A Plasmodium yoelii yoelii erythrocyte binding protein that uses Duffy binding-like domain for invasion: a rodent model for studying erythrocyte invasion

A Plasmodium yoelii yoelii erythrocyte binding protein that uses Duffy binding-like domain for invasion: a rodent model for studying erythrocyte invasion
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  Molecular & Biochemical Parasitology 128 (2003) 101–105 Short communication A  Plasmodium yoelii yoelii  erythrocyte binding protein that usesDuffy binding-like domain for invasion: a rodent modelfor studying erythrocyte invasion C. Durga Prasad, Agam Prasad Singh, Chetan E. Chitnis 1 , Amit Sharma ∗  Malaria Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India Received 10 November 2002; received in revised form 20 January 2003; accepted 20 January 2003 Keywords:  Erythrocyte invasion; Erythrocyte binding protein;  Plasmodium yoelii yoelii ; Duffy antigen; Rodent malaria Malaria continues to be a global health problem and isthe cause of tremendous morbidity and mortality. Invasionof erythrocytes by merozoites is one of the key steps inlife cycle of malaria parasites. This invasion process ismediated by specific receptor–ligand interactions betweenparasite-encoded proteins and erythrocyte surface recep-tors [1]. The human malaria parasite  Plasmodium vivax  iscompletely dependent on the Duffy blood group antigen forinvasion [2], while  Plasmodium falciparum  uses both sialicacid-dependent and -independent pathways for erythrocyteinvasion [3–7]. The related simian malaria parasite  Pasmod-ium knowlesi  is able to use the Duffy antigen, sialic acidresidues as well as other as yet unidentified receptors toinvade rhesus erythrocytes [8–10]. A family of erythrocytebinding proteins (EBPs), which includes  P. vivax  and  P.knowlesi  Duffy binding proteins (DBPs), the  P. knowlesi   and    proteins,  P. falciparum  EBA-175 and its homologues,mediate interactions with these receptors during erythrocyteinvasion [11]. Based on sequence homology, the extracel-lular domains of these EBPs have been divided into sixsegments (referred to as regions I–VI). Each of these EBPscontains two conserved cysteine-rich regions (regions II andVI) [12]. Expression of different regions of these EBPs onthe surface of COS cells and subsequent erythrocyte bindingassays (EBAs) revealed that the N-terminal cysteine-rich re-gion (region II, also referred to as Duffy binding-like (DBL)domain) had specific erythrocyte binding activity in each ∗ Corresponding author. Tel.:  + 91-11-26711731;fax:  + 91-11-26711731.  E-mail addresses: (C.E. Chitnis), (A. Sharma). 1 Co-corresponding author. case [13,14]. Members of the EBP family are located in micronemes of invasive merozoites and interact with differ-ent erythrocyte surface receptors to mediate invasion [15].EBPs have been identified from primate malaria parasites,such as  P. falciparum ,  P. vivax ,  Plasmodium reichnowi ,  P.knowlesi  and  Plasmodium cynomolgi  [1,16–19]. Rodentmalaria parasite species provide an accessible model systemto study parasite biology, and have been used extensivelyto study the various stages of the malaria parasite infectionin the mammalian host. In addition, these rodent modelshave been utilized for the identification and evaluation of potential vaccine candidates. So far, EBPs containing DBLdomains have only been identified in primate malaria para-sites [1]. The identification of EBP homologues from rodent malaria parasite species has not yet been reported.Earlier, in vivo erythrocyte invasion studies of   Plasmod-ium yoelii yoelii  using wild-type and Duffy knockout micerevealed that  P. yoelii yoelii  uses the Duffy blood group anti-gen to invade mature erythrocytes, and an unidentified re-ceptor to invade reticulocytes [20]. In this paper, we reportthe identification of a  P. yoelii yoelii  EBP that uses the proto-typical DBL domain to bind mature mouse erythrocytes. Weshow that the  P. yoelii yoelii  EBP is a close homologue of   P.vivax  and  P. knowlesi  DBPs. We further show that region IIof the  P. yoelii yoelii  EBP (PyRII) binds mouse erythrocyteswith specificity. With the identification of an EBP homo-logue in  P. yoelii yoelii , it is now possible to study the roleof EBPs in erythrocyte invasion using the widely accessiblerodent malaria parasite model. It will also enable evaluationof receptor-blocking strategies targeting DBL domains.We searched the PlasmoDB using amino acid sequence of the N-terminal cysteine-rich region (region II, PvRII) of   P.vivax  DBP (PvDBP) as a query to identify a homologue of  0166-6851/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved.doi:10.1016/S0166-6851(03)00040-9  102  C.D. Prasad et al./Molecular & Biochemical Parasitology 128 (2003) 101–105  C.D. Prasad et al./Molecular & Biochemical Parasitology 128 (2003) 101–105  103 DBL domain in  P. yoelii yoelii . This BLASTP search identi-fieda295residueproteindomainin P.yoeliiyoelii .Theiden-tified domain showed ∼ 30% sequence identity with PvRII,along with conservation of the 12 cysteine residues confirm-ing it as a DBL domain. Subsequent to release of the com-plete genome sequence of   P. yoelii yoelii  [21], we searchedthe genomic database using the 295 residue  P. yoelii yoelii DBL domain as a query. This yielded a protein sequence of 786 amino acids (accession number AABL01001466) en-coded by  P. yoelii yoelii . The corresponding nucleotide se-quence is contained in the contig MALPY01471. Analysisof the protein sequence with the program SignalP identifieda signal sequence at the N-terminus of the protein. However,analysis with the program PSORT indicated lack of a trans-membrane region at the C-terminus. Independent searchesof the EST database, using the nucleotide sequence of the786 residue protein, identified a 669bp EST sequence (ac-cession number BM166444). This EST sequence showed a401bp overlap with the DNA sequence encoding the 786residue protein from the contig MALPY01471. Comparisonof the EST and genomic sequences allowed assignment of exon/intronboundaries(Fig.1A).TranslationoftheESTandgenomic sequences allowed us to generate an 839 residueprotein,witha21residueputativetransmembraneregionanda 38 residue putative cytoplasmic domain at the C-terminus(Fig. 1A). The  P. yoelii yoelii  gene consists of five exonsand four introns (Fig. 1A). Exon 1 encodes a 12 residuesignal sequence. Exon 2 encodes a 762 residue protein thatcontains two cysteine-rich regions (Fig. 1A). The transmem-brane region is encoded by exon 3 while the cytoplasmicdomain is encoded by exons 4 and 5. The overall structureand organization of the assembled gene is very similar togenes encoding  P. vivax  and  P. knowlesi  DBPs [12].Comparison of the identified 839 residue  P. yoelii yoelii protein with  P. vivax  and  P. knowlesi  DBPs allowed us todivide the extracellular domain of the  P. yoelii yoelii  pro-  Fig. 1. The overall structure of genes encoding EBPs. (A) The gene and primary protein sequence structure of PyEBP. The identified DNA sequence(3189bp) of PyEBP is present in the contig MALPY01471 (chromosome 1, total contig length  = 4927bp). The exon (Ex) boundaries are 1–36 (Ex1),240–2526 (Ex2), 2694–2772 (Ex3), 2929–3002 (Ex4) and 3146–3189 (Ex5). The five exons are represented by boxes while the introns are shown aslines and are drawn to scale. Within exon 2, regions from I to VI are demarcated. The 5 ′ cysteine-rich domain (region II, also called DBL, in gray)and the 3 ′ cysteine-rich domain (region VI) are shown, along with the signal sequence and the transmembrane regions. (B) CLUSTALW-based multiplesequence alignment of the Duffy binding domains from  Plasmodium yoelii yoelii ,  Plasmodium vivax  and  Plasmodium knowlesi  EBPs. Homology searcheswere performed using BLASTP algorithm at NCBI ( and PlasmoDB ( Multiple alignment of amino acid sequences was done using CLUSTALW. Prediction of the transmembrane domain and the signal sequence was done using the programs PSORT andSignalP accessed at (C) Alignment of the corresponding region VI of   P. yoelii yoelii ,  P. vivax ,  P. knowlesi  EBPs and  P. yoelii yoelii  YM MAEBL. Shading of identical and conserved residues was done using the program BOXSHADE. (D) Transcription of the gene encoding the P. yoelii yoelii  EBP in the blood stages. BALB/c mice were injected intraperitoneally with  P. yoelii yoelii  (MR4-Cat. No. MRA-312) and parasite RNAwas purified using standard protocols. This RNA was used in the subsequent RT-PCR experiments. Lane 1: 1kb plus DNA ladder (Invitrogen); lane2: RT-PCR amplification of the 885bp fragment corresponding to the DBL domain of   P. yoelii yoelii  EBP. Lane 3: negative control for the RT-PCRreaction which is lacking the enzyme reverse transcriptase. No product indicates absence of genomic DNA contamination in the RNA preparation. Lane4: negative control for the PCR reaction without the genomic DNA template; and lane 5: PCR amplification of the 885bp fragment corresponding to theDBL domain of   P. yoelii yoelii  EBP with genomic DNA as a template. The following forward and reverse primers were used for the PCR amplificationof the 885bp fragment: PyDBLf: 5 ′ AACCAGCTGGTTAATGAATGTAAGGAA 3 ′ and PyDBLr: 5 ′ AACGGGCCCAGAACAAACACACAATC 3 ′ . Lane6: molecular weight markers, lane 7: PCR using  P. yoelii yoelii  cDNA library from MR4. Lane 8: PCR with  P. yoelii yoelii  genomic DNA using thesame primers, showing the existence of introns in the PyEBP gene. The following forward and reverse primers were used in these PCR amplifications:PyEx1f: 5 ′ CAGTATTCGCTTATCACATGCA 3 ′ and PyEx2r: 5 ′ CTCCTTCACGTGCTGCATCT 3 ′ . These anneal to exons 1 and 2, respectively. tein into regions I–VI (Fig. 1A). The  P. yoelii yoelii  se-quence shares high sequence homology with the  P. vivax and  P. knowlesi  DBPs in the cysteine-rich regions II and VI(Fig. 1B and C). Overall, the  P. yoelii yoelii  DBL domainshows ∼ 30% sequence identity to region II of   P. vivax  and P. knowlesi  DBPs (Fig. 1B). There are no gaps in the se-quence alignment and all 12 cysteines and 8 tryptophans areconserved. Analysis of   P. yoelii yoelii  region VI indicateshomology to region VI of   P. vivax  and  P. knowlesi  DBPs(Fig. 1C). The  P. yoelii yoelii  region VI also shows homol-ogy to the region VI of   P. yoelii yoelii  MAEBL, a chimericEBP found in rodent malaria parasites [22,23]. Given thepresence of a DBL domain in region II and a conserved re-gion VI, we propose that this 839 residue  P. yoelii yoelii  pro-tein belongs to the EBP family and name it  P. yoelii yoelii EBP (PyEBP). Our analysis suggests the conservation of erythrocyte binding function across phylogenetically distantmalaria species.PCRamplificationusingoligonucleotideprimersbasedonthe PyEBP DBL domain and  P. yoelii yoelii  genomic DNAas template generated a single fragment of 885bp (Fig. 1D).To determine if the PyEBP gene was transcribed in the bloodstages, we extracted RNA from  P. yoelii yoelii  blood stageparasites. The RNA was reverse-transcribed using randomhexamers. The resulting cDNA was used as a template forPCR with primers specific for DBL domain from PyEBP(Fig. 1D). This RT-PCR reaction yielded two fragments of 885 and  ∼ 400bp, respectively (Fig. 1D, lane 2). No PCRproducts were seen when RNA was used as a template inPCR without the addition of reverse transcriptase, indicatingthat there was no genomic DNA contamination (Fig. 1D,lane 3). The 885 and ∼ 400bp DNA fragments were clonedand sequenced. Analysis of the 885bp fragment sequenceshowed an exact match with the DBL domain of PyEBP. The ∼ 400bp fragment neither had any open reading frames of significant length, nor showed homology to DBL sequences.  104  C.D. Prasad et al./Molecular & Biochemical Parasitology 128 (2003) 101–105 Fig. 2. (A) Binding of erythrocytes to transfected COS-1 cells expressing the DBL domain of   Plasmodium yoelii yoelii  EBP. Erythrocyte binding can beseen as rosettes of erythrocytes bound to transfected COS-1 cells. (B) The number of COS-1 cells with rosettes of erythrocytes was counted in 50 fieldsat a magnification of 200. Data for three independent EBA experiments are shown. The transfection efficiency of COS-1 cells varied from 0.5 to 1%, asdetermined by immunofluorescence assays. These RT-PCR experiments indicate that the PyEBP gene istranscribed in the blood stages of the  P. yoelii yoelii  parasitelife cycle. We also performed PCR reactions on genomicDNA and cDNA using specific primers that amplified acrossan intron boundary (Fig. 1D). There is a predicted intronof 0.2kb between exons 1 and 2. Correct splicing yielded aPCR product of 0.56kb using cDNA template and a productof 0.76kb using genomic DNA template (Fig. 1D, lanes7 and 8). These PCR experiments produced the expectedresults and indicated both expression and correct splicing of the PyEBP gene in the blood stage.To test binding to erythrocytes, the DBL domain of PyEBP (henceforth called PyRII) was expressed on thesurface of mammalian COS cells as a fusion with HSV gDand tested for binding to erythrocytes as described before[13,24]. EBAs were conducted with mouse, human, rhesusand rabbit red cells. In these experiments, PyRII bound tomouse erythrocytes but not to rhesus or rabbit erythrocytes(Fig. 2A). Very low levels of binding were observed withhuman erythrocytes (Fig. 2B). These results suggested thatPyRII specifically bound mouse erythrocytes. It has beenshown that  P. yoelii yoelii  uses the Duffy antigen on maturemouse erythrocytes as a receptor for invasion [20]. To testwhether the DBL domain identified in this work was theligand for the Duffy antigen, we tested binding of PyRII tomouse erythrocytes treated with various enzymes, such aschymotrypsin, trypsin and neuraminidase. It has previouslybeen shown that chymotrypsin, but not trypsin, specificallycleaves the mouse Duffy blood group antigen while neu-raminidase selectively removes sialic acid residues fromproteoglycans [20]. Chymotrypsin-treated mouse erythro-cytes did not bind PyRII expressed on the surface of COS-1cells (Fig. 2). Further, both trypsin and neuraminidase treat-ments had little or no effect on binding of PyRII to mouseerythrocytes (Fig. 2A). These results suggest that PyRIIbinds with specificity to the mouse Duffy antigen duringinvasion.The recent completion of the rodent malaria parasitegenome sequencing project [21] assisted us in the rapididentification of a  P. yoelii yoelii -encoded EBP. Our iden-tification of an EBP from  P. yoelii yoelii  provides a ro-dent malaria model system that can be utilized to studyDBL–Duffy interactions involved in erythrocyte invasion.Recombinant malaria vaccines are being developed based  C.D. Prasad et al./Molecular & Biochemical Parasitology 128 (2003) 101–105  105 on the DBL domains of   P. vivax  DBP and  P. falciparum EBA-175 [25–27]. The rodent malaria model can now beused to evaluate whether immune responses elicited againstDBL domains of EBPs can block erythrocyte invasion andthereby provide protection against blood stage infection.In addition, the testing of receptor-blocking peptidomimicsthat block the DBL–Duffy interaction can now be under-taken in a rodent malaria model system. Acknowledgements We thank past and present members of the Malaria Group,ICGEB, for laboratory assistance. We thank Indu Sharmaand Ravi Chandra for help with isolation of parasite mate-rial. We also thank Drs. Gary Cohen and Roselyn Eisenbergfor providing the plasmid pRE4 and the monoclonal anti-body DL6. Dr. C. Chitnis is a Howard Hughes InternationalResearch Scholar. Dr. C. Chitnis and Dr. A. Sharma aresupported by International Wellcome Trust Senior ResearchFellowships. References [1] Chitnis CE. Molecular insights into receptors used by malaria para-sites for erythrocyte invasion. 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