RESUMO
BACKGROUND: Different proteins derived from the membrane or the apical organelles become involved in malarial parasite invasion of host cells. Among these, the rhoptry neck proteins (RONs) interact with a protein component of the micronemes to enable the formation of a strong bond which is crucial for the parasite's successful invasion. The present study was aimed at identifying and characterizing the RON5 protein in Plasmodium vivax and evaluating its ability to bind to reticulocytes. METHODS: Taking the Plasmodium falciparum and Plasmodium knowlesi RON5 amino acid sequences as template, an in-silico search was made in the P. vivax genome for identifying the orthologous gene. Different molecular tools were used for experimentally ascertaining pvron5 gene presence and transcription in P. vivax VCG-1 strain schizonts. Polyclonal antibodies against PvRON5 peptides were used for evaluating protein expression (by Western blot) and sub-cellular localization (by immunofluorescence). A 33 kDa PvRON5 fragment was expressed in Escherichia coli and used for evaluating the reactivity of sera from patients infected by P. vivax. Two assays were made for determining the RON5 recombinant fragment's ability to bind to reticulocyte-enriched human umbilical cord samples. RESULTS: The pvron5 gene (3,477 bp) was transcribed in VCG-1 strain schizonts and encoded a ~133 kDa protein which was expressed in the rhoptry neck of VCG-1 strain late schizonts, together with PvRON2 and PvRON4. Polyclonal sera against PvRON5 peptides specifically detected ~85 and ~30 kDa fragments in parasite lysate, thereby suggesting proteolytic processing in this protein. Comparative analysis of VCG-1 strain PvRON5 with other P. vivax strains having different geographic localizations suggested its low polymorphism regarding other malarial antigens. A recombinant fragment of the PvRON5 protein (rPvRON5) was recognized by sera from P. vivax-infected patients and bound to red blood cells, having a marked preference for human reticulocytes. CONCLUSIONS: The pvron5 gene is transcribed in the VCG-1 strain, the encoded protein is expressed at the parasite's apical pole and might be participating in merozoite invasion of host cells, taking into account its marked binding preference for human reticulocytes.
Assuntos
Plasmodium vivax/genética , Proteínas de Protozoários/genética , Reticulócitos/parasitologia , Humanos , Dados de Sequência Molecular , Plasmodium vivax/crescimento & desenvolvimento , Plasmodium vivax/metabolismo , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Reticulócitos/metabolismo , Esquizontes/crescimento & desenvolvimento , Esquizontes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Identifying the minimal functional regions of the proteins which the malaria parasite uses when invading its host cells constitutes the first and most important approach in an effective design for a chemically synthesised, multi-antigen, multi-stage, subunit-based vaccine. This work has been aimed at identifying the PfRh1 protein binding regions (residues 1-2580) belonging to the reticulocyte binding-like (RBL or P. falciparum Rh [PfRh]) family implicated in the parasite's alternative target cell invasion routes. Eighteen peptide regions (called high activity binding peptides - HABPs) binding to red blood cells (RBC) were identified in peptides mapped in a highly robust, specific and sensitive receptor-ligand assay. These HABPs were saturable in the experimental conditions assayed here and most had an alpha helix structure. Polymorphism studies revealed that only six of the eighteen HABPs identified had changes at amino acid level amongst the seven P. falciparum strains evaluated. Most HABPs' specific binding became altered when RBC were treated with neuraminidase, chymotrypsin and trypsin, suggesting differing sensitivity for RBC membrane receptors. After ascertaining that the Rh1 gene was transcribed and expressed in late-stage schizonts of the FCB-2 strain, invasion inhibition assays were carried out. When most of these HABPs were assayed in P. falciparum in vitro culture they were able to inhibit high percentages of FVO strain invasion compared to low inhibition percentages observed with the FCB-2 strain. This data shows small Rh1 regions' participation during invasion and suggests that these units should be included in further immunological and structural studies.
Assuntos
Vacinas Antimaláricas/química , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/química , Sequência de Aminoácidos , Eritrócitos/parasitologia , Interações Hospedeiro-Patógeno , Humanos , Malária/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Plasmodium falciparum/classificação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/imunologia , Esquizontes/metabolismo , Vacinas de Subunidades Antigênicas/metabolismoRESUMO
Chromosome ends have been implicated in the default silencing of clonally variant gene families in the human malaria parasite Plasmodium falciparum. These chromosome regions are organized into heterochromatin, as defined by the presence of a repressive histone H3 lysine 9 trimethylated marker and heterochromatin protein 1. Here, we show that the non-coding subtelomeric region adjacent to virulence genes forms facultative heterochromatin in a cell cycle-dependent manner. We demonstrate that telomere-associated repeat elements (TAREs) and telomeres are transcribed as long non-coding RNAs (lncRNAs) during schizogony. Northern blot assays revealed two classes of lncRNAs: a ~4-kb transcript composed of telomere sequences and a TARE-3 element, and a >6-kb transcript composed of 21-bp repeats from TARE-6. These lncRNAs are transcribed by RNA polymerase II as single-stranded molecules. RNA-FISH analysis showed that these lncRNAs form several nuclear foci during the schizont stage, whereas in the ring stage, they are located in a single perinuclear compartment that does not co-localize with any known nuclear subcompartment. Furthermore, the TARE-6 lncRNA is predicted to form a stable and repetitive hairpin structure that is able to bind histones. Consequently, the characterization of the molecular interactions of these lncRNAs with nuclear proteins may reveal novel modes of gene regulation and nuclear function in P. falciparum.
Assuntos
Núcleo Celular/metabolismo , Plasmodium falciparum/genética , RNA de Protozoário/metabolismo , RNA não Traduzido/metabolismo , Telômero/metabolismo , Animais , Northern Blotting , Núcleo Celular/genética , Cromossomos/genética , Cromossomos/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Hibridização in Situ Fluorescente/métodos , Plasmídeos/genética , Plasmídeos/metabolismo , Plasmodium falciparum/metabolismo , Ligação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Estabilidade de RNA , RNA de Protozoário/genética , RNA não Traduzido/genética , Sequências Repetitivas de Ácido Nucleico , Esquizontes/citologia , Esquizontes/metabolismo , Telômero/genética , Transcrição GênicaRESUMO
The 2-C-methyl-D-erythritol-4-phosphate and shikimate pathways were found to be active in Plasmodium falciparum and both can result in vitamin E biosynthesis in plants and algae. This study biochemically confirmed vitamin E biosynthesis in the malaria parasite, which can be inhibited by usnic acid. Furthermore, we found evidence pointing to a role of this vitamin in infected erythrocytes. These findings not only contribute to current understanding of P. falciparum biology but also reveal a pathway that could serve as a chemotherapeutic target.
Assuntos
Eritrócitos/parasitologia , Estágios do Ciclo de Vida , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Vitamina E/biossíntese , Animais , Benzofuranos/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Estágios do Ciclo de Vida/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/fisiologia , Esquizontes/efeitos dos fármacos , Esquizontes/metabolismo , Vitamina E/análise , alfa-Tocoferol/análise , alfa-Tocoferol/metabolismo , gama-Tocoferol/análise , gama-Tocoferol/metabolismoRESUMO
Gathering knowledge about the proteins involved in erythrocyte invasion by Plasmodium merozoites is the starting point for developing new strategies to control malarial disease. Many of these proteins have been studied in Toxoplasma gondii, where some belonging to the Moving Junction complex have been identified. This complex allows a strong interaction between host cell and parasite membranes, required for parasite invasion. In this genus, four rhoptry proteins (RON2, RON4, RON5 and RON8) and one micronemal protein (TgAMA-1) have been found as part of the complex. In Plasmodium falciparum, RON2 and RON4 have been characterized. In the present study, we identify PfRON5, a ~110 kDa protein which is expressed in merozoite and schizont stages of the FCB-2 strain.
Assuntos
Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Sequência de Bases , Expressão Gênica , Merozoítos/metabolismo , Dados de Sequência Molecular , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Esquizontes/metabolismoRESUMO
Plasmodium falciparum rhoptry-associated membrane antigen (RAMA) peptides used in normal red blood cell (RBC) binding assays revealed that peptides 33426 (79NINILSSVHRKGRILYDSF97) and 33460 (777HKKREKSISPHSYQKVSTKVQ797) bound with high activity, presenting nanomolar affinity constants. Such high binding activity peptides (HABPs) displayed helicoid and random coil structures as determined by circular dichroism. HABPs inhibited P. falciparumin vitro invasion of normal RBC by up to 61% (depending on concentration), suggesting that some RAMA protein regions could be involved in P. falciparum invasion of RBC. The nature and localisation of receptors on RBC surface responsible for HABP binding were studied using enzyme-treated erythrocytes and structural analysis.