PfRON3 is an erythrocyte-binding protein and a potential blood-stage vaccine candidate antigen
© Zhao et al.; licensee BioMed Central Ltd. 2014
Received: 16 September 2014
Accepted: 9 December 2014
Published: 12 December 2014
Erythrocyte invasion by merozoites is an essential step in Plasmodium falciparum infection and leads to subsequent disease pathology. Proteins both on the merozoite surface and secreted from the apical organelles (micronemes, rhoptries and dense granules) mediate the invasion of erythrocytes; some of the molecules have been regarded as targets in the development of an anti-malaria vaccine. Recently, a subgroup of rhoptry neck proteins (PfRON2, PfRON4 and PfRON5) associated with the microneme protein apical membrane antigen AMA1 has been described as components of the moving junction complex that assists merozoite invasion into erythrocytes. However, unlike PfRON2, PfRON4 and PfRON5, the latest study suggested that PfRON3 might be located in the rhoptry bulb and participates in a novel PfRON complex (PfRON2, 3 and 4), but does not form a complex with AMA1. Additionally, the full-length PfRON3 protein possesses three transmembrane regions at the N-terminus, which is highly conserved among RON3 orthologues in the genus Plasmodium, Toxoplasma gondii and Eimeria tenella. Overall, these findings suggest that PfRON3 may play an important role in merozoite invasion into erythrocytes.
PfRON3 was primarily expressed during the late trophozoite stage, with a peak in transcription levels at 40 hours post-invasion. The subcellular localization of PfRON3 was confirmed that it is a merozoite rhoptry bulb protein. Additionally, the recombinant form of PfRON3 protein bound to the erythrocyte and was recognized by sera collected from malaria endemic areas in Africa, and anti-PfRON3 antibodies significantly inhibited merozoite invasion into erythrocytes.
The expression of PfRON3 was analysed via real-time quantitative PCR, and the recombinant PfRON3 proteins were generated with an Escherichia coli expression system. The subcellular localization of PfRON3 was assessed with immunoelectron microscopy and immunofluorescence assay (IFA). The recognition PfRON3 by malaria immune sera was analysed with an enzyme-linked immunosorbent assay (ELISA). Erythrocyte-binding assays were performed using recombinant PfRON3 proteins and invasion inhibition assays were carried out with PfRON3-specific antibodies.
This study confirmed that PfRON3 is a rhoptry protein with an erythrocyte-binding property, which is likely associated red blood cell invasion. PfRON3 is a potential vaccine candidate.
Among the malaria parasites that infect humans, Plasmodium falciparum is the most virulent parasite and was responsible for over 600,000 deaths in 2013 worldwide . The pathogenesis of malaria occurs after the invasion of erythrocytes by merozoites and the replication of the obligate asexual intracellular parasites in the host erythrocytes. The erythrocyte invasion process begins with the attachment and proper re-orientation of the merozoite adhered to red blood cell surface via several merozoite surface proteins [2, 3]. Subsequently, an electron dense structure known as moving junction formed between the merozoite apical end and the erythrocyte membrane, involving rhoptry neck proteins and apical membrane antigen 1 (AMA1) . Using the actin-myosin machinery, the merozoite pulls itself through the moving junction and eventually forms a parasitophorous vacuole inside the host erythrocyte . During merozoite invasion into the erythrocyte, several rhoptry neck proteins are released and translocated onto the erythrocyte membrane acting as AMA1 receptors [6–8]. Meanwhile, AMA1 is secreted and inserted into the parasite plasma membrane to form the RON-AMA1 complex. Recently, three of the rhoptry neck proteins (PfRON 2, PfRON4 and 5) have been reported to be involved in the complex formation during invasion and bound to the micronemal protein apical membrane antigen PfAMA1 in P. falciparum[9–11]. In Toxoplasma gondii, four of the rhoptry neck proteins (TgRON2, TgRON4, TgRON5 and TgRON8) were found to associate with the moving junction and bind to TgAMA1 [12–14]. However, the interaction of P. falciparum RON proteins with AMA1 in the context of erythrocyte invasion remains further investigations.
A recent study has indicated that PfRON3 does not form complex with AMA1 but participates in a novel PfRON complex (PfRON2, PfRON3 and PfRON4). Its localization in the rhoptry body has been implicated , but its association with merozoite invasion is unclear. In this study, the expression of PfRON3 and its erythrocyte-binding activity was systematically investigated. It was found that PfRON3 bound to human red blood cell and specific antibodies to PfRON3 inhibited parasite invasion into erythrocytes.
Plasmodium falciparum (strain 3D7) asexual stages were maintained in human O+ erythrocytes with 5% serum and 0.25% AlbuMAXII according to standard procedures . The parasites were synchronized by three rounds of treatment with 5% sorbitol at 4 hours post-invasion and parasites at 8, 16, 24, 32, 40, and 48 hours post-infection were harvested.
Transcription analysis of the PfRON3 gene via real-time quantitative PCR
Real-time quantitative PCR was carried out as previously described . Briefly, RNA corresponding to six time points of post-invasion of the highly synchronized parasites was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Following DNase treatment (TaKaRa, Dalian, China), cDNA was synthesized with AMV reverse transcriptase using an oligo(dT) primer. The following primers were used for quantitative RT-PCR: forward, 5-TTC GCT TCC TTC ATC GGT GC and reverse, 5-TCG TAA AAT TCG GTT GGG GC. Quantitative RT-PCR was performed using an ABI PRISM® 7500 Real-Time PCR System (Applied Biosystems) with SYBR® Premix Ex TaqTM (TaKaRa). The seryl-tRNA synthetase gene (PF3D7_1205100) which is stably expressed during the erythrocytic stage of the parasite was selected as the internal control and used for normalization . Analysis of the transcript levels relative to the average level of the internal control gene was calculated as 2-ΔΔCt(PfRON3 gene). The experiment was repeated three times, and the mean and standard error were determined.
Generation of PfRON3-specific antibodies
To generate specific antibodies to MSP-1-42, the gene fragment was amplified with primer MSP-1-42-F (5′- gaattc CCA CAA CTG AAG ATG GGG GTC AC-3′) and MSP-1-42-R (5′- ctcgag TGT AGA TGA TGT TCC AGT TA-3′). The gene fragment was cloned into pET-32a, and the recombinant protein (His-tagged PfMSP1-42) was expressed and purified as described above. Four female Wister rats respectively received four immunizations with 100 μg of purified His-tagged PfMSP1-42 protein emulsified with Freund’s adjuvant. The anti-PfMSP-1 antibodies were used as controls. Specific IgG fractions of the immunized rat and rabbit sera were affinity-purified with Protein G SepharoseTM 4 Fast Flow (GE Healthcare) and Protein A SepharoseTM 4 Fast Flow (GE Healthcare) according to the manufacturer’s protocols.
Expression analysis of PfRON3 with SDS-PAGE and Western blot assays
Schizont-rich parasites were harvested after 1% saponin treatment. The parasite proteins were then solubilized in SDS-PAGE loading buffer (250 mM Tris, 1.92 M glycine, and 1% SDS), incubated at 98°C for 5 min and subjected to electrophoresis under reducing conditions in a 12% polyacrylamide gel. The proteins were transferred onto 0.2 μm nitrocellulose membranes (Bio-Rad, CA, USA) using a semi-dry blotting system (Bio-Rad, CA, USA). The membranes were blocked with 5% defatted milk (Sigma, St Louis, USA) for one hour and then incubated with anti-PfRON3 IgG (1:1,000 dilution). The membranes were further incubated with horseradish peroxidase conjugated secondary antibody (GE Healthcare) and visualized with Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) using a LAS 4000 mini luminescent image analyzer (GE Healthcare).
Localization of PfRON3 with specific antibodies in immunofluorescence assay (IFA)
Thin smears of schizont stage P. falciparum-infected erythrocytes were fixed with methanol at -80°C for 5 min, washed with PBST (PBS containing 0.1% Triton X-100) at room temperature (RT) for 15 min, and then blocked with PBS containing 5% defatted milk at 37°C for one hour. The smears were then incubated with rabbit anti-PfRON3 antibody (1:50 dilution) and a control rat anti-MSP1-42 antibody at 37°C for one hour, followed by incubation with both Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen) and Alexa Fluor 594-conjugated goat anti-rat IgG (Life) secondary antibody (1:1,000) at 37°C for one hour. The parasite nuclei were stained with DAPI (Roche, Basel, Switzerland) at ambient temperature for 5 min. High-resolution images were captured with a fluorescence microscope (Olympus, BX 53).
Localization of PfRON3 with immunoelectronic microscopy
Immunoelectronic microscopy was carried out as previously described . Briefly, parasites were fixed for 15 min on ice in a mixture of 1% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The fixed specimens were washed, dehydrated and embedded in LR White resin (Fluka, 82882-1EA-F) as described previously [21, 22]. The ultra-thin sections were blocked in PBS containing 5% defatted milk. The grids were then incubated at 4°C overnight with rabbit anti-PfRON3 or control IgG. After washing, the grids were incubated at 37°C for one hour with goat anti-rabbit IgG conjugated to 5 nm gold particles (Sigma) with 1:40 dilutions. The grids were then rinsed with distilled water, dried and stained with uranyl acetate and lead citrate. The samples were examined using a transmission electronic microscope (Hitachi H-7650, Japan).
RBC-binding activity of PfRON3
To investigate the erythrocyte-binding activity of PfRON3, GST-tagged PfRON3-I, II, III and IV (Figure 1), and GST (negative control) were expressed in E. coli BL21  and purified on glutathione - sepharose. Primer pairs for region I are PfRON3-I-F (5′- gga tcc AAT CTC AGA CAA TTA TAT AGA A) and PfRON3-I-R (5′-ctcgag ACT GGA ATA TGG ATT ATT CAA TG). The primers for region II are PfRON3-II-F (5′-ggatcc GAA AGA TAT GGT GTT TTA AAA C) and PfRON3-II-R (5′-ctcgag GGT ACC TTG GTG ATA AGT TTG TT). The primers for region III are PfRON3-III-F (5′-ggatcc TTT ACT AAT TTC TTT TTA AGA AAC T) and PfRON3-III-R (5′-ctcgag TTT TGT TCC ATA GTT TTC TGT ATT T), and the primers for region IV are PfRON3-IV-F (5′-gaattc GAT CAA AGT ACA ACC GCT GATG) and PfRON3-IV-R (5′-ctcgag TGG TCC ACC ATA TGC TTT TTC AC). Next, 200 μl of human erythrocytes were washed three times and resuspended in 200 μl of PBS. Subsequently, 100 μg of recombinant proteins were mixed with the erythrocytes respectively, and incubated at RT for three hours. After incubation, the erythrocytes were washed with PBS three times. Eventually, the proteins that bound to erythrocytes were dissolved on a 10% SDS-PAGE gel and detected via Western blot using anti-GST IgG (Sungene Biotech, 1:5,000 dilution), which was used to characterize GST-tagged proteins.
To test the erythrocyte-binding activity of the native PfRON3, parasites at approximately 44 h post-invasion were harvested by centrifugation at 1500 rpm and washed 3 times with cold PBS. The parasites were lysed by sonication in the presence of a cocktail of protease inhibitors (Sigma). The solution was centrifuged 10 min at 12000 rpm. The supernatant was collected and incubated with 100 μl erythrocytes at 37°C for 1 hour. The cells were washed 3 times with PBS. The protein that bound to erythrocytes was analysed by SDS-PAGE followed by Western-blot using PfRON3 specific antibodies as described above.
Immunorecognition of PfRON3 by human sera
The recognition of His-PfRON3 by human serum samples collected from malaria-endemic regions in Africa [23, 24] was examined. ELISA plates (Nunc, Rochester, NY, USA) were coated with the recombinant protein (5 μg/ml, 50 μl per well) at 4°C overnight. The plates were washed four times with PBS containing 0.05% Tween 20. The coated wells were then blocked with 3% BSA in PBS for one hour at 37°C and subsequently washed four times. The malaria-infected serum samples (n = 10) were added at 1:100, 1:200, 1:400 and 1:800 dilutions and incubated for one hour at 37°C. After washing four times, an alkaline phosphatase-conjugated goat anti-human secondary antibody (Sigma, St Louis, USA) was added at a 1:20,000 dilution and incubated at 37°C for one hour. Finally, a substrate solution containing pNPP [4-Nitrophenyl phosphate disodium salt hexahydrate] (Sigma, St Louis, USA), 9.7% diethanolamine (pH 9.8) and 0.1 M magnesium chloride was added to the wells. A human negative serum from a Chinese individual was used as a negative control and TBST solution was used as a blank control. The cutoff point of OD value for a positive sample was set to be at least two times higher than that of the negative sample at any dilution point. The plates were read using a Biotek 93 micro-ELISA auto-reader 808 at 405 nm. The experiment was repeated three times and the OD405 values were represented by the mean and standard error values of the three experiments.
Invasion inhibition assay with PfRON3 antibodies
The invasion inhibition activity of PfRON-specific antibodies was tested in vitro as previously described . Briefly, PfRON3 specific IgG was purified from rabbit antisera with Protein A-sepharose 4 Fast Flow (GE Healthcare) as described above. Synchronized P. falciparum cultures (ring stage) with 0.3% to 0.5% parasitaemia and 5% haematocrit were incubated with affinity-purified anti-PfRON3 IgG, anti-MSP1-42 IgG and healthy rabbit IgG (negative control). The antibody concentrations were 250, 150, 100, 50, and 10 μg/ml. The inhibitory activity of the antibodies on merozoite invasion was tested over two cycles of parasite replication. Parasitaemia was determined via flow cytometry using a FACS (BD FACS Aria cell sorter, CA, USA) and 50,000 cells were analysed in each experiment. The parasite nuclei were stained with ethidium bromide (EB) and tested in triplicates, the mean and standard errors were determined. The invasion inhibition efficiency of the control rabbit IgG was arbitrarily set as 0%, and the inhibition efficiencies of the PfRON3 specific as well as that of the MSP-1-42 specific antibodies were calculated by comparing the parasitemia in the culture with the control IgG.
Results and discussion
The PfRON3 gene is primarily transcribed at the late developmental stage of P. falciparum
One study early  indicated PfRON3 protein was expressed at late stage of P. falciparum. To determine the transcription of the PfRON3 gene during the blood-stage of P. falciparum 3D7 strain, real-time quantitative PCR analyses were performed by collecting highly synchronized parasite cultures at eight-hour intervals. Transcription of the PfRON3 gene was determined to start at approximately 24 hours and reached a peak at approximately 40 hours post-invasion (Additional file 1: Figure S1). The data further confirmed the earlier reports that the PfRON protein family is predominantly expressed at late schizont stage [10, 26].
The purification of recombinant proteins
The expression and localization of PfRON3
A previous report indicated that PfRON3 may be located in the rhoptry body . In this study, the location of PfRON3 was re-analysed with immunoEM. Parasite sections in late schizont stages were incubated with RON3-specific IgG and subsequently with a secondary antibody labelled with gold particles. Gold particle signals were clearly detected in the bulb portion of the rhoptries (Figure 3C). The data further confirmed that PfRON3 is localized in the rhoptry bulb rather than the rhoptry neck.
RON3 binds to human erythrocytes
PfRON3 was recognized by the antibodies of individuals living in malaria-endemic regions and anti-PfRON3 antibodies inhibited merozoite invasion
The data of this study indicate that the PfRON3 gene was primarily transcribed at the late schizont stage, and the encoded PfRON3 is mainly localized in the rhoptry bulb, which is likely to be released and bind erythrocyte upon the interaction of the anterior end of a merozoite with the surface of an erythrocyte. PfRON3 is recognized by the antibodies of individuals living in malaria endemic areas. Importantly, PfRON3-specifc antibodies inhibited the parasite invasion into erythrocytes, thereby suggesting that PfRON3 could be a potential malaria vaccine candidate.
We appreciate very much the human sera provided by Professors Klavs Berzins and Marita Troye-Blomberg at Stockholm University. This study was financially supported by the National Natural Science Foundation of China to QC (grant number 81130033 and 81420108023) and to NJ (grant number 81171592).
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