Molecular & Biochemical Parasitology 142 (2005) 158–169
Identification, expression, localization and serological characterization of a tryptophan-rich antigen from the human malaria parasite Plasmodium vivax夽 Rashmi Jalah a , Ritu Sarin a , Neetu Sud a , Mohammad Tauqeer Alam a , Neha Parikh a , Taposh K. Das b , Yagya D. Sharma a,∗ a
Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India b Department of Anatomy, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India Received 7 June 2004; received in revised form 11 January 2005; accepted 26 January 2005 Available online 26 April 2005
Abstract Plasmodium vivax is most common but non-cultivable human malaria parasite which is poorly characterized at the molecular level. Here, we describe the identification and characterization of a P. vivax Tryptophan-Rich Antigen (PvTRAg) which contains unusually high (8.28%) tryptophan residues and is expressed by all blood stages of the parasite. The pvtrag gene comprises a 978 bp open reading frame interrupted by two introns. The first intron is located in the 5 -untranslated region while the second one is positioned 174 bp downstream to the ATG codon. The encoded ∼40 kDa protein contains a transmembrane domain near the N-terminus followed by a tryptophan-rich domain with significantly high surface probability and antigenic index. It is localized in the parasite cytoplasm as well as in the cytoplasm of the parasitized erythrocyte. The purified E. coli expressed recombinant PvTRAg protein showed a very high seropositivity rate for the presence of antibodies amongst the P. vivax patients, indicating that the antigen generates significant humoral immune response during the natural course of P. vivax infection. Analysis of various field isolates revealed that the tryptophan-rich domain is highly conserved except for three-point mutations. The PvTRAg could be a potential vaccine candidate since similar tryptophan-rich antigens of P. yoelii have shown protection against malaria in murine model. © 2005 Elsevier B.V. All rights reserved. Keywords: Vivax malaria; Tryptophan-rich protein; Humoral immune response; Gene expression; Protein purification; Immunolocalization
Abbreviations: PvTRAg, Plasmodium vivax tryptophan-rich antigen; PypAg, Plasmodium yoelii particulate antigen; CVC, caveola vesicle complex 夽 Note: Nucleotide sequence data reported in this paper are available in the GenBankTM , EMBL and DDBJ databases under the accession numbers Y18842 (1J2 cDNA), AY575008 (isolate Pv594 from Uttar Pradesh), AY575009 (isolate Pv686 from Uttar Pradesh), AY570515 (isolate Pv905 from Uttar Pradesh), AY570516 (isolate 834 from Assam), AY575010 (isolate Pv835 from Assam), AY570517 (isolate Pv836 from Assam), AY576437 (isolate Pv838 from Assam), AY575011 (isolate Pv840 from Assam), AY753149 (isolate Pv841 from Assam), AY570518 (isolate Pv851 from Assam), AY575012 (isolate Pv852 from Assam), AY570519 (isolate Pv861 from Assam), AY575013 (isolate Pv862 from Assam), AY753150 (isolate Pv866 from Assam), AY753166 (isolate Pv1023 from Goa), AY753151 (isolate Pv1024 from Goa), AY753167 (isolate Pv1026 from Goa), AY753152 (isolate Pv1030 from Goa), AY753153 (isolate Pv1031 from Goa), AY753154 (isolate Pv1032 from Goa), AY753155 (isolate Pv1035 from Goa), AY753156 (isolate Pv1036 from Goa), AY753157 (isolate Pv1037 from Goa), AY753158 (isolate Pv1040 from Goa), AY753159 (isolate Pv1042 from Goa), AY753160 (isolate Pv1043 from Goa), AY753161 (isolate Pv1044 from Goa), AY753162 (isolate Pv1049 from Goa), AY753163 (isolate Pv1050 from Goa), AY753168 (isolate Pv1085 from Madhya Pradesh), AY753164 (isolate Pv1086 from Madhya Pradesh), AY753165 (isolate Pv1127 from Delhi). ∗ Corresponding author. Tel.: +91 11 26588145; fax: +91 11 26589286. E-mail address:
[email protected] (Y.D. Sharma). 0166-6851/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2005.01.020
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1. Introduction Plasmodium vivax is the second most common human malaria parasite which accounts for over half of all malaria cases outside Africa with an estimated 75 million acute episodes every year [http://www.malariavaccine.org/ files/vivax-factsheet.pdf]. Although it does not result in a high mortality as P. falciparum, it inflicts debilitating morbidity and consequent economic impact in endemic communities. This parasite has also started showing resistance towards antimalarial drugs, thus necessitating the development of newer drugs and vaccines for effective control of the disease. The development of such newer therapeutics requires identification and characterization of newer parasite target molecules. In this regard, an enormous progress has been made for P. falciparum but the same is not true for P. vivax probably due to its non-cultivable nature [1–3]. Nevertheless, several P. vivax molecules from different stages of the parasite have been characterized [4–13]. Of them, various merozoite surface proteins (e.g., MSP-1, MSP-3, MSP-4, MSP-5 and MSP-9), apical membrane protein-1 (AMA-1), duffy binding protein (DBP), Pvs25 and Pvs28, circumsprozoite protein (CSP), and thrombospondin-related anonymous protein (TRAP) have been studied in greater details and are potential vaccine candidates [6–10,12,13]. Vaccine trials for some of them (e.g., CSP, MSP-1, Pvs25 and Pvs28) have already started and are at their early stages [14–16]. Since malaria vaccine is proposed to be a multivalent vaccine and the above candidates are yet to be proven to provide effective protection against the disease, search for newer and more potential candidate antigens of this parasite should continue. In this regard efforts should be made to characterize those antigens which are associated with the parasite-induced structures. This is because several of the parasite molecules involved in host–parasite interactions, and thus causing the disease pathogenesis and immunity, are localized in them [17–21]. Best example is the P. falciparum induced knobs on the surface of its infected erythrocyte [17,18]. Parasite antigens localized on the knobs are known to bind to endothelial cells thus assist parasitized RBC to sequester in internal organs and escape its destruction by spleen [17–19]. In case of P. vivax, the parasite induces cytoplasmic clefts and caveola–vesicle complexes (CVC) in the infected erythrocyte [20–23]. These structures are involved in transportation of the parasite material from parasite to erythrocyte membrane or to the out side medium [21,22,24]. Indeed, several P. vivax antigens (e.g., 17, 28, 45, 54, 64, 70, 85 and 90 kDa) have been localized in these structures [22,25–28]. Blockade of the transport of such parasite molecules should prove harmful to the parasite. In our continued efforts to study P. vivax molecules involved in host–parasite interaction, we had earlier described cloning and characterization of some of the enzymes and structural proteins of this parasite [29–33]. Here, we describe the cDNA cloning and characterization of a P. vivax Tryptophan-Rich Antigen (PvTRAg) which seems to get as-
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sociated with CVC structures. This may be an important parasite molecule because tryptophan-rich proteins found in several infectious microorganisms play significant role in host cell invasion and efflux of toxic material [34–38]. Such characteristic features of molecules could be useful for the development of therapeutic reagents. PvTRAg may also be exploited for vaccine development since similar tryptophan-rich proteins of P. yoelii have shown protection in murine model and had been proposed as vaccine candidates [39,40].
2. Materials and methods 2.1. Parasites Blood samples were collected from malaria patients living in northern (Delhi, Uttar Pradesh), northeastern (Assam), central (Madhya Pradesh), and western (Goa) states of India. Malaria is prevalent in all these states where it is caused by Plasmodium falciparum or Plasmodium vivax. Latter parasite is prevalent in all these states except northeastern state of Assam where P. falciparum is the predominant species. Sample collection sites are quite far from each other except Delhi and Ghaziabad (Uttar Pradesh). The relative distances of Sonapur (Assam), Jabalpur (Madhya Pradesh), Panjim (Goa), and Ghaziabad (Uttar Pradesh) from Delhi are ∼2400, ∼800, ∼1900 and ∼15 km, respectively. Patients with fever were attending malaria clinics of the primary health care centres at Sonapur (Assam), Ghaziabad (Uttar Pradesh), Panjim (Goa), Jabalpur (Madhya Pradesh) and New Delhi (Delhi). Their thick and thin blood smears were examined for the presence of malaria parasite by light microscopy after Geimsa staining. About 100 to 200 l of heparinized blood was collected from those patients who were found P. vivax positive after their full consent. These individuals were informed about the study. The ethical guidelines of the institute were followed for blood collection. Patients were treated with prescribed dose of chloroquine and primaquine as per National Drug Policy. The P. falciparum positive cases were given chloroquine dose of 10 mg/kg body weight at day 0 and day 1 followed by 5 mg/kg body weight for third day (total adult dose of 1500 mg). They also received 0.75 mg/kg body weight (total adult dose of 45 mg) of primaquine as a single dose. The P. vivax malaria cases were treated with a single dose of chloroquine (10 mg/kg body weight, i.e., 600 mg adult dose) on day 0 followed by primaquine (0.25 mg/kg body weight) daily for 5 days (total adult dose of 75 mg spread over 5 days, i.e., 15 mg per day). 2.2. cDNA cloning and sequencing The previously isolated P. vivax mRNA from patients living in Uttar Pradesh was used to synthesize the cDNA by reverse transcriptase [33]. The cDNA library was constructed in the lambda ZAP II from the Stratagene and following their instructions (Stratagene Inc., La Jolla, CA,
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USA). The library was screened with human cDNA prepared from the mRNA of the peripheral blood mononuclear cells (PBMC). The plaques that were not showing any hybridization (i.e., negative) were randomly picked and purified. The phagemid containing cDNA insert was excised out and used for nucleotide sequencing from both ends with universal primers on the automated DNA sequencer (ABI PRISM 373 A, Perkin Elmer, Applied Biosystems Inc., Foster City, CA, USA). Various subclones of the cDNA were also generated in pGEM3Z for sequencing (Promega Corp., Wisconsin, MD, USA). Sequences thus obtained were aligned together using DNASIS and LasergeneSeqman softwares. Homologies with the online databank sequences were determined using the BLAST and FASTA algorithms available at the http://www.ncbi.nlm.nih.gov and http://www.plasmodb.org websites. Multiple sequence alignments (using ClustalW at http://www.ebi.ac.uk), physicochemical parameter’s prediction (using ProtParam) and transmembrane segment prediction (using TMPred) were performed at the http://us.expasy.ch website.
oil immersion in Nikon Eclipse E600 microscope (Nikon, Tokyo, Japan). 2.5. Indirect immunofluorescence assay The heparinized P. vivax patient’s blood was centrifuged at 700 × g for 10 min at 4 ◦ C. The pellet containing the P. vivax infected RBCs was washed and resuspended in PBS. Thin blood smears were prepared, air dried and fixed with chilled acetone–methanol (1:1) for 20 min at −20 ◦ C. The smears were blocked with 1% BSA in PBS, washed with PBS and then incubated with 1:100 dilution of either pre-immune or antisera raised in rabbits against the 95 kDa E. coli expressed fusion protein. After washing with PBS, the smears were air-dried and incubated with fluorescein isothiocyanate labeled swine anti-rabbit IgG at a 1:30 dilution (Dakopatts, Glostrup, Denmark). Slides were viewed under UV and visible light (100× oil immersion) using Nikon Eclipse E600 microscope. 2.6. Immunoelectronmicroscopy
2.3. Expression and purification of recombinant protein The 978 bp ORF was PCR amplified from the 1193 bp cDNA insert of 1J2 clone by using Platinum Pfx DNA Polymerase (Invitrogen Life Technologies (GIBCO-BRL), Carlsbad, CA, USA) with following primers: forward 5 ATAGCCCATATGGAAGCAGCTAGAG-3 and reverse 5 GCAGCTTTAAGCTTTGCGGCCAAG-3 . The PCR product was purified, cloned into the SmaI site of pGEM3Z, and subsequently subcloned into the NdeI and SalI sites of pTYB12 expression vector (New England Biolabs Inc., Beverly, MA, USA). Expression of fusion protein was induced by adding IPTG to a final concentration of 1 mM. The recombinant protein was purified using chitin bead affinity chromatography as per the manufacturer’s protocol (New England Biolabs Inc.). 2.4. In situ mRNA hybridization This was performed to check the transcription of the pvtrag gene during different blood stages of the parasite, by following the procedure described earlier [41]. Briefly, thin smears from fresh P. vivax infected patient’s blood were prepared on poly-l-lysine coated slides (Sigma Aldrich, San Louis, MO, USA). The slides were fixed and hybridized with fluorescein labeled RNA probe using RNA color kit from Amersham and their protocol (Amersham Biosciences, Buckinghamshire, England, UK). The RNA probe was synthesized using a 345 bp of pvtrag in pGEM3Z (EcoRV subclone). The in vitro transcription from this clone using T7 and SP6 promoter was carried out in the presence of fluorescein 11-UTP to generate labeled sense and antisense probes. Signals were detected using anti-FITC alkaline phosphatase conjugated antibody followed by NBT-BCIP substrate color reaction. The slides were mounted with DPX and viewed under 100×
Heparinized P. vivax patient’s blood was subjected to Ficoll–Hypaque centrifugation to remove peripheral blood mononuclear cells. The remaining cells were passed through a 55% (v/v) Percoll solution in PBS to purify the trophozoiteand schizont-containing erythrocytes. These purified infected erythrocytes were washed once with 0.1 M PBS [pH 7.4] and fixed for 4 h in 0.5% glutaraldehyde and 2% paraformaldehyde (in 0.1 M sodium phosphate buffer [pH 7.2]) at 4 ◦ C. The pellets were dehydrated by passaging through gradually increasing concentrations of ethanol and embedded in LR white resin. Ultra thin sections (60–90 nm) were cut and lifted on to Formvar-coated Nickel grids. For immunolabeling, the sections were blocked with 2% skimmed milk followed by treatment with above polyclonal antibodies (1:100 dilutions) for 18 h at 4 ◦ C. The samples were washed with 0.1 M sodium phosphate buffer, pH 7.2 (containing 0.1% BSA and 0.01% Tween 20) and incubated with 1:100 dilution of goat anti-rabbit gold conjugate for 2 h. The sections were stained in 3% aqueous uranyl acetate, dried and viewed at 80 kV acceleration voltages under a Morgani 268 D transmission electron microscope (FE1, Eindhoven, The Netherlands). 2.7. Enzyme-linked immunosorbant assay ELISA was performed under the same conditions as described earlier [42] using purified recombinant protein and sera from P. vivax—infected and uninfected healthy individuals. Briefly, 50 ng of the purified recombinant PvTRAg protein was coated in each well of a 96-well microtiter plate. Wells were blocked with 5% BSA in PBS and then allowed to react with P. vivax infected patient or normal healthy human serum (1:400 dilution) followed by secondary antibody, i.e., goat anti-human IgG horseraddish peroxidase conjugate
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(Sigma Aldrich, San Louis, MO, USA) at a 1:3000 dilution. oPhenylenediamine hydrochloride (OPD) substrate was used to develop the color. The OD was recorded at 492 nm in a microplate ELISA reader (Molecular Devices Corp., Sunnyvale, CA, USA). The OD of the control wells (i.e., no antigen, no primary antibody, no secondary antibody and no substrate controls) was subtracted from the average OD of each of the respective samples to give the final reading. The average OD plus 3 standard deviations of the 30 normal healthy human serum samples was used as the cutoff value for positive reactivity.
were carried out under following conditions: denaturation at 94 ◦ C, annealing at 60 ◦ C and extension at 72 ◦ C for 1 min each. Final extension was carried out for 20 min at 72 ◦ C. The PCR products were analyzed on 1.5% agarose gel and sequenced using these primers on an automated DNA sequencer (as described above).
2.8. Western blot analysis
A multistagic P. vivax cDNA library in lambda ZAP II vector was constructed and hybridized with human cDNA probe to discard the clones of human origin. The non-binding clones were randomly sequenced from the ends. Two of the cDNA clones were selected for further characterization since they had the same insert size of 1193 bp and also the same nucleotide sequence at the ends. The entire nucleotide sequence of both the clones was also identical to each other. The cDNA sequence has 61% A + T content. Upon comparison of this cDNA sequence with the genomic DNA sequence (available at http://www.tigr.org), the presence of two introns was detected in this gene (Fig. 1a). The first intron (167 bp) is situated in the 5 -untranslated region while the second intron (223 bp) is located 174 bp down stream of the ATG codon and thereby splitting the coding region into two parts (Fig. 1a). The consensus splice site sequence GU/AG exists at the intron–exon junction at both places. The cDNA contains a 978 bp open reading frame which encodes a 326 aa long protein with a calculated molecular mass of 40.2 kDa. The encoded protein is rich in charged amino acids (39.26%) and contains unusually high tryptophan residues (8.28%) but lacks cysteine. It has significantly high-predicted surface probability and antigenic index. A single transmembrane domain exists at its N-terminus (from aa residues 38 to 56) followed by a sequence (from aa residues 87 to 308) containing 27 tryptophan residues distributed over a 221 aa domain (Fig. 1b). Due to the presence of an unusually high number of tryptophan residues, this protein has been named as PvTRAg. Homology searches at the nucleotide level did not yield any significant results. However, at the amino acid level, significant homologies were obtained with a number of proteins from various Plasmodium species (Fig. 1c). Interestingly, all these proteins are also rich in charged amino acids and have the characteristic tryptophan-rich domains. Multiple sequence alignment of PvTRAg revealed that these tryptophan residues are positionally conserved amongst all of them (Fig. 1c). PvTRAg shares 42% identity (65% homology) with hypothetical protein of P. yoelii in a 230 aa overlap, 34% identity (60% homology) in a 235 aa overlap with PypAg-3; 33% identity (60% homology) in a 253 aa overlap identity with PfTryThrA, and 28% identity (52% homology) in a 248 aa overlap with PypAg-1. We have also noticed several ORFs encoding tryptophan-rich proteins in the P. vivax genome available at the database (http://www.plasmodb.org).
Total E. coli lysate or the purified recombinant protein was subjected to 10% SDS–PAGE. The antigens were transferred to nitrocellulose membrane and blocked with 3% BSA. All the serum samples were pre-adsorbed with pTYB12 containing E. coli lysate to remove E. coli antibodies. The cleaned human serum samples were used at a dilution of 1:100. Rabbit anti-human IgG alkaline phosphatase conjugated secondary antibody (Dakopatts) was used at a 1:1500 dilution. The color was developed with nitroblue tetrazolium and 5-bromo-4chloro-3-indolylphosphate (NBT-BCIP) substrate (Promega Corp.). For immunoblotting of induced E. coli cell lysates with cleaned rabbit antisera (1:100 dilution), a similar protocol was followed except that an alkaline phosphatase conjugated goat anti-rabbit secondary antibody was used at a dilution of 1:25,000 (Pierce Chemical Company, Rockford, IL, USA). To determine the size of the PvTRAg in P. vivax, the total parasite extracts were prepared from the Percoll purified infected erythrocytes. The IRBCs were treated with saponin and then centrifuged at 10,000 × g for 10 min at 4 ◦ C. The pellet containing free parasites was washed four times with PBS and sonicated. The sample was resolved on 12.5% SDS–PAGE and the proteins were transferred onto nitrocellulose paper. The filter was reacted with pre-cleaned rabbit anti-PvTRAg or pre-immune sera (1:100 dilutions) followed by horseraddish peroxidase-conjugated anti-rabbit IgG secondary antibodies (Amersham Biosciences, Buckinghamshire, England, UK) at a 1:15,000 dilution. Detection was performed by using the enhanced chemiluminescence system from Amersham as per their instructions. The erythrocytes from uninfected healthy controls were processed similarly except the Percoll gradient. 2.9. Genetic variation The DNA from the P. vivax-infected blood was isolated by using a DNeasy tissue kit from Qiagen and following their instructions (Qiagen, GmbH, Germany). This DNA was subjected to PCR using primers: forward 5 -ACTTCCTAGGAGTCGAATCCGATG-3 and reverse 5 -TTTTTATTTAATGCAGCTTTCGCCT-3 . The parasite DNA was initially denatured at 94 ◦ C for 10 min. Forty cycles of amplification
3. Results 3.1. Isolation and sequence analysis of the pvtrag gene
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Fig. 1. Sequence analysis of the PvTRAg. (a) Genomic organization of the pvtrag gene. The protein coding regions are shown by boxes. Position of the introns is shown by wavy lines while the solid lines represent the untranslated regions in 5 - and 3 -ends. TM, transmembrane domain (dark shaded box); TRD, tryptophan-rich domain (hatched box); INT1, intron 1; INT2, intron 2; CR1, coding region 1; CR2, coding region 2. Scale in bp is indicated. (b) The deduced amino acid sequence of PvTRAg. The positions of amino acids are indicated on the left-hand side. The tryptophan residues are shown in bold. The putative transmembrane domain (38–56 aa) is double underlined. The asterisk indicates stop codon. (c) Multiple sequence alignment of PvTRAg with various tryptophan-rich proteins of other Plasmodium species using ClustalW program (available at http://www.ebi.ac.uk). The positionally conserved tryptophan residues are shown in bold and are shaded grey. Stars (“*”) indicate the identical amino acids while double (“:”) and single (“.”) dots indicate the conserved and semi-conserved substitutions, respectively. Numbers on the right-hand side indicate the amino acid residue number. The sequences have been shown in descending order of their respective homologies to PvTRAg. PvTRAg, P. vivax tryptophan-rich antigen (accession no. Y18842); Py1, P. yoelii hypothetical protein-1 (accession no. AABL01001059); PypAg-3, P. yoelii particulate antigen-3 (accession no. AF250029); PfTryThrA, P. falciparum tryptophan and threonine-rich antigen (accession no. AY027491); PypAg-1, P. yoelii particulate antigen-1 (accession no. AF103869) and Py2, P. yoelii hypothetical protein-2 (accession no. AABL01001059).
3.2. Expression and purification of the recombinant PvTRAg The cDNA of PvTRAg was expressed in E. coli to produce a 95 kDa N-terminal fusion protein with the 55 kDa CBD–intein tag (lane 4 in Fig. 2a). Initially, the expression levels were quite low but addition of 1% glucose to the growth media improved the expression levels. The use of BL21 codon plus (DE3-RP) strain not only resulted in better yield of the
expressed recombinant PvTRAg protein (i.e., around 8–10% of the total cellular protein; lane 4 in Fig. 2a) but also produced around 20% of the protein in the soluble form when induction was carried out at lower temperatures of 18–22 ◦ C (lane 5 in Fig. 2a). The partially soluble protein was purified to an yield of about 2–3 mg/l of induced culture and a purity of ≥90%, in a single chromatographic step (lane 7 in Fig. 2a). The use of non-denaturing concentrations of nonionic detergents (0.2% Triton X-100 and 0.2% Tween 20)
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Fig. 2. Expression and purification of recombinant PvTRAg. (a) The 10% SDS–PAGE analysis was performed to check the expression of the pvtrag derived protein in E. coli vector pTYB12. The total E. coli lysate (lanes 1–4) was loaded from the induced vector (lane 2) and recombinant clone containing ORF of PvTRAg (lane 4). The total E. coli lysate from the uninduced cultures containing vector alone (lane 1) and recombinant clone (lane 3) were also separated. Lanes 5 and 6 contains the soluble fraction and insoluble inclusion bodies respectively of the induced recombinant clone. The purified recombinant PvTRAg (without CBD–intein tag) by chitin bead affinity chromatography was loaded in lane 7. Sizes of protein markers are shown on left hand side. Arrows show the position of fusion protein (∼95 kDa), CBD–intein (∼55 kDa) and the purified recombinant protein without tag (∼40 kDa) (b) Western blot analysis of the total E. coli lysate containing uninduced (lane 1), induced (lane 2) recombinant clone and the purified recombinant PvTRAg without tag (lane 3). The blot was allowed to react with polyclonal rabbit antibodies raised against the CBD–intein–PvTRAg fusion protein. Arrows indicate the protein bands of CBD–intein–PvTRAg (∼ 95 kDa), CBD–intein (∼55 kDa) and PvTRAg (∼40 kDa). All three bands are visible in lane 2 due to the partial cleavage of the fusion protein by DTT present in sample solubilization buffer. Sizes of protein markers are indicated on right hand side.
and high salt concentrations (1 M NaCl) in the wash buffers disrupted the non-specific interactions and hence decreased the host protein contaminants in the purified protein. The identity of the purified PvTRAg was further confirmed by its reactivity to the anti-PvTRAg sera which gave a single band of ∼40 kDa (lane 3 in Fig. 2b). These antibodies reacted to the 95 kDa recombinant CBD–intein–PvTRAg fusion protein in the total induced E. coli lysate along with 55 kDa CBD and ∼40 kDa PvTRAg bands (lane 2 in Fig. 2b), while the uninduced lysate did not show any reaction (lane 1 in Fig. 2b). These extra bands were derived from the fusion protein due to partial cleavage induced by DTT at the intein protein splice site. 3.3. In vivo expression of the pvtrag gene by P. vivax Since we have cloned cDNA for PvTRAg, it was evident that the gene is transcriptionally active in the parasite. The pvtrag transcript was detected in all the blood stages of the parasite by in situ mRNA hybridization (Panel I in Fig. 3a). The sense probe, which was used as a negative control, did not show any color reaction (Panel II in Fig. 3a). That this transcript was translated into protein was confirmed by indirect immunofluorescence assay on the P. vivax-infected blood smears using polyclonal anti-PvTRAg antibodies. All these blood stages of the parasite showed positive reactivity with this antibody in this assay (Panel I in Fig. 3b). The preimmune serum did not show any reaction (Panel II in Fig. 3b). These results thus confirmed that the pvtrag is expressed by
all the erythrocytic stages of the parasite. The fluorescence pattern was similar to PypAg-3 and PypAg-1 of P. yoelii [37,39]. 3.4. Localization of PvTRAg in the parasite The immunoelectron microscopy was performed to localize PvTRAg in the P. vivax-infected erythrocyte (Fig. 4). The gold particles were seen in the parasite as well as in the red cell cytoplasm of the parasitized RBC (Fig. 4a). Immunogold particles were also seen associated with the parasitophorous vacuolar membrane, caveola–vesicle complex (CVC) and membrane of the parasitized erythrocyte (Fig. 4a–d). The labeling was specific since uninfected erythrocytes did not show deposition of gold particles, nor did pre-immune serum show any reaction to the infected or uninfected erythrocytes (data not shown). 3.5. Size of the parasite synthesized PvTRAg To determine the size of the native PvTRAg in P. vivax, the polyclonal anti-PvTRAg antibodies were allowed to react with total P. vivax parasite lysate. A single protein band of ∼40 kDa was recognized by this antibody (Fig. 5). The size of the native PvTRAg in P. vivax matches with that of the calculated as well as the E. coli expressed recombinant protein. This also indicates that there may be no major posttranslational or processing events occurring for the native PvTRAg in the parasite. The RBC proteins did not react with
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Fig. 3. Expression of PvTRAg by blood stages of the parasite. (a) The mRNA in situ hybridization was performed on the P. vivax patient’s blood smears. The 345 bp fragment of the pvtrag gene, cloned in pGEM 3Z vector, was used to synthesize sense and antisense RNA probes for hybridization. Panel I: hybridization with riboprobe derived from antisense strand, Panel II: hybridization with riboprobe derived from the sense strand. R: ring; T: trophozoite; S: schizont. The bar indicates 10 m. (b) The indirect immunofluorescence assay was carried out on thin smears of the P. vivax-infected blood. Smears were allowed to react with the polyclonal rabbit antibody raised against the fusion protein CBD–intein–PvTRAg (Panel I) and the pre-immune rabbit serum (Panel II). The slides were developed with FITC-labeled secondary antibody. Slides were viewed under UV and visible light. R: ring; T: trophozoite; S: schizont. The bar indicates 10 m.
the anti-PvTRAg sera. Furthermore, this antibody is specific and does not recognize any other tryptophan-rich protein of the same parasite. This is similar to P. yoelii and P. falciparum tryptophan-rich proteins where the respective antibody recognized only a single band on Western blot [37–39]. This indicates that antigenic epitopes among the tryptophan-rich proteins of the same parasite are different. 3.6. Conserved sequence of tryptophan-rich domain of PvTRAg among P. vivax isolates We have investigated the positionally conserved nature of tryptophan residues in the tryptophan-rich region of PvTRAg and polymorphism therein, if any, among field isolates. We have analyzed 33 clinical isolates of P. vivax obtained from Assam (north-east), Delhi, Uttar Pradesh (north), Madhya Pradesh (central) and Goa (western) states of India which are far apart from each other except Delhi and Ghaziabad. The
784 bp fragment of the pvtrag gene (codons 64–361) covering the entire region of tryptophan-rich domain was amplified and analyzed. The results showed no length polymorphism among these samples as all isolates yielded same sized PCR product. The nucleotide sequence of all these isolates was also identical to each other except three changes. These changes were observed at codons 80, 186 and 293 of the pvtrag gene (Table 1). Majority of Indian isolates had CTC at codon 80 (28 of 33 isolates), AAC at codon 186 (32 of 33 isolates), and ACC at codon 293 (32 of 33 isolates) coding for Leu, Asn and Thr, respectively. Five isolates (2 from Uttar Pradesh, 2 from Goa and 1 from Madhya Pradesh) had ATC (Ile) at codon 80 while only one isolate (from Madhya Pradesh) had GCC (Ala) at codon 293. Indeed this isolate from Madhya Pradesh had the same sequence as Salvador I strain at all these three codons (Table 1). The nucleotide sequence also revealed that all the tryptophan residues in this domain are highly conserved among field isolates.
Table 1 Sequence analysis of pvtrag gene from clinical isolates of P. vivaxa Isolates
1J2 cDNA Pv594, Pv1023, Pv1026 Pv686, Pv905, Pv834, Pv835, Pv836, Pv838, Pv840, Pv841, Pv851, Pv852, Pv861, Pv862, Pv866, Pv1024, Pv1030, Pv1031, Pv1035, Pv1036, Pv1037, Pv1040, Pv1042, Pv1043, Pv1044, Pv1049, Pv1050, Pv1086, Pv1127 Pv1085, Salvador Ib a
Allele type
Changes in nucleotide and amino acid sequence Codon 80 (amino acid)
Codon 186 (amino acid)
Codon 293 (amino acid)
I II III
ATC (Ile) ATC (Ile) CTC (Leu)
AAA (Lys) AAC (Asn) AAC (Asn)
ACC (Thr) ACC (Thr) ACC (Thr)
IV
ATC (Ile)
AAC (Asn)
GCC (Ala)
The 784 bp fragment of pvtrag gene (from codons 64 to 361) spanning the tryptophan-rich domain was PCR amplified from clinical isolates and sequenced. Only the position of variant codons (amino acid) is shown here. Rest of the sequence was same. Bold letter shows the mutated nucleotide with respect to 1J2cDNA. b Sequence for Salvador I strain is taken from the Plasmodb database (http://www.plasmodb.org).
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Fig. 4. Localization of PvTRAg by immunoelectron microscopy. The P. vivax-infected erythrocytes were fixed in glutaraldehyde and allowed to react with polyclonal anti-PvTRAg antibodies followed by gold-labeled secondary antibody. The gold particles can be seen in the parasite cytoplasm (a). They are also seen to be associated with the parasitophorous vacuolar membrane (a and b) and caveola–vesicle complex (a–d). The antigen is seen associated with the caveola–vesicle complexes present in plasmalemma (b) or those which are getting fused with the erythrocyte membrane (c and d). Caveola–vesicle complex containing gold particles are indicated by arrows. EM: erythrocyte membrane; EC: erythrocyte cytoplasm; PVM: parasitophorous vacuolar membrane; P: parasite; CVC: caveola–vesicle complex. Size of scale bar is indicated.
3.7. Humoral immune response to PvTRAg among P. vivax patients The protein was highly immunogenic as it induced high titer antibodies in rabbits. In order to determine the serologic response in individual patients, the purified recombinant PvTRAg was allowed to react with individual samples of P. vivax-infected and uninfected human sera on ELISA and Western blots. The healthy controls were from lab personnel who lived in malaria endemic area of Delhi but had no malaria episode in the recent past. All the 30 P. vivax patient sera showed reactivity with the recombinant PvTRAg by both the techniques (Fig. 6). Some of the patients were found to contain higher level of antibodies than the others. Also, the reactivity of each serum sample in the ELISA was similar in relative intensity to that of the immunoblot. The results thus indicate the natural immunogenicity and antigenicity of PvTRAg in patients. However, presence of cross-reacting an-
tibodies from recent P. falciparum infections in some of these sera cannot be ruled out at this stage. This is because the P. falciparum molecule PfTryThrA shares 60% homology with PvTRAg and thus may share cross-reacting antigenic epitopes. Further studies are required to dissect out this immune response.
4. Discussion Identification and characterization of the potential malaria vaccine candidate antigen(s) among large number of parasiteencoded proteins is a difficult task. Nevertheless, using various approaches, many potential P. vivax vaccine candidate antigens have been identified and their protective efficacy is being studied [4–16]. Since malaria vaccine is expected to be a multivalent subunit vaccine, the identification and characterization of additional candidate antigens may be required.
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Fig. 5. Size of parasite synthesized PvTRAg. Total lysate of P. vivax-infected (lane 2) and uninfected (lane 1) erythrocytes were separated on SDS–PAGE, transferred to nitrocellulose and reacted with the rabbit polyclonal antibody raised against the recombinant fusion protein CBD–intein–PvTRAg. Arrow indicates the protein band (∼40 kDa) recognized by the antibody in the infected lane. The sizes of protein markers are shown on left hand side.
The P. vivax genome project may yield many more such potential drug targets and vaccine candidates. However, there is a lack of technology to test these candidates for their protective nature. Mice malaria model, although not completely compatible with human malaria, yields valuable information on homologous molecules. In this regard, Burns et al. have successfully used the particulate blood stage antigens of P. yoelii for immunization of mice to study their protective effect against malaria [40]. This protection was found to be B-cell dependent, which was related to the production of IgG antibodies against six to eight membrane-associated P. yoelii antigens. Later, two of these membrane-associated antigens were characterized and they were found to contain higher amount of tryptophan residues [37,39]. The protective epitopes were identified in the tryptophan-rich domain of these antigens [39]. The tryptophan-rich proteins of the malaria parasite were therefore proposed to be the potential vaccine candidate antigens [37,39]. We describe here, for the first time, the identification and characterization of a tryptophan-rich antigen from the human malaria parasite P. vivax (PvTRAg) whose EST we had sequenced earlier (accession number Y18842). This antigen shares homology to the tryptophan-rich proteins of P. falciparum and P. yoelii (Fig. 1) where most of the tryptophan residues were found to be positionally conserved [37,39]. Yet, PvTRAg seems to be different from these antigens not only due to its sequence differences but also because of the different pattern of its gene expression during blood stages of the parasite. For example, the PypAg-1 is expressed by schizonts, PyPAg-3 by trophozoites, PypAg-2 by merozoite and trophozoites, MaTraA of P. falciparum in merozoites [37,39,43],
but PvTRAg is expressed by all blood stages (Fig. 3) like PfTryThrA of P. falciparum [38]. Our immunolocalization data indicates that PvTRAg is transported from the parasite to the red cell membrane (Fig. 4). The PvTRAg may exit from the parasite through PVM and then travel to the red cell membrane through cytoplasm (Fig. 4a). En route it gets associated with the CVC (Fig. 4b). This is because PvTRAg containing CVCs are present in the plasmalemma of infected erythrocyte (Fig. 4b) and also seen fused with the red cell membrane (Fig. 4c and d). The localization of PvTRAg in the fused vesicle of CVC (Fig. 4d) seems to suggest that PvTRAg is being placed at the surface or released to the outside medium from the infected erythrocyte. It may be stated here that PypAg-1 and PypAg-3 of P. yoelii with similar localization are known to interact with normal RBC thereby assisting in the reinvasion process during rosette formation [37]. Association of PvTRAg with CVCs may also suggest another possibility that it may be involved in assisting the transport of parasite material somewhat similar to the tryptophan-rich sensory protein of Rhodobacter sphaeroides [22,34]. The latter is localized in outer membrane of bacteria and implicated in the transport or efflux of the tetrapyrrole intermediates of the heme biosynthetic pathway [34]. Indeed CVCs are known to be involved in the transportation of material from the parasite to the out side medium through red cell cytoplasm [21,22,24]. Previous studies have also localized several P. vivax antigens in them [22,25–28]. The presence of positionally conserved tryptophan residues amongst PvTRAg of various P. vivax isolates (Table 1) and its various homologues (Fig. 1c) may be related to certain common features associated to their structure and function. It is quite likely that the conformation of these proteins is largely constrained by the number and hydrophobic nature of these tryptophan residues since they lack cysteine. Such tryptophan-rich domains have been reported in a variety of transmembrane surface proteins, where they play a role in membrane association [44] and anchorage of the protein in the membrane [45]. They have also been proved to be involved in host membrane binding and fusion reactions for various pathogens, which lead to their infectivity, e.g., the Trp-rich region of HIV transmembrane glycoprotein gp41 [36,46]. Infact various pore forming toxins and antimicrobial peptides have been found to contain tryptophan residues, where they are involved in target membrane binding [35,47–50]. Tryptophan residues have also been shown to play a role in protein–protein interactions and signal transduction [34,51–54]. The high surface probability, homology of PvTRAg to known malarial transmembrane proteins and its immunolocalization patterns indicate that like most tryptophan-rich proteins it may be involved in similar protein and membrane binding interactions. Further studies are in progress to elucidate such functions of PvTRAg. The sequence analysis also predicted a high antigenic index for PvTRAg which was reflected by the high seropositivity rate for the presence of anti-PvTRAg antibodies among
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Fig. 6. Humoral immune response to PvTRAg among P. vivax patients. (a) ELISA was performed for each individual serum by coating the 96-well microtiter ELISA plates with the purified recombinant PvTRAg. Thirty individual sera from P. vivax patients and 30 uninfected healthy controls were used. Each bar represents the OD value for individual serum. Pp: pooled patient sera; Np: pooled sera from healthy controls. (b) Western blot analysis was performed on the purified recombinant PvTRAg, separated on 10% PAGE and electroblotted onto nitrocellulose. The blot was reacted with all the individual 30 P. vivax patient serum samples, pooled sera of these 30 patients (Pp), and the pooled sera from 30 healthy uninfected individuals (Np). The serologic activity of the ∼40 kDa recombinant PvTRAg band is indicated by the arrow.
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the P. vivax patients (Fig. 6). This humoral immune response is similar to the B-cell response generated against the protective particulate blood stage antigens of P. yoelii in mice [39,40]. PvTRAg thus generates B-cell response during the natural course of P. vivax infection. It also does not exhibit antigenic polymorphism which is desirable for a candidate malaria vaccine antigen. In conclusion, we present here the identification along with molecular and immunological characterization of a tryptophan-rich antigen from one of the common human malarial parasite P. vivax. Based on its properties and the protective nature of its counterparts in P. yoelii, PvTRAg can be considered as an appropriate component of the multi-subunit vaccines that are being designed for malaria.
[10]
[11]
[12]
[13]
[14]
Acknowledgements The financial support (to Y.D.S.) came from the Department of Biotechnology (Government of India). Senior Research Fellowships (to R.J. and N.S.), Junior Research Fellowship (to M.T.A.) were from the Council of Scientific and Industrial Research and Senior Research Fellowship (to R.S.) was from University Grants Commission. We thank Dr. H.K. Prasad, Dr. Nutan Nanda, Dr. S.T. Pasha, Mr. D.S. Rawat, Dr. Sukla Biswas for their help and Dr. S.S. Chauhan for critical evaluation of the manuscript. We also thank Dr. M.A. Ansari, Dr. Vas Dev, Dr. Ashwani Kumar and Dr. Neeru Singh for providing us the parasites. We are grateful to the Bioinformatics facilities of BTIS.
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