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

Abstract

BackgroundDifferent 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.MethodsTaking 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.ResultsThe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0619-1) contains supplementary material, which is available to authorized users.

Receptor-ligand and parasite protein-protein interactions inPlasmodium vivax: Analysing rhoptry neck proteins 2 and 4

“…It cannot be ruled out that proteins must first interact with proteins on host cell membrane for this to occur, as seen in this Pv RON4 study (Figure ). It has been found that one Pv RON5 region and some Pf RON5 20 residue‐long peptides specifically interact with RBC membrane (Arevalo‐Pinzon et al, ; Curtidor et al, ).…”

“…Using P. vivax strains adapted in non‐human primates (Pico de Coana et al, ) or infected patients' blood (Bozdech et al, ) has enabled comparative approaches with other Plasmodium species (Patarroyo, Calderon, & Moreno‐Perez, ) or using omic sciences (Moreno‐Perez, Degano, Ibarrola, Muro, & Patarroyo, ; Venkatesh et al, ) to identify a significant amount of proteins expressed in P. vivax schizonts (Sch) and Mz (Patarroyo et al, ). Pv RON2 (Arevalo‐Pinzon, Curtidor, Patino, & Patarroyo, ), Pv RON4 (Arevalo‐Pinzon, Curtidor, Abril, & Patarroyo, ), and Pv RON5 (Arevalo‐Pinzon, Bermudez, Curtidor, & Patarroyo, ) have been identified recently; these are homologous to those identified in P. falciparum , which are located in the apical extreme of P. vivax Colombia Guaviare 1 (VCG‐1) strain Sch. Different biochemical techniques have been used to report a conserved Pv RON5 fragment located towards the carboxyl‐terminal extreme interacting with RBC, having a preference for CD71 + cells (Arevalo‐Pinzon et al, ), suggesting that RONs could be participating in host–parasite interactions, similar to that found in P. falciparum .…”

“…Recombinant plasmids encoding Pv AMA1 ectodomain, Domains I and II ( Pv AMA1‐DI‐II), Domains II and III ( Pv AMA‐DII‐III), and the C‐terminal fragment of Pv RON5 were taken from previous studies to express such fragments in E. coli (Arevalo‐Pinzon et al, ; Arevalo‐Pinzon et al, ). The pHVDR22 plasmid was used to express the Duffy‐binding Region II on COS‐7 cell membrane as positive control ( Pv DBP‐RII; Chitnis & Miller, ).…”

“…Recombinant proteins r Pv AMA‐1, r Pv AMA‐DI‐II, and r Pv AMA‐DII‐III were obtained in soluble form, following previously described conditions (Arevalo‐Pinzon et al, ); r Pv RON4, r Pv RON5, r Pv RON2‐RI, and r Pv RON2‐RII were expressed and purified according to a previous study (Arevalo‐Pinzon et al, ), with some modifications. Briefly, 4–5 hr after IPTG induction, cell cultures were spun at 10,000× g for 30 min at 4 °C, and the bacterial pellet containing the protein in inclusion bodies was washed twice with buffer A (20‐mM Tris–HCl pH 8.0, 1‐mM EDTA, 1‐mM iodoacetamide, 1‐mM PMSF, and 1‐μg/ml leupeptin) supplemented with lysozyme, followed by cell disruption using a sonicator (Branson); 20‐mM EDTA, 2% Triton X100, and 0.5‐M NaCl were then added.…”

“…PvRON2 (Arevalo-Pinzon, , PvRON4 (Arevalo-Pinzon, Curtidor, Abril, & Patarroyo, 2013), and PvRON5 (Arevalo-Pinzon, Bermudez, have been identified recently; these are homologous to those identified in P. falciparum, which are located in the apical extreme of P. vivax Colombia Guaviare 1 (VCG-1) strain Sch. Different biochemical techniques have been used to report a conserved PvRON5 fragment located towards the carboxyl-terminal extreme interacting with RBC, having a preference for CD71 + cells (Arevalo-Pinzon et al, 2015), suggesting that RONs could be participating in host-parasite interactions, similar to that found in P. falciparum.…”