Estudio de interacciones Hospedero-Patógeno y Proteína-Proteína en Plasmodium vivax: evaluación de las Proteínas del Cuello de Roptrias -2, -4 y -5 y del Antígeno Apical de Membrana-1

[ES] La malaria es una de las enfermedades tropicales transmitidas por vectores más importantes a nivel mundial, donde Plasmodium vivax representa una de las especies más ampliamente distribuidas, afectando ~13.8 millones de personas por año. Pese a ello, el progreso aparentemente lento de la infección y los niveles bajos de parasitemia en el humano, comparados a lo reportado en Plasmodium falciparum, han llevado a clasificar a la infección por P. vivax erróneamente como benigna. Esto, sumado a los retos experimentales que trae consigo el cultivo de este parásito, obstaculizan en gran medida el conocimiento a nivel biológico, celular y molecular, necesario para el desarrollo de métodos de control efectivos contra P. vivax. Hoy en día, se conoce que un inadecuado diagnóstico, el mal manejo terapéutico y/o el retraso en el tratamiento, pueden llevar a recaídas y estados de enfermedad grave, similares a los reportados para la malaria producida por P. falciparum, lo que impone retos en la búsqueda de nuevas alternativas específicas contra esta especie. Teniendo en cuenta la necesidad de identificar posibles blancos terapéuticos contra P. vivax, este trabajo se enfocó en estudiar interacciones del tipo receptor-ligando y proteína-proteína de moléculas de P. vivax localizadas en los organelos apicales de esquizontes intraeritrocíticos. Basados en estudios previos en P. falciparum y Toxoplasma gondii, donde se describe la importancia funcional de las proteínas localizadas en el cuello de las roptrias (RONs) -2, -4 y 5 y del antígeno apical de membrana 1 (AMA1), se caracterizó en P. vivax la unión de cada una de estas proteínas con reticulocitos humanos y se evaluó la capacidad de PvRON2 para establecer interacciones con las proteínas PvRON4, PvRON5 y PvAMA1. Para esto, inicialmente se caracterizó mediante herramientas bioinformáticas y experimentales la presencia de los genes pvron4 y pvron5 en el genoma y transcriptoma de esquizontes de la cepa Vivax Colombia Guaviare 1 (VCG-1) de P. vivax. Estos dos genes codifican proteínas de alto peso molecular que se expresan en el polo apical de esquizontes y co-localizan con proteínas presentes en las roptrias. Para evaluar la capacidad de interacción de las proteínas PvRON2, PvRON4, PvRON5 y PvAMA1 con receptores sobre la membrana de reticulocitos humanos, todas ellas fueron producidas de forma recombinante y purificadas mediante cromatografía de afinidad. Se encontró que la proteína recombinante PvRON5 se unió tanto a normocitos como a reticulocitos CD71+, con una marcada preferencia por reticulocitos humanos. Por su parte, las proteínas recombinantes que incluyen los dominios I y II de PvAMA-1 (PvAMA-DI-DII), la región central de la proteína PvRON2 (PvRON2-RI) y la región carboxi-terminal de PvRON4, interactúan específicamente con reticulocitos CD71+CD45-. Los estudios de competencia de unión con péptidos sintéticos que cubren las secuencias de las proteínas recombinantes mostraron que los péptidos 21270 (derivado del DI de PvAMA1), 40305 (de PvRON4) y 40595 (de PvRON2-RI) fueron capaces de inhibir la unión de las proteínas recombinantes a reticulocitos CD71+CD45-, lo que sugiere que estas secuencias peptídicas contienen parte de las propiedades de unión de cada una de las proteínas de las que derivan. Los tres péptidos se unen específicamente y con alta afinidad a eritrocitos con porcentajes de unión mayores al 2% (obtenidas de las curvas de unión específica), permitiendo catalogarlos como péptidos con alta capacidad de unión a eritrocitos (HABPs, del inglés High Activity Binding Peptides). La unión de las proteínas PvAMA1 y PvRON4 a eritrocitos humanos fue sensible al tratamiento de los eritrocitos con diferentes enzimas (tripsina, quimotripsina y/o neuraminidasa), sugiriendo que la naturaleza de los receptores para estas proteínas es de tipo proteico. Estos resultados destacan la función de adhesina de las proteínas evaluadas y revelan las regiones mínimas de interacción con la célula hospedera, que sumado a la expresión de estas proteínas en esquizontes intraeritrocíticos y su localización en organelos apicales, sugieren una fuerte participación de estas moléculas durante el proceso de invasión de los merozoitos de P. vivax a reticulocitos humanos. Finalmente, mediante resonancia de plasmones de superficie, se caracterizaron las interacciones entre la proteína PvRON2 con las proteínas PvRON4, PvRON5 y PvAMA1. Los análisis revelaron que la región carboxi-terminal de la proteína PvRON2 (PvRON2-RII) y PvRON2-RI interactúan específicamente y con alta afinidad con el dominio II y III de PvAMA1 (PvAMA-DII-DIII) y con una menor afinidad con las proteínas PvAMA-DI-DII, PvRON4 y PvRON5. Al modificar algunos residuos de la proteína PvAMA1, reportados en P. falciparum como críticos en la interacción RON2-AMA1, no se encontraron diferencias importantes en los valores de las velocidades de asociación (Kon), disociación (Koff) y en la constante de disociación de la interacción (kD). Esto sugiere que, si bien existen interacciones proteína-proteína (IPP) conservadas entre estos parásitos (Pv-Pf), cada parásito utiliza distintas regiones de las proteínas para interactuar, lo que resalta su capacidad para especializarse o restringirse para invadir un tipo de célula específica y pone de manifiesto aún más la necesidad de diseñar medidas de control específicas contra P. vivax. [EN] Malaria is one of the most important tropical diseases transmitted by vectors worldwide; Plasmodium vivax represents one of the most widely distributed species (affecting ~ 13.8 million people worldwide per year). Despite this, the apparently slow progress of infection and low parasitaemia levels in humans compared to those reported in Plasmodium falciparum have erroneously led to P. vivax infection being classified as benign. Added to this, the experimental challenges involved in culturing this parasite greatly hinder accumulating the biological, cellular and molecular knowledge necessary for developing effective control methods against P. vivax. It is known nowadays that unsuitable diagnosis, poor therapeutic management and/or delayed treatment can lead to relapses and severe disease states similar to those reported for P. falciparum malaria, thereby imposing challenges regarding the search for new, specific, alternative approaches to tackling this species. The present work has been focused on studying receptor-ligand and protein-protein interactions of P. vivax molecules located in intra-erythrocyte schizonts’ apical organelles regarding the need for identifying therapeutic targets against P. vivax. Protein interaction with human reticulocytes was characterised in P. vivax and PvRON2 ability to establish interactions with PvRON4, PvRON5 and PvAMA1 was evaluated, based on previous P. falciparum and Toxoplasma gondii studies, describing the functional importance of rhoptry neck proteins. Work began by using bioinformatics and experimental tools for predicting pvron4 and pvron5 genes in the P. vivax VCG-1 (Vivax Colombia Guaviare 1) strain’s genome and schizonts transcriptome. These two genes encode high molecular weight proteins which are expressed at schizonts’ apical poles and co-localise with proteins in the rhoptries. Such proteins were produced recombinantly and purified by affinity chromatography for evaluating PvRON2, PvRON4, PvRON5 and PvAMA1 ability to interact with receptors on human reticulocyte membrane. Recombinant PvRON5 bound to both CD71+ normocytes and reticulocytes, having a marked preference for human reticulocytes. PvAMA1 domains I and II (PvAMA-DI-DII), PvRON2 central region (PvRON2-RI) and PvRON4 carboxy-terminal region specifically interacted with CD71+CD45- reticulocytes. Competition studies with synthetic peptides covering recombinant protein sequences showed that PvAMA1-derived peptide 21270, PvRON4-derived 40305 and PvRON2-RI-derived 40595, were capable of inhibiting recombinant protein binding to CD71+CD45- reticulocytes, suggesting that these peptide sequences contained some of the evaluated proteins’ binding properties. The three peptides bound specifically and with high affinity to erythrocytes having higher (2%) binding percentages (obtained from specific binding curves), thereby allowing their classification as high erythrocyte binding capacity peptides (HABPs). PvAMA1 and PvRON4 binding to human erythrocytes was sensitive to erythrocytes treatment with different enzymes (trypsin, chymotrypsin and/or neuraminidase), suggesting the receptors’ protein type nature. These results highlighted the adhesin function of the proteins evaluated and revealed minimum host cell interaction regions suggesting these molecules’ active participation during P. vivax merozoite invasion of human reticulocytes (along with these proteins’ expression in intra-erythrocytic schizonts and location in apical organelles). Surface plasmon resonance was used for characterising PvRON2 interactions with PvRON4, PvRON5 and PvAMA1. This revealed that PvRON2-RI and carboxy-terminal regions (PvRON2- RII) specifically interacted and with great affinity with PvAMA1 domain II and III (PvAMA-DIIDIII) but with less affinity with PvAMA-DI-DII, PvRON4 and PvRON5. No significant differences were found in interaction association (Kon) or dissociation (Koff) rates or dissociation constant (kD) values when modifying some PvAMA1 residues reported as being critical in the P. falciparum RON2-AMA-1 interaction, suggesting that although conserved interactions between these parasites (Pv-Pf) have been observed, each parasite uses different regions to interact, thereby highlighting their ability to specialise or restrict themselves to invading a specific cell type and the need for designing specific control measures against P. vivax

PvRON2, a new Plasmodium vivax rhoptry neck antigen

<p>Abstract</p> <p>Background</p> <p>Rhoptries are specialized organelles from parasites belonging to the phylum <it>Apicomplexa</it>; they secrete their protein content during invasion of host target cells and are sorted into discrete subcompartments within rhoptry neck or bulb. This distribution is associated with these proteins' role in tight junction (TJ) and parasitophorous vacuole (PV) formation, respectively.</p> <p>Methods</p> <p><it>Plasmodium falciparum </it>RON2 amino acid sequence was used as bait for screening the codifying gene for the homologous protein in the <it>Plasmodium vivax </it>genome. Gene synteny, as well as identity and similarity values, were determined for <it>ron2 </it>and its flanking genes among <it>P. falciparum</it>, <it>P. vivax </it>and other malarial parasite genomes available at PlasmoDB and Sanger Institute databases. <it>Pvron2 </it>gene transcription was determined by RT-PCR of cDNA obtained from the <it>P. vivax </it>VCG-1 strain. Protein expression and localization were assessed by Western blot and immunofluorescence using polyclonal anti-<it>Pv</it>RON2 antibodies. Co-localization was confirmed using antibodies directed towards specific microneme and rhoptry neck proteins.</p> <p>Results and discussion</p> <p>The first <it>P. vivax </it>rhoptry neck protein (named here <it>Pv</it>RON2) has been identified in this study. <it>Pv</it>RON2 is a 2,204 residue-long protein encoded by a single 6,615 bp exon containing a hydrophobic signal sequence towards the amino-terminus, a transmembrane domain towards the carboxy-terminus and two coiled coil α-helical motifs; these are characteristic features of several previously described vaccine candidates against malaria. This protein also contains two tandem repeats within the interspecies variable sequence possibly involved in evading a host's immune system. <it>Pv</it>RON2 is expressed in late schizonts and localized in rhoptry necks similar to what has been reported for <it>Pf</it>RON2, which suggests its participation during target cell invasion.</p> <p>Conclusions</p> <p>The identification and partial characterization of the first <it>P. vivax </it>rhoptry neck protein are described in the present study. This protein is homologous to <it>Pf</it>RON2 which has previously been shown to be associated with <it>Pf</it>AMA-1, suggesting a similar role for <it>Pv</it>RON2.</p

PvRON2, a new Plasmodium vivax rhoptry neck antigen

Background: Rhoptries are specialized organelles from parasites belonging to the phylum Apicomplexa; they secrete their protein content during invasion of host target cells and are sorted into discrete subcompartments within rhoptry neck or bulb. This distribution is associated with these proteins’ role in tight junction (TJ) and parasitophorous vacuole (PV) formation, respectively. Methods: Plasmodium falciparum RON2 amino acid sequence was used as bait for screening the codifying gene for the homologous protein in the Plasmodium vivax genome. Gene synteny, as well as identity and similarity values, were determined for ron2 and its flanking genes among P. falciparum, P. vivax and other malarial parasite genomes available at PlasmoDB and Sanger Institute databases. Pvron2 gene transcription was determined by RT-PCR of cDNA obtained from the P. vivax VCG-1 strain. Protein expression and localization were assessed by Western blot and immunofluorescence using polyclonal anti-PvRON2 antibodies. Co-localization was confirmed using antibodies directed towards specific microneme and rhoptry neck proteins. Results and discussion: The first P. vivax rhoptry neck protein (named here PvRON2) has been identified in this study. PvRON2 is a 2,204 residue-long protein encoded by a single 6,615 bp exon containing a hydrophobic signal sequence towards the amino-terminus, a transmembrane domain towards the carboxy-terminus and two coiled coil a-helical motifs; these are characteristic features of several previously described vaccine candidates against malaria. This protein also contains two tandem repeats within the interspecies variable sequence possibly involved in evading a host’s immune system. PvRON2 is expressed in late schizonts and localized in rhoptry necks similar to what has been reported for PfRON2, which suggests its participation during target cell invasion. Conclusions: The identification and partial characterization of the first P. vivax rhoptry neck protein are described in the present study. This protein is homologous to PfRON2 which has previously been shown to be associated with PfAMA-1, suggesting a similar role for PvRON2

Trabajo de Grado Maestría

La búsqueda de nuevas alternativas terapéuticas contra el cáncer es un área en progreso, dado que los tratamientos convencionales presentan efectos colaterales indeseables y además las células tumorales desarrollan resistencia frente a la terapia convencional. En esta área, los compuestos derivados de productos naturales han jugado un papel importante a lo largo de los arios y siguen siendo una buena alternativa en la búsqueda de nuevos medicamentos que puedan ser empleados directamente en la terapia, o que sirvan corno sensibilizantes de las drogas ya existentes. En este trabajo se usaron las fracciones provenientes de las plantas Caesalpinia spinosa (P2Et) y Petiveria alliacea (FAST-8) como tratamiento directo o sensibilizadores de la terapia convencional. Con estas fracciones se evalúo en líneas celulares leucémicas y de cáncer de seno humano y de ratón, la actividad citotóxica, la modulación de proteínas relacionadas con la resistencia o susceptibilidad al tratamiento corno la HSP70 y la vía de muerte activada. Adicionalmente, la influencia de estas fracciones en la modulación de las concentraciones de los fármacos antitumorales convencionales con diferente blanco molecular fue evaluada. Los resultados obtenidos permiten concluir que las fracciones P2Et y FAST-8 tienen citotoxicidad sobre las células tumorales, modulan la HSP70 e inducen muerte por mecanismos que son dependientes del tipo celular. Con este trabajo además pudimos observar que fracciones complejas obtenidas de plantas pueden ser usadas en combinación con fármacos convencionales pueden promover la actividad de la droga o algunas veces, aumentar la resistencia de las células tumorales. La sensibilización al tratamiento alopático, mediado por bajas concentraciones de las fracciones podría ser utilizada en un futuro para diminuir la toxicidad de las terapias antitumorales, a condición que se estudien los mecanismos moleculares implicados

Estudio de interacciones hospedero-patógeno y proteína-proteína en Plasmodium Vivax : evaluación de las proteínas del cuello de optrias -2, -4 y -5 y del antígeno apical de membrana-1

La malaria es una de las enfermedades tropicales transmitidas por vectores más importantes a nivel mundial, donde Plasmodium vivax representa una de las especies más ampliamente distribuidas, afectando ~13.8 millones de personas por año. Pese a ello, el progreso aparentemente lento de la infección y los niveles bajos de parasitemia en el humano, comparados a lo reportado en Plasmodium falciparum, han llevado a clasificar a la infección por P. vivax erróneamente como benigna. Esto, sumado a los retos experimentales que trae consigo el cultivo de este parásito, obstaculizan en gran medida el conocimiento a nivel biológico, celular y molecular, necesario para el desarrollo de métodos de control efectivos contra P. vivax. Hoy en día, se conoce que un inadecuado diagnóstico, el mal manejo terapéutico y/o el retraso en el tratamiento, pueden llevar a recaídas y estados de enfermedad grave, similares a los reportados para la malaria producida por P. falciparum, lo que impone retos en la búsqueda de nuevas alternativas específicas contra esta especie. Teniendo en cuenta la necesidad de identificar posibles blancos terapéuticos contra P. vivax, este trabajo se enfocó en estudiar interacciones del tipo receptor-ligando y proteína-proteína de moléculas de P. vivax localizadas en los organelos apicales de esquizontes intraeritrocíticos. Basados en estudios previos en P. falciparum y Toxoplasma gondii, donde se describe la importancia funcional de las proteínas localizadas en el cuello de las roptrias (RONs) -2, -4 y 5 y del antígeno apical de membrana 1 (AMA1), se caracterizó en P. vivax la unión de cada una de estas proteínas con reticulocitos humanos y se evaluó la capacidad de PvRON2 para establecer interacciones con las proteínas PvRON4, PvRON5 y PvAMA1. Para esto, inicialmente se caracterizó mediante herramientas bioinformáticas y experimentales la presencia de los genes pvron4 y pvron5 en el genoma y transcriptoma de esquizontes de la cepa Vivax Colombia Guaviare 1 (VCG-1) de P. vivax. Estos dos genes codifican proteínas de alto peso molecular que se expresan en el polo apical de esquizontes y co-localizan con proteínas presentes en las roptrias. Para evaluar la capacidad de interacción de las proteínas PvRON2, PvRON4, PvRON5 y PvAMA1 con receptores sobre la membrana de reticulocitos humanos, todas ellas fueron producidas de forma recombinante y purificadas mediante cromatografía de afinidad. Se encontró que la proteína recombinante PvRON5 se unió tanto a normocitos como a reticulocitos CD71+, con una marcada preferencia por reticulocitos humanos. Por su parte, las proteínas recombinantes que incluyen los dominios I y II de PvAMA-1 (PvAMA-DI-DII), la región central de la proteína PvRON2 (PvRON2-RI) y la región carboxi-terminal de PvRON4, interactúan específicamente con reticulocitos CD71+CD45-. Los estudios de competencia de unión con péptidos sintéticos que cubren las secuencias de las proteínas recombinantes mostraron que los péptidos 21270 (derivado del DI de PvAMA1), 40305 (de PvRON4) y 40595 (de PvRON2-RI) fueron capaces de inhibir la unión de las proteínas recombinantes a reticulocitos CD71+CD45-, lo que sugiere que estas secuencias peptídicas contienen parte de las propiedades de unión de cada una de las proteínas de las que derivan. Los tres péptidos se unen específicamente y con alta afinidad a eritrocitos con porcentajes de unión mayores al 2% (obtenidas de las curvas de unión específica), permitiendo catalogarlos como péptidos con alta capacidad de unión a eritrocitos (HABPs, del inglés High Activity Binding Peptides). La unión de las proteínas PvAMA1 y PvRON4 a eritrocitos humanos fue sensible al tratamiento de los eritrocitos con diferentes enzimas (tripsina, quimotripsina y/o neuraminidasa), sugiriendo que la naturaleza de los receptores para estas proteínas es de tipo proteico. Estos resultados destacan la función de adhesina de las proteínas evaluadas y revelan las regiones mínimas de interacción con la célula hospedera, que sumado a la expresión de estas proteínas en esquizontes intraeritrocíticos y su localización en organelos apicales, sugieren una fuerte participación de estas moléculas durante el proceso de invasión de los merozoitos de P. vivax a reticulocitos humanos. Finalmente, mediante resonancia de plasmones de superficie, se caracterizaron las interacciones entre la proteína PvRON2 con las proteínas PvRON4, PvRON5 y PvAMA1. Los análisis revelaron que la región carboxi-terminal de la proteína PvRON2 (PvRON2-RII) y PvRON2-RI interactúan específicamente y con alta afinidad con el dominio II y III de PvAMA1 (PvAMA-DII-DIII) y con una menor afinidad con las proteínas PvAMA-DI-DII, PvRON4 y PvRON5. Al modificar algunos residuos de la proteína PvAMA1, reportados en P. falciparum como críticos en la interacción RON2-AMA1, no se encontraron diferencias importantes en los valores de las velocidades de asociación (Kon), disociación (Koff) y en la constante de disociación de la interacción (kD). Esto sugiere que, si bien existen interacciones proteína-proteína (IPP) conservadas entre estos parásitos (Pv-Pf), cada parásito utiliza distintas regiones de las proteínas para interactuar, lo que resalta su capacidad para especializarse o restringirse para invadir un tipo de célula específica y pone de manifiesto aún más la necesidad de diseñar medidas de control específicas contra P. vivax.Malaria is one of the most important tropical diseases transmitted by vectors worldwide; Plasmodium vivax represents one of the most widely distributed species (affecting ~ 13.8 million people worldwide per year). Despite this, the apparently slow progress of infection and low parasitaemia levels in humans compared to those reported in Plasmodium falciparum have erroneously led to P. vivax infection being classified as benign. Added to this, the experimental challenges involved in culturing this parasite greatly hinder accumulating the biological, cellular and molecular knowledge necessary for developing effective control methods against P. vivax. It is known nowadays that unsuitable diagnosis, poor therapeutic management and/or delayed treatment can lead to relapses and severe disease states similar to those reported for P. falciparum malaria, thereby imposing challenges regarding the search for new, specific, alternative approaches to tackling this species. The present work has been focused on studying receptor-ligand and protein-protein interactions of P. vivax molecules located in intra-erythrocyte schizonts’ apical organelles regarding the need for identifying therapeutic targets against P. vivax. Protein interaction with human reticulocytes was characterised in P. vivax and PvRON2 ability to establish interactions with PvRON4, PvRON5 and PvAMA1 was evaluated, based on previous P. falciparum and Toxoplasma gondii studies, describing the functional importance of rhoptry neck proteins. Work began by using bioinformatics and experimental tools for predicting pvron4 and pvron5 genes in the P. vivax VCG-1 (Vivax Colombia Guaviare 1) strain’s genome and schizonts transcriptome. These two genes encode high molecular weight proteins which are expressed at schizonts’ apical poles and co-localise with proteins in the rhoptries. Such proteins were produced recombinantly and purified by affinity chromatography for evaluating PvRON2, PvRON4, PvRON5 and PvAMA1 ability to interact with receptors on human reticulocyte membrane. Recombinant PvRON5 bound to both CD71+ normocytes and reticulocytes, having a marked preference for human reticulocytes. PvAMA1 domains I and II (PvAMA-DI-DII), PvRON2 central region (PvRON2-RI) and PvRON4 carboxy-terminal region specifically interacted with CD71+CD45- reticulocytes. Competition studies with synthetic peptides covering recombinant protein sequences showed that PvAMA1-derived peptide 21270, PvRON4-derived 40305 and PvRON2-RI-derived 40595, were capable of inhibiting recombinant protein binding to CD71+CD45- reticulocytes, suggesting that these peptide sequences contained some of the evaluated proteins’ binding properties. The three peptides bound specifically and with high affinity to erythrocytes having higher (2%) binding percentages (obtained from specific binding curves), thereby allowing their classification as high erythrocyte binding capacity peptides (HABPs). PvAMA1 and PvRON4 binding to human erythrocytes was sensitive to erythrocytes treatment with different enzymes (trypsin, chymotrypsin and/or neuraminidase), suggesting the receptors’ protein type nature. These results highlighted the adhesin function of the proteins evaluated and revealed minimum host cell interaction regions suggesting these molecules’ active participation during P. vivax merozoite invasion of human reticulocytes (along with these proteins’ expression in intra-erythrocytic schizonts and location in apical organelles). Surface plasmon resonance was used for characterising PvRON2 interactions with PvRON4, PvRON5 and PvAMA1. This revealed that PvRON2-RI and carboxy-terminal regions (PvRON2-RII) specifically interacted and with great affinity with PvAMA1 domain II and III (PvAMA-DII-DIII) but with less affinity with PvAMA-DI-DII, PvRON4 and PvRON5. No significant differences were found in interaction association (Kon) or dissociation (Koff) rates or dissociation constant (kD) values when modifying some PvAMA1 residues reported as being critical in the P. falciparum RON2-AMA-1 interaction, suggesting that although conserved interactions between these parasites (Pv-Pf) have been observed, each parasite uses different regions to interact, thereby highlighting their ability to specialise or restrict themselves to invading a specific cell type and the need for designing specific control measures against P. vivax.Fundación Instituto de Inmunología de ColombiaColcienciasCentro de Investigación del Cáncer - Universidad de Salamanc

From a basic to a functional approach for developing a blood stage vaccine against Plasmodium vivax

Introduction: Numerous challenges have hampered developing an anti-malarial vaccine against the most widespread malarial parasite worldwide: Plasmodium vivax. Despite the progress achieved in studying proteins in short-term in vitro culture or in experimental models, there is still no clear method for defining which antigens or their regions should be prioritized for including them in a vaccine. Areas covered: The methods used by research groups so far which have focused on the functional analysis of P. vivax blood stage antigens have been reviewed here. A logical strategy orientated toward resolving two of the most commonly occurring problems in designing vaccines against this species has thus been proposed (i.e. the search for candidates and evaluating/ascertaining their functional role in the invasion of such molecules). Expert commentary: Advances in knowledge regarding P. vivax biology have been extremely slow. Only two key receptor–ligand interactions concerning merozoite entry to reticulocytes have been reported during the last 20 years: PvDBP1-DARC and PvRBP2b-CD71. Despite increasing knowledge about the parasite’s intimate preference for its host cells, it has yet to be determined which regions of the merozoite molecules characterized to date meet the requirement of inducing protective immune responses effectively blocking heterologous parasite entry to human cells. © 2020, © 2020 Informa UK Limited, trading as Taylor and Francis Group

The Plasmodium vivax rhoptry neck protein 5 is expressed in the apical pole of Plasmodium vivax VCG-1 strain schizonts and binds to human reticulocytes

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. © 2015 Arévalo-Pinzón et al.; licensee BioMed Central

Annotation and characterization of the Plasmodium vivax rhoptry neck protein 4 (PvRON4)

Background: The tight junction (TJ) is one of the most important structures established during merozoite invasion of host cells and a large amount of proteins stored in Toxoplasma and Plasmodium parasites’ apical organelles are involved in forming the TJ. Plasmodium falciparum and Toxoplasma gondii apical membrane antigen 1 (AMA-1) and rhoptry neck proteins (RONs) are the two main TJ components. It has been shown that RON4 plays an essential role during merozoite and sporozoite invasion to target cells. This study has focused on characterizing a novel Plasmodium vivax rhoptry protein, RON4, which is homologous to PfRON4 and PkRON4.\ud \ud Methods: The ron4 gene was re-annotated in the P. vivax genome using various bioinformatics tools and taking PfRON4 and PkRON4 amino acid sequences as templates. Gene synteny, as well as identity and similarity values between open reading frames (ORFs) belonging to the three species were assessed. The gene transcription of pvron4, and the expression and localization of the encoded protein were also determined in the VCG-1 strain by molecular and immunological studies. Nucleotide and amino acid sequences obtained for pvron4 in VCG-1 were compared to those from strains coming from different geographical areas.\ud \ud Results: PvRON4 is a 733 amino acid long protein, which is encoded by three exons, having similar transcription and translation patterns to those reported for its homologue, PfRON4. Sequencing PvRON4 from the VCG-1 strain and comparing it to P. vivax strains from different geographical locations has shown two conserved regions separated by a low complexity variable region, possibly acting as a “smokescreen”. PvRON4 contains a predicted signal sequence, a coiled-coil α-helical motif, two tandem repeats and six conserved cysteines towards the carboxyterminus and is a soluble protein lacking predicted transmembranal domains or a GPI anchor. Indirect immunofluorescence assays have shown that PvRON4 is expressed at the apical end of schizonts and co-localizes at the rhoptry neck with PvRON2