RESUMEN
Malaria proteases are attractive anti-malarial targets because of their roles in parasite development and infection. Falcipain-2 (FP-2), a food vacuole cysteine protease in Plasmodium falciparum, is involved in hemoglobin degradation and cleavage of cytoskeletal elements. To understand the route of trafficking and identify the signals involved in trafficking to food vacuole, we have generated transgenic parasites expressing green fluorescent protein (GFP) fusion proteins comprising of N-terminal regions of falcipain-2 fused to GFP. Using falcipain2-GFP chimeras and anti-falcipain-2 antibody, we show that falcipain-2 is trafficked through a classical vesicle mediated secretory pathway involving endoplasmic reticulum and Golgi-like apparatus. Photobleaching and confocal microscopy techniques reveal that falcipain-2 is carried to the food vacuole in the form of cytostomal vesicles. We identify an N-terminal sequence (1-120aa) of falcipain-2, sufficient for its transport to the food vacuole. Analysis of sequences of few other food vacuole targeted proteins suggests a common mechanism for protein trafficking to food vacuole of malaria parasite.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Eritrocitos/parasitología , Plasmodium falciparum/enzimología , Transporte de Proteínas , Vacuolas/enzimología , Animales , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Confocal , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoAsunto(s)
Genes Protozoarios , Plasmodium falciparum/genética , alfa Carioferinas/genética , beta Carioferinas/genética , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Núcleo Celular/metabolismo , Clonación Molecular , Modelos Genéticos , Datos de Secuencia Molecular , Plasmodium falciparum/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Homología de Secuencia de Aminoácido , alfa Carioferinas/análisis , beta Carioferinas/análisisRESUMEN
The C-terminal 42-kDa fragment of the merozoite surface protein-1 of Plasmodium falciparum (PfMSP-1(42)) was expressed as a recombinant protein in Escherichia coli and purified to near homogeneity. We tested the immunogenicity of recombinant PfMSP-1(42) in three clinically acceptable adjuvants (Montanide ISA 720, alum and MF59) in mice and in rabbits. High antibody responses were obtained with two adjuvant formulations with IgGl being the predominant immunoglobulin isotype. Significant T-cell proliferation responses were also observed. Competitive enzyme linked immunosorbant assay (ELISA) showed the presence of both invasion and processing inhibitory antibodies in sera obtained from the immunized rabbits. Passive immunizations of mice with anti-PfMSP-1(42) IgG purified from the rabbit-sera were found to be protective against a parasite challenge with P. berghei/P. falciparum chimeric line (Pb-PfM19) that expresses Plasmodium falciparum MSP-1(19). These findings may be useful for the development of a malaria vaccine based on Plasmodium falciparum MSP-1(42).
Asunto(s)
Antígenos de Protozoos/administración & dosificación , Vacunas contra la Malaria/administración & dosificación , Malaria Falciparum/prevención & control , Proteína 1 de Superficie de Merozoito/administración & dosificación , Plasmodium falciparum/genética , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Inmunoglobulina G/biosíntesis , Vacunas contra la Malaria/genética , Proteína 1 de Superficie de Merozoito/genética , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
A differential immunoscreening of the lambdagt11 Plasmodium falciparum genomic expression library was carried out using anti-P. yoelii sera (convalescent-phase mouse sera) and immune sera collected from healthy adults, to identify novel cross-reactive and possibly protective antigens of the parasite. One clone, with an insert size of 1132 bp that reacted strongly with both the sera was selected. The insert was found to be a part of the P. falciparum karyopherin beta (PfKbeta) homologue. RT-PCR and Northern blot analysis confirmed the expression of PfKbeta in the blood stages of the parasite. The approximately 110 kDa protein was localized in the cytoplasm at the ring and trophozoite, and in the parasitophorous vacuole at the schizont stage. Two large fragments of PfKbeta representing the N- and C-terminal halves were expressed in E. coli. The recombinant proteins were highly immunogenic in mice, and also found to be the target for immune response in natural infections of Plasmodium spp. Anti-sera against the protein showed a low level of anti-parasitic activity. Immunization with recombinant PfKbeta fragments was only partially protective against a heterologous challenge infection in mice. Our results show that the parasite releases a highly immunogenic, cytoplasmic protein into the host which may not contribute to the development of protective immunity.
Asunto(s)
Antígenos de Protozoos/inmunología , Malaria Falciparum/inmunología , Malaria/inmunología , Plasmodium yoelii/inmunología , beta Carioferinas/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/genética , Antígenos de Protozoos/metabolismo , Enfermedades Endémicas , Biblioteca de Genes , Humanos , Sueros Inmunes/inmunología , Inmunización , Malaria/parasitología , Malaria/prevención & control , Malaria Falciparum/parasitología , Ratones , Ratones Endogámicos BALB C , Plasmodium falciparum/inmunología , Plasmodium yoelii/crecimiento & desarrollo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMEN
The process of merozoite release in Plasmodium falciparum involves rupture of the parasitophorous vacuole membrane and erythrocyte plasma membrane. Through the use of protease inhibitors that halt the merozoite release, a number of parasite proteases, especially serine, aspartic, and cysteine proteases, have been implicated in the schizont rupture. To understand the precise role of cysteine proteases in the merozoite release, in the present study, we treated P. falciparum cultures with siRNAs corresponding to falcipain-1, falcipain-2, and falcipain-3, the three papain-family proteases of the parasite. Treatment of malaria parasites with either of the falcipain siRNAs considerably reduced parasite growth. Morphological examination of the siRNA treated parasite cultures revealed that most of the parasites in falcipain-2 siRNA treated cultures were arrested at schizont stage. Analysis of a transgenic P. falciparum line expressing chimeric-GFP upon treatment with falcipain-2 siRNA revealed block in the rupture of erythrocyte membrane at the time of merozoite egression. These results suggest that falcipain-2 is an important parasitic protease that participates in hemoglobin degradation and in the merozoite release.
Asunto(s)
Cisteína Endopeptidasas/fisiología , Membrana Eritrocítica/parasitología , Eritrocitos/parasitología , Plasmodium falciparum/fisiología , Animales , Células Cultivadas , Cisteína Endopeptidasas/genética , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Humanos , Plasmodium falciparum/enzimología , ARN Interferente Pequeño/genéticaRESUMEN
RNA interference (RNAi) has emerged as a specific and efficient tool to silence gene expression in a variety of organisms and cell lines. An important prospect for RNAi technology is its possible application in the treatment of diseases using short interfering RNAs (siRNAs). However, the effect of siRNAs in adult animals and their potential to treat or prevent diseases are yet to be fully investigated. The main goal of the present study is to find out whether it was possible to carry out RNAi on circulating malaria parasite in vivo. To trigger RNAi in mouse malaria parasite, we used siRNAs corresponding to cysteine protease genes of Plasmodium berghei (berghepain-1 & 2). Intravenous injections of berghepains' siRNAs in infected animal resulted in characteristic enlargement of food vacuole in circulating parasites. Protein analysis of these treated parasites showed substantial accumulation of hemoglobin, which is reminiscent of the effect observed upon treating Plasmodium falciparum with different cysteine protease inhibitors. Parasites treated with berghepain 1 & 2 siRNAs showed marked reduction in the levels of their cognate mRNAs, thereby suggesting specific inhibition of berghepains' gene expression in vivo. We also observed the generation of approximately 25 nt RNA species from berghepains' mRNAs in the treated parasites, which is a characteristic of an RNAi phenomenon. These results thus provide evidence that beyond its value for validation of gene functions, RNAi may provide a new approach for disease therapy.
Asunto(s)
Malaria/parasitología , Plasmodium berghei/genética , ARN Protozoario/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Cisteína Endopeptidasas/genética , Femenino , Genes Protozoarios , Malaria/patología , Malaria/terapia , Ratones , Ratones Endogámicos BALB C , Plasmodium berghei/citología , Plasmodium berghei/enzimología , Interferencia de ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Protozoario/química , Vacuolas/ultraestructuraRESUMEN
Malaria remains a public health problem of enormous magnitude, affecting over 500 million people every year. Lack of success in the past in the development of new drug/vaccines has mainly been attributed to poor understanding of the functions of different parasite proteins. Recently, RNA interference (RNAi) has emerged as a simple and incisive technique to study gene functions in a variety of organisms. In this study, we report the results of RNAi by double-stranded RNA of cysteine protease genes (falcipain-1 and -2) in the malaria parasite, Plasmodium falciparum. Using RNAi directed towards falcipain genes, we demonstrate that blocking the expression of these genes results in severe morphological abnormalities in parasites, inhibition of parasite growth in vitro and substantial accumulation of haemoglobin in the parasite. The inhibitory effects produced by falcipain double-stranded (ds)RNAs are reminiscent of the effects observed upon administering E-64, a cysteine protease inhibitor. The parasites treated with falcipain's dsRNAs also show marked reduction in the levels of corresponding endogenous falcipain mRNAs. We also demonstrate that dsRNAs of falcipains are broken into short interference RNAs approximately 25 nucleotides in size, a characteristic of RNAi, which in turn activates sequence-specific nuclease activity in the malaria parasites. These results thus provide more evidence for the existence of RNAi in P. falciparum and also suggest possibilities for using RNAi as an effective tool to determine the functions of the genes identified from the P. falciparum genome sequencing project.