagation at rates indistinguishable from those in physiological media. These conditions abolish long-known increases in intracellular Na(+) via parasite-induced channels, excluding a requirement for erythrocyte cation remodelling. We also dissected Na(+) , K(+) and Cl(-) requirements and found that unexpectedly low concentrations of each ion meet the parasite's demands. Surprisingly, growth was not adversely affected by up to 148 mM K(+) , suggesting that low extracellular K(+) is not an essential trigger for erythrocyte invasion. At the same time, merozoite egress and invasion required a threshold ionic strength, suggesting critical electrostatic interactions between macromolecules at these stages. These findings provide insights into transmembrane signalling in malaria and reveal fundamental differences between host and parasite ionic requirements.
Asunto(s)
Cationes/farmacología , Malaria/parasitología , Parásitos/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Animales , Cloruros/farmacología , Medios de Cultivo/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Eritrocitos/ultraestructura , Interacciones Huésped-Parásitos , Humanos , Merozoítos/efectos de los fármacos , Merozoítos/crecimiento & desarrollo , Concentración Osmolar , Parásitos/crecimiento & desarrollo , Fosfatos/metabolismo , Plasmodium falciparum/crecimiento & desarrollo , Potasio/farmacología , Sodio/farmacología , Sacarosa/farmacología , Trofozoítos/efectos de los fármacos , Trofozoítos/crecimiento & desarrollo , Trofozoítos/ultraestructuraRESUMEN
Sequestration of Plasmodium falciparum-infected erythrocytes (Pf-iEs) in the microvasculature of vital organs plays a key role in the pathogenesis of life-threatening malaria complications, such as cerebral malaria and malaria in pregnancy. This phenomenon is marked by the cytoadhesion of Pf-iEs to host receptors on the surfaces of endothelial cells, on noninfected erythrocytes, and in the placental trophoblast; therefore, these sites are potential targets for antiadhesion therapies. In this context, glycosaminoglycans (GAGs), including heparin, have shown the ability to inhibit Pf-iE cytoadherence and growth. Nevertheless, the use of heparin was discontinued due to serious side effects, such as bleeding. Other GAG-based therapies were hampered due to the potential risk of contamination with prions and viruses, as some GAGs are isolated from mammals. In this context, we investigated the effects and mechanism of action of fucosylated chondroitin sulfate (FucCS), a unique and highly sulfated GAG isolated from the sea cucumber, with respect to P. falciparum cytoadhesion and development. FucCS was effective in inhibiting the cytoadherence of Pf-iEs to human lung endothelial cells and placenta cryosections under static and flow conditions. Removal of the sulfated fucose branches of the FucCS structure virtually abolished the inhibitory effects of FucCS. Importantly, FucCS rapidly disrupted rosettes at high levels, and it was also able to block parasite development by interfering with merozoite invasion. Collectively, these findings highlight the potential of FucCS as a candidate for adjunct therapy against severe malaria.
Asunto(s)
Antimaláricos/farmacología , Sulfatos de Condroitina/farmacología , Merozoítos/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Animales , Antimaláricos/efectos adversos , Células Cultivadas , Sulfatos de Condroitina/efectos adversos , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Células Hep G2 , Humanos , Pepinos de Mar/químicaRESUMEN
The commitment of Plasmodium merozoites to invade red blood cells (RBCs) is marked by the formation of a junction between the merozoite and the RBC and the coordinated induction of the parasitophorous vacuole. Despite its importance, the molecular events underlying the parasite's commitment to invasion are not well understood. Here we show that the interaction of two parasite proteins, RON2 and AMA1, known to be critical for invasion, is essential to trigger junction formation. Using antibodies (Abs) that bind near the hydrophobic pocket of AMA1 and AMA1 mutated in the pocket, we identified RON2's binding site on AMA1. Abs specific for the AMA1 pocket blocked junction formation and the induction of the parasitophorous vacuole. We also identified the critical residues in the RON2 peptide (previously shown to bind AMA1) that are required for binding to the AMA1 pocket, namely, two conserved, disulfide-linked cysteines. The RON2 peptide blocked junction formation but, unlike the AMA1-specific Ab, did not block formation of the parasitophorous vacuole, indicating that formation of the junction and parasitophorous vacuole are molecularly distinct steps in the invasion process. Collectively, these results identify the binding of RON2 to the hydrophobic pocket of AMA1 as the step that commits Plasmodium merozoites to RBC invasion and point to RON2 as a potential vaccine candidate.
Asunto(s)
Merozoítos/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Sitios de Unión , Secuencia Conservada/genética , Cisteína/metabolismo , Citocalasina D/farmacología , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Merozoítos/efectos de los fármacos , Merozoítos/ultraestructura , Modelos Biológicos , Datos de Secuencia Molecular , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/ultraestructura , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Protozoarias/química , Relación Estructura-ActividadRESUMEN
Diclazuril has long been used as an effective benzeneacetonitrile anticoccidial for the control of Eimeria tenella that causes coccidiosis. However, the molecular mechanism underlying the anticoccidial effects of diclazuril remains elusive. In this study, a proteomic analysis of the effect of diclazuril on second-generation merozoites of E. tenella was performed. Using two-dimensional gel electrophoresis and real-time quantitative polymerase chain reaction (RT-PCR), 13 target proteins were found to be significantly affected by diclazuril treatment, with 11 of these proteins being identified as annotated proteins from E. tenella or other Apicomplexa parasites. These proteins contribute to various functions, including metabolism, protein synthesis, and host cell invasion. Using RT-PCR, we identified the potential pattern of transcriptional regulation induced by diclazuril, and we suggest some promising targets for the intervention of E. tenella infection.