Identification, characterization and antigenicity of the Plasmodium vivax rhoptry neck protein 1 (Pv RON1)
© Moreno-Perez et al; licensee BioMed Central Ltd. 2011
Received: 14 July 2011
Accepted: 24 October 2011
Published: 24 October 2011
Plasmodium vivax malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as Pf RONs, Pf 92, Pf 38, Pf 12 and Pf 34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the P. vivax rhoptry neck protein 1 (Pv RON1) and examine its antigenicity in natural P. vivax infections.
The Pv RON1 encoding gene, which is homologous to that encoding the P. falciparum apical sushi protein (ASP) according to the plasmoDB database, was selected as our study target. The pvron1 gene transcription was evaluated by RT-PCR using RNA obtained from the P. vivax VCG-1 strain. Two peptides derived from the deduced P. vivax Sal-I Pv RON1 sequence were synthesized and inoculated in rabbits for obtaining anti-Pv RON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs) by Western blot, and its localization by immunofluorescence assays. The antigenicity of the Pv RON1 protein was assessed using human sera from individuals previously exposed to P. vivax malaria by ELISA.
In the P. vivax VCG-1 strain, RON1 is a 764 amino acid-long protein. In silico analysis has revealed that Pv RON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The Pv RON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with P. vivax.
This study shows the identification and characterization of the P. vivax rhoptry neck protein 1 in the VCG-1 strain. Taking into account that Pv RON1 shares several important characteristics with other Plasmodium antigens that play a functional role during RBC invasion and, as shown here, it is antigenic, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability in the Aotus monkey model are thus recommended.
Malaria remains one of the prevailing health problems worldwide. According to the World Health Organization (WHO) , nearly 225 million people are infected annually; about 785,000 of them die as a direct consequence of this disease, of which 85% are children aged less than five years. Although malaria in humans is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi, the first two species represent about 90% of the clinical cases reported . P. falciparum is responsible for causing the disease's highest mortality rates while P. vivax represents significant morbidity having socioeconomic implications . In spite of international control strategies and policies having been implemented during the last fifty years, mortality figures are still alarming; therefore, developing an efficient vaccine to combat this imminent threat has become an urgent need.
Invasion of red blood cells (RBC) by Plasmodium parasites involves highly coordinated events which are directed by a set of proteins secreted from the apical organelles (rhoptries and micronemes) . It has been shown that several rhoptry proteins, such as Pf RON2, -4, and the Pf AMA-1 antigen (secreted by micronemes), are involved in tight junction formation between the parasite and its target cell [5–7]; it has also been found that some others (such as Pf RON1, Pf 92, Pf 38, Pf 12 and Pf 34) are associated with detergent-resistant membrane microdomains (DRM) through glycosylphosphatidylinositol (GPI)-anchor sequences , which are considered organizing centers for the assembly of molecules implicated in cell signaling [9, 10]. To date, several of these DRM proteins have been shown to play an active role in host cell interaction and to trigger antibody responses in the host [11–15].
Rhoptry neck protein 1 (RON1), initially described in Toxoplasma gondii (Tg RON1) , has been a particularly interesting protein. It is a highly-conserved antigen amongst Apicomplexa members. Different tgron1 homologous genes have also been found in members of the Plasmodium genus, such as P. falciparum[16, 17]. Pf RON1 is also known as the apical sushi protein (ASP), exhibiting a prominent transcriptional peak towards the end of the intraerythrocyte lifecycle . This protein has 731 amino acids encoded by 4 exons and ~85.46 kDa molecular mass. It has been previously demonstrated that Pf ASP undergoes proteolytic processing, resulting in ~50 kDa and ~30 kDa polypeptides . The protein also contains a signal peptide, a GPI-anchor sequence and a complement control protein (CCP) or sushi-like conserved domain ; this domain typically contains ~60 residues and represents a protein module involved in protein-protein interactions and/or cell adhesion, containing 4 cysteines, proline residues and highly conserved acidic amino acids (aspartic and glutamic acid). Furthermore, it has been recently found that Pf RON1 has 4 high activity binding peptide sequences (HABPs) to erythrocytes (manuscript in preparation), and could thus be important for parasite entry to target cells.
In the search for an effective anti-malarial vaccine several studies have been focused on identifying antigens expressed during the parasite's asexual stage due to the fact that some of these antigens are mainly responsible for invasion of RBC [20, 21]. Several proteins included in developing a promising vaccine against P. falciparum have been functionally characterized to date . However, studies carried out with P. vivax have been limited due to the difficulty in maintaining an in vitro continuous parasite culture. Taking this into account and considering the degree of conservation observed between the genetic content of different Plasmodium species [23, 24], some bioinformatics tools have been used for homology searches of the complete P. vivax genome for antigens already described in P. falciparum. To date, a considerable number of surface [25–28] and rhoptry proteins [22, 29–35] have been experimentally identified and characterized in P. vivax using both its genome  and intraerythrocyte lifecycle transcriptome  as well as an Aotus-adapted strain . These proteins belong to a list of antigens, which are being tested as vaccine candidates in the Aotus monkey model.
This study shows the identification and characterization of the Pv RON1 protein which is homologous to Pf ASP. Different aspects such as Pv RON1 transcription and expression at the end of the intraerythrocyte life cycle, the subcellular localization pattern and the ability to trigger an immune response in patients who have presented active episodes of P. vivax malarial infection have been determined by molecular biology and immunochemistry techniques.
The P. vivax ron1 gene sequence (which is homologous to the Pf ASP encoding gene) was obtained from the plasmoDB database ; likewise, pfasp orthologous genes in other malarial species were searched. Identity and similarity values between P. falciparum - P. vivax and P. vivax - P. knowlesi hypothetical protein products were determined using ClustalW software . The presence of a signal peptide, repeat sequences and a GPI-anchor sequence was determined by SignalP 3.0 , XSTREAM repeat  and GPI-anchor  bioinformatics tools, respectively. Putative domains were assessed using the Interpro database . Antheprot software  was used for determining two peptides throughout the Pv RON1 hypothetical sequence (PlasmoDB accession number: PVX_000945) that could be good B-cell epitopes, taking the highest average hydrophilicity (>4.9), solvent accessibility (>162) and Parker's antigenicity (>37.8) values into account (calculated in a 20 residue sliding window), according to previously established parameters [45–47].
The experimental handling of animals used here was carried out in accordance with Colombian Law 84/1989 and resolution 504/1996. Aotus monkeys kept at FIDIC's primate station (Leticia, Amazon) and New Zealand rabbits provided by the Instituto Nacional de Salud (Bogotá, Colombia) were handled following the guidelines for the care and use of laboratory animals (National Institute of Health, USA) under the constant supervision of a veterinarian. Immunization and bleeding procedures for Aotus monkeys had been previously approved by our institute's ethics committee, and were carried out in agreement with the conditions stipulated by CorpoAmazonia (resolution 00066, September 13th2006). A monkey from the Aotus genus was experimentally infected with the VCG-1 strain (Vivax Colombia Guaviare 1) and subjected to daily monitoring to assess the progress of infection throughout the whole study (up to day 18) using acridine orange staining. The monkey was treated with pediatric doses of chloroquine (10 mg/kg on the first day and 7.5 mg/kg/day until the fifth day) and primaquine (0.25 mg/kg/day from the third to the fifth day) at the end of the study to guarantee total blood parasite clearance. Once experiments were over, CorpoAmazonia officers supervised the primate's return to its natural habitat in excellent health conditions.
Isolating the P. vivax parasite
The VCG-1 strain was obtained according to a previously described methodology . Mature forms of the parasite, mainly schizonts, were purified from a blood sample (3 mL) infected with P. vivax using a Percoll discontinuous gradient (GE Healthcare, Uppsala, Sweden), according to a previously established protocol . This schizont-enriched sample was then used for performing all of the following procedures: RNA, genomic DNA or parasite total protein extraction, as well as indirect immunofluorescence assays.
RNA extraction and cDNA synthesis
Total RNA from the schizont-enriched sample was extracted using the Trizol method and treated with RNase-free RQ1 DNase (Promega, Wisconsin, USA), according to the manufacturer's instructions. 5 μL RNA were used to synthesize cDNA by RT-PCR, using the SuperScript III enzyme (Invitrogen, California, USA). Complementary DNA synthesis was carried out in the following conditions: 65°C for 5 mins, 50°C for 1 hour and 70°C for 15 mins. After a 15-min incubation period with RNase (Promega, USA) at 37°C, the product was stored at -20°C until its use.
Cloning and sequencing
Three specific primer sets were designed to cover the whole gene sequence and produce two smaller-sized recombinant fragments (Forward 5'-ACAGAAGAGAAGAGAACAGA-3', Reverse 5'-GTTCACACATGCGGTCAC-3'; Forward 5'- ATGGCGAAGGAGCCCAAGTG -3', Reverse 5'- ATCCCTAGCAATGCTTCG -3'; Forward 5'-ATGCTGCTAGTGCCACCCG-3', Reverse 5'- CTGAACACCATCGAAATCG -3'). After PCR amplification using the Platinum Pfx DNA polymerase enzyme (Invitrogen, California, USA), each product was purified by Wizard PCR preps kit (Promega), ligated to the pEXP5 CT/TOPO vector (Invitrogen) and cloned in Escherichia coli TOP10 bacteria (Invitrogen), following the manufacturer's instructions. Recombinant DNA was purified using an UltraClean mini plasmid prep purification kit (MO BIO laboratories, California, USA) and each sequence's integrity was confirmed by sequencing two clones obtained from independent PCRs, using the automated genetic analyzer ABI PRISM 310 (PE Applied Biosystems, California, USA).
Peptide synthesis and polyclonal antibody production
Two 20-amino-acid-long peptides designed on the Pv RON1 deduced sequence (Sal-1 strain) (CG-KHKEGGAAKRHKKEPHEQRG-GC and CG-EDASVADKDGQPGERGQDGQ-GC) were synthesized according to a previously established methodology , polymerized, lyophilized and characterized by RP-HPLC and MALDI-TOF MS. Afterwards, a 150 μg dose of a mixture from both synthetic peptides, emulsified in Freund's complete adjuvant (FCA) (Sigma, Missouri, USA), was inoculated into New Zealand rabbits on day 0, while the same mixture emulsified in Freund's incomplete adjuvant (FIA) was inoculated on days 21 and 42. Sera were collected before the first immunization (pre-immune sera) and 20 days after the last booster dose (post III sera). Rabbit sera were absorbed with E. coli proteins coupled to a Sepharose column and then stored at -20°C until use, according to the manufacturer's instructions (Amersham Biosciences, Buckinghamshire, UK).
Parasite protein extraction
A parasite pellet was treated with 0.2% saponin, then washed six times with PBS and homogenized in lysis buffer (5% SDS, 10 mM PMSF, 10 mM iodoacetamide, 1 mM EDTA). The total lysate was quantified by using a micro BCA protein assay kit (Thermo scientific) and resolved in SDS-PAGE in reducing and non-reducing conditions. Immunoblot detection was carried out using sera from rabbits previously immunized with synthetic peptides.
Isolating detergent-resistant microdomains (DRMs)
Parasite mature forms extracted by the Percoll method were treated with 0.2% saponin in PBS for 5 minutes. After six washes in PBS, the pellet was suspended in a 200 μL TNET solution (1% Triton X-100, 25 mM Tris-HCl, 150 mM NaCl and 1 mM EDTA) which contained protease inhibitors (1 mM PMSF, 1 mM iodoacetamide, 1 mM EDTA and 1 mg/mL leupeptin). The sample was poured into two aliquots which were then treated in the following conditions: a 100 μL aliquot was incubated at 4°C for 30 minutes and subsequently centrifuged at 7,550 rpm for 10 minutes; the remaining sample was kept at 37°C for 30 minutes and then centrifuged in the same conditions described above. The supernatant was recovered in a new Eppendorf tube and the remaining pellet from each assay was suspended in lysis buffer (5% SDS, 10 mM PMSF, 10 mM iodoacetamide, 1 mM EDTA). Each fraction was quantified by Micro BCA protein assay kit (Thermo scientific) and resolved in 12% SDS-PAGE.
Expression and purification of recombinant protein fragments
Once the sequence from cloned inserts had been confirmed, these were transformed in the E. coli BL21-AI strain (Invitrogen), according to the manufacturer's recommendations. In brief, cells were grown overnight at 37°C and then inoculated in Terrific broth medium supplemented with 0.1 mg/mL ampicillin and 0.1% (w/v) D-glucose. Once the cells had reached their stationary phase (0.6 OD600), 0.2% L-arabinose (w/v) was added as inductor and these were incubated again in the same conditions described above for 4 hours. The culture was centrifuged at 13,000 rpm for 30 minutes and the pellet was suspended in extraction buffer (6 M Urea, 12 mM imidazole, 10 mM Tris-Cl, 100 mM NaH2PO4 and 10 mg/mL lysozyme) supplemented with protease inhibitors (1 mM PMSF, 1 mM iodoacetamide, 1 mM EDTA and 1 mg/mL leupeptin) and lysed by sonication. rPv RON1-a and rPv RON1-c recombinant fragment expression was assessed on 12% SDS-PAGE. pEXP5 CT/TOPO vector was used to facilitate purification and recognition by monoclonal anti-histidine antibodies. This vector adds a six-histidine tag towards the C-terminus of each fragment, which is used for purifying the recombinant protein by affinity chromatography using a Ni+2-NTA resin (Qiagen, California, USA). Briefly, the extraction buffer described above was used to elute the non-retained proteins and then the same buffer plus 500 mM imidazole was used to elute the recombinant protein. All individually collected fractions were analyzed by SDS-PAGE and Western blot. Pure fractions were dialyzed in PBS pH 7.0. The protein was ultrafiltered and concentrated using an Amicon filtration system (LabX scientific marketplace, Midland Canada) and quantified with a micro BCA protein assay kit (Thermo Fisher scientific, Pittsburgh, PA, USA).
SDS-PAGE and Western blot
5 μg of each recombinant and 50 μg of both parasite total lysate and the fractions obtained in DRMs were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. These membranes were blocked with 5% skimmed milk in 0.05% PBS-Tween for one hour. After three washes with 0.05% PBS-Tween, each membrane was cut into strips which were individually analyzed as follows: strips with recombinant protein were incubated for 1 hour at room temperature in 5% skimmed milk in 0.05% PBS-Tween, including a 1:100 dilution of rabbit (pre-immune and post-III) or human sera. Strips with parasite total lysate and those containing DRM fractions were incubated for 1 hour at room temperature in 5% skimmed milk in 0.05% PBS-Tween, including a 1:100 dilution of anti-Pv RON1 antibodies. After 3 washes, strips were incubated for 1 hour with goat anti-rabbit IgG coupled to phosphatase as secondary antibody in a 1:4,500 dilution at room temperature, or goat anti-human IgG in a 1:5,000 dilution. Western blot for recombinant proteins was carried out as follows: a nitrocellulose strip was used as positive control; this strip was incubated with a peroxidase-coupled monoclonal anti-histidine antibody diluted 1:4,500 in skimmed milk plus 0.05% PBS-Tween. Blots were revealed with a substrate peroxidase VIP kit (Vector Laboratories, Burlingame, Canada) or BCIP/NBT color development substrate kits (Promega), according to the manufacturer's instructions.
Indirect immunofluorescence assay (IFA)
Blood smears from the Aotus monkey infected with P. vivax were fixed with 4% (v/v) formaldehyde. Slides were washed thrice with PBS and blocked at 37°C for 45 minutes using a 1% (v/v) bovine serum albumin (BSA) solution in PBS. After 3 washes, slides were incubated at 37°C for 1 hour with a mixture of anti-Pv RON1 rabbit sera in 1:30 dilution and anti-Pv RhopH3 mice sera (previously obtained in our institute ) in 1:40 dilution, suspended in PBS -1% BSA - 0.1% Triton X-100 solution. After three washes, plates were incubated with FITC-conjugated anti-rabbit IgG antibody (Sigma) and Red-conjugated anti-mouse IgG antibody (Millipore) in previously described conditions. Slides were stained with DAPI (2 μg/mL) for 20 minutes at room temperature and then observed under an Olympus BX51 fluorescence microscope, using 100× oil immersion objective.
Accession number: the nucleotide and amino acid sequences used here have been reported in the GenBank database, under accession number JN188400.
Results and Discussion
Pvron1 gene identification and localization in a syntenic chromosome region
Pv RON1 characterization in silico
XSTREAM analysis revealed a tandem repeat (TR) between amino acids 148 to 171 (Figure 2A). According to the alignment performed with other homologous proteins in Plasmodium species (P. falciparum, P. knowlesi and P. chabaudi) this region has been shown to be exclusive for P. vivax (Figure 2B). The TR consists of four blocks of eight amino acids having the EKLPIGGG consensus sequence. Antheprot software prediction has shown that this could be a good B-cell lineal epitope. Another two blocks having a similar sequence to that of the TR were found between amino acids 133 to 147; these sequences showed substitutions or deletions of some residues, suggesting that this region might be under selective pressure. It has been previously described that the polymorphic repeat sequences found in some P. falciparum malarial antigens, such as the circumsporozoite protein (CSP), the ring-infected erythrocyte surface antigen (RESA) and the S-antigen, can affect antibody affinity maturation and mask critical epitopes involved in invasion from being recognized [51–54]; the present analysis has thus led us to suggesting that the TR regions found in Pv RON1 could be part of a mechanism used by the parasite to evade the host's humoral immune response. However, further experimental evidence is needed to confirm this hypothesis.
The pvron1 gene is transcribed during the blood stage
The presence of ron1 gene transcripts in the P. vivax VCG-1 strain was assessed by PCR, using cDNA as template. Figure 2C shows the amplification products obtained from RT-PCR and genomic DNA, indicating that the pvron1 gene is transcribed in schizont-enriched samples. These results agreed with previously reported studies showing a peak transcription from 35 to 40 hours of the P. vivax Sal-1 strain intraerythrocyte cycle  and between 35 and 48 hours in the P. falciparum 3D7 strain . Once genomic and complementary DNA sequences were aligned, it was found that the pvron1 gene was encoded by four exons, similar to the homologous pfasp gene , the fourth of them containing a sushi domain. Two nucleotide substitutions and a thirty-two base pair deletion corresponding to a repeat block (EKLPIGGG) were observed when the pvron1 gene sequences from Sal-1 reference strain and VCG-1 Aotus- adapted strain were compared. These mutations were non-synonymous and produced changes in the following amino acids: glutamic acid for glycine in position 148 and isoleucine for threonine in position 740. Furthermore, 17 Cys residues were shown to be conserved between these two strains. When comparing these residues from the Pv RON1 sequence with its P. falciparum homologue (Pf ASP), these presented an identical localization, suggesting that there was probably a degree of conservation in the 3D structure of such molecules.
Pv RON1 expression and association with DRMs
To evaluate Pv RON1 presence in DRMs, a parasite lysate was solubilized at different temperatures and used for rabbit sera recognition assays. Consistent with the results that recognized the protein in the parasite lysate (Figure 3B; Line 2), polyclonal antibodies detected bands at ~100 kDa, ~57 kDa and ~42 kDa in the pellet soluble fraction at 4°C, and in the treatment soluble fraction at 37°C (Figure 3B; lines 8 and 10); these bands disappeared when the pellet was treated with the non-ionic detergent at 37°C (Figure 3B; line 12). Interestingly, sera revealed bands in the soluble fraction at 4°C (Figure 3B; line 6), which can be attributed to the high protein expression levels during schizont stage. Together, these results led to confirming that PvR ON1 antigen was associated with DRMs, differing from what has been reported in the DRM proteome study .
Localization in rhoptries
Pv RON1 antigenicity
This study has described the identification and characterization of a DRM-associated rhoptry protein. Taking into account the multiple particularities of Pv RON1 identified here (i.e. the gene's transcription, the protein's expression towards the end of the intraerythrocyte lifecycle and its localization and the broad recognition presented by sera from individuals infected with P. vivax), it can be suggested that this protein is a good vaccine candidate. The presence of secretory and GPI-anchor signals and a domain probably involved in invasion also suggested that the Pv RON1 antigen could play a functional role during RBC invasion; further immunogenicity and protection-inducing ability studies are thus needed in the Aotus experimental model to confirm the value of this protein or its products, as components of a multi-epitope, subunit-based vaccine against P. vivax malaria.
We would like to thank Rafael Areiza and Nohora Guayacan for their technical support and Jason Garry for translating and reviewing this manuscript.
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