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

Abstract

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.