Pv RON2, a new Plasmodium vivax rhoptry neck antigen

Abstract

BackgroundRhoptries 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.MethodsPlasmodium 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 discussionThe 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 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. 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.ConclusionsThe 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.

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

“…Each protein's main characteristics are shown. Signal sequence (dark blue), transmembrane domains (black), coiled‐coil domains (grey), and cysteine residues conserved amongst Plasmodium vivax strains (dotted red lines) (Arevalo‐Pinzon et al, ; Arevalo‐Pinzon, Curtidor, Abril, & Patarroyo, ). The locations are shown of two Pv RON2 fragments ( Pv RON2‐RI and Pv RON2‐RII) and one Pv RON4 fragment, which were cloned in vectors to be expressed in COS‐7 cells and E scherichia coli .…”

“…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 .…”

Characterizing Pv ARP, a novel Plasmodium vivax antigen

“…The sequence was seen to be exclusive for P. vivax and had mutations (two substitutions and four deletions), thereby suggesting that it was under pressure from the immune system. TR have been common in several P. vivax antigens described to date, which are mainly located on the surface or in apical organelles; these would include the circumsporozoite protein (CSP) [33], merozoite surface protein 9 (MSP-9) [34], Pv 34 [35] and rhoptry neck proteins 1 and 2 [23,36]. Even though several studies have shown that the tandem repeats of Pv CSP trigger an immune response when inoculated in primates and humans [33,37,38], the response so produced did not completely inhibit infection caused by the parasite.…”

Annotation and characterization of the Plasmodium vivax rhoptry neck protein 4 (Pv RON4)

“…The anti- Pv RON4 antisera also recognized the protein on VCG-1 late-stage schizonts, having the typical apical organelle punctate pattern (Figure  3C) common to other apical proteins [8,18]. Co-localization studies using antibodies against Pv RON4 and Pv 12 showed that while Pv 12 was expressed on merozoite surface with a bunch of grapes-like pattern, Pv RON4 had a punctate pattern, not overlapping Pv 12 staining (Figure  4).…”

“…Among the various strategies being used to overcome this problem, comparative analysis with other species, such as P. falciparum and adapting P. vivax strains to Aotus monkeys [7], have allowed the identification during the last few years of several antigens in P. vivax which could be participating in invasion [8-11]. …”