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. 2011 Mar 14:10:60.
doi: 10.1186/1475-2875-10-60.

PvRON2, a new Plasmodium vivax rhoptry neck antigen

Gabriela Arévalo-Pinzón  1 , Hernando Curtidor, Liliana C Patiño, Manuel A Patarroyo
Affiliations

PvRON2, a new Plasmodium vivax rhoptry neck antigen

Gabriela Arévalo-Pinzón et al. Malar J. .

Abstract

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 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.

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.

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Figures

Figure 1
Figure 1
Schematic representation of ron2 chromosomal localization (grey) and adjacent ORFs (white) in Plasmodium falciparum, vivax, knowlesi, chabaudi, berghei and yoelii. The accession numbers for each ORF in PlasmoDB are shown. The length of the represented chromosomal fragment and its localization within the genome in each species is shown on the right. Identity and similarity values at the amino acid level between P. falciparum and P. vivax, as well as between P. vivax and P. knowlesi are pointed out.
Figure 2
Figure 2
cDNA amplification and PvRON2 schematic representation. A. PCR amplification from pvron2 gene RT-PCR product, with three sets of primers as described in the Materials and Methods section. Lane 1. pvron2 region I (~2,176 bp). Lane 2. pvron2 region II (~2,580 bp). Lane 3. pvron2 region III (~2,061 bp). Lane 4. molecular weight pattern. Lane 5. PvAMA-1 ectodomain amplification (positive control). Lane 6. Negative control. B. PvRON2 protein representation. The signal peptide is shown in blue, the transmembrane domain (TMD) in dark grey, coiled-coil motifs in light grey and red lines indicate conserved cysteines between Pf, Pv, Pk, Pc, Pb and Py. * represents polymorphic sites between Sal-1 (reference) and VCG-1 strains. The localization and sequence of inoculated peptides is marked.
Figure 3
Figure 3
PvRON2 expression and apical localization. A. Anti-PvRON2 rabbit polyclonal antibodies detected two bands at around ~220 and ~185 kDa in parasite lysate by Western blot. Lane 1: pre-immune serum. Lane 2: hyper-immune serum. B. P. vivax schizonts incubated with anti-PvRON2 polyclonal antibodies and revealed with FITC-labelled anti-rabbit IgG (green). Parasite nuclei were stained with DAPI (blue). C. Co-localization study: schizonts were simultaneously incubated with anti-PvRON2 and anti-PvAMA-1 (top) or anti-PvRhopH3 (bottom) and detected with FITC-labelled anti-rabbit and with rhodamine-labelled anti-mouse. Arrows indicate the typical dotted pattern displayed by apical organelles.
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