Targeted deletion of Plasmodium knowlesi Duffy binding protein confirms its role in junction formation during invasion

Authors

  • Agam P. Singh,

    1. Malaria Research Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India.
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    • Present address: MSB♯ 131, Michael Heidelberger Division of Immunology, Department of Pathology, New York University Medical Centre, New York, NY, 10016, USA.

  • Hastings Ozwara,

    1. Parasitology Department, Biomedical Primate Research Centre (BPRC), Rijswijk, The Netherlands.
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  • Clemens H. M. Kocken,

    1. Parasitology Department, Biomedical Primate Research Centre (BPRC), Rijswijk, The Netherlands.
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  • Sunil K. Puri,

    1. Central Drug Research Institute (CDRI), Lucknow, India.
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  • Alan W. Thomas,

    1. Parasitology Department, Biomedical Primate Research Centre (BPRC), Rijswijk, The Netherlands.
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  • Chetan E. Chitnis

    Corresponding author
    1. Malaria Research Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India.
      E-mail cchitnis@icgeb.res.in; Tel. (+91) 112 618 7695; Fax (+91) 112 618 7695.
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E-mail cchitnis@icgeb.res.in; Tel. (+91) 112 618 7695; Fax (+91) 112 618 7695.

Summary

Red cell invasion by Plasmodium merozoites involves multiple steps such as attachment, apical reorientation, junction formation and entry into a parasitophorous vacuole. These steps are mediated by specific molecular interactions. P. vivax and the simian parasite P. knowlesi require interaction with the Duffy blood group antigen to invade human erythrocytes. P. vivax and P. knowlesi Duffy binding proteins (PvDBP and PkDBP), which bind the Duffy antigen during invasion, share regions of sequence homology and belong to a family of erythrocyte binding proteins (EBPs). By deletion of the gene that encodes PkDBP, we demonstrate that interaction of PkDBP with the Duffy antigen is absolutely necessary for invasion of human erythrocytes by P. knowlesi. Electron microscopy studies reveal that PkDBP knockout parasites are unable to form a junction with human erythrocytes. The interaction of PkDBP with the Duffy antigen is thus necessary for the critical step of junction formation during invasion. These studies provide support for development of intervention strategies that target EBPs to inhibit junction formation and block erythrocyte invasion by malaria parasites.

Introduction

The clinical symptoms of malaria are primarily attributed to the blood-stage of the parasite life cycle, which results from repeated rounds of erythrocyte invasion, parasite multiplication, red cell lysis and release of free merozoites. The ability of Plasmodium merozoites to invade and replicate within human erythrocytes is thus central to malaria pathogenesis. The invasion of erythrocytes by malaria parasites is a complex multi-step process (Ward et al., 1994). Following initial interaction with host erythrocytes, merozoites reorient so that their apical ends, marked by the presence of membrane bound organelles called rhoptries and micronemes, face the erythrocyte membrane (Ward et al., 1994). A critical step following reorientation is the formation of an irreversible junction between the apical end of the merozoite and red cells, which commits the parasite to invasion (Aikawa et al., 1978; Miller et al., 1979). The junction appears in electron micrographs as a thickening of the red cell membrane at its point of contact with the apical end of the merozoite (Aikawa et al., 1978; Miller et al., 1979). As the parasite invades the red cell, the junction moves around the merozoite as a circumferential ring that pinches closed at the posterior end of the merozoite so that the parasite finds itself in a parasitophorous vacuole when invasion is complete (Aikawa et al., 1978; Miller et al., 1979).

Although the morphology of red cell invasion by malaria parasites has been described, the molecular interactions and mechanisms that mediate this complex process are poorly understood. The invasion specificity of different Plasmodium species suggests that red cell invasion is mediated by highly specific molecular interactions between erythrocyte receptors and parasite ligands (Chitnis, 2001). For example, P. vivax requires interaction with the Duffy blood group antigen and only invades Duffy-positive human erythrocytes (Miller et al., 1975; 1976). The related malaria parasite P. knowlesi commonly infects rhesus macaque monkeys under natural conditions but has recently been shown to also cause naturally acquired human infections (Singh et al., 2004). Like P. vivax, P. knowlesi is also dependent on interaction with the Duffy antigen for invasion of human erythrocytes (Miller et al., 1975) but invades rhesus erythrocytes using the rhesus Duffy antigen as well as alternative receptors (Haynes et al., 1988). P. falciparum, the parasite responsible for the bulk of malaria-associated mortality, commonly uses sialic acid residues on glycophorin A as receptors for invasion (Miller et al., 1977; Pasvol et al., 1982; Friedman et al., 1984). However, like P. knowlesi, P. falciparum also uses multiple invasion receptors and can invade erythrocytes by alternative sialic acid-glycophorin A independent pathways (Mitchell et al., 1986; Hadley et al., 1987; Perkins and Holt, 1988; Dolan et al., 1994; Okoyeh et al., 1999). Parasite proteins that mediate interaction with these red cell receptors share similar features and belong to a family of erythrocyte binding proteins (EBPs). The EBP family includes P. vivax and P. knowlesi Duffy binding proteins (PvDBP and PkDBP), P. knowlesiβ and γ proteins, which bind receptors other than Duffy antigen on rhesus erythrocytes, P. falciparum erythrocyte binding antigen EBA-175, which binds sialic acid residues on glycophorin A, and its paralogues, EBA-140 and EBA-181, which bind receptors other than sialic acid-glycophorin A (Camus and Hadley, 1985; Haynes et al., 1988; Wertheimer and Barnwell, 1989; Adams et al., 1992; Mayer et al., 2001; 2002; 2004; Gilberger et al., 2003; Maier et al., 2003). The extracellular regions of each of these EBPs contain two conserved cysteine-rich regions, referred to as regions II and VI (Adams et al., 1992; Chitnis, 2001). The functional receptor-binding domains of EBPs have been mapped to their N-terminal conserved cysteine-rich regions, regions II, which are also referred to as Duffy-binding-like (DBL) domains after the first binding domains identified from PvDBP and PkDBP (Chitnis and Miller, 1994; Sim et al., 1994). Although EBPs that bind red cell receptors during invasion have been identified, the precise functional roles they play in the complex invasion process remain to be determined. Here, we describe a genetic approach to study the function of EBPs in red cell invasion by P. knowlesi.

Using transfection technology we have disrupted the P. knowlesiα gene (Fig. 1), which encodes PkDBP, and have studied the invasion phenotype of the resulting P. knowlesiα gene knockout (PkαKO) parasites. PkαKO parasites are unable to invade human erythrocytes demonstrating the critical role of the PkDBP–Duffy antigen interaction in red cell invasion. Importantly, we also show that PkαKO parasites are unable to form a junction with human erythrocytes and invasion is arrested at this step. The interaction of PkDBP with the Duffy antigen is thus essential for junction formation during invasion. These studies identify the interaction of EBPs with their receptors as valid targets for development of intervention strategies that block erythrocyte invasion and prevent malaria.

Figure 1.

Disruption of P. knowlesiα gene by homologous recombination.
A. Structure of the P. knowlesiα gene. The exon (boxes)–intron (thin lines) structure of the P. knowlesiα gene, which encodes PkDBP, is shown. The extracellular domain of PkDBP is divided into six regions (I to VI) based on sequence homology with other erythrocyte binding proteins. The receptor-binding domain of PkDBP maps to region II. Positions of primers P1, P3 and P4, which were used to confirm deletion of P. knowlesiα gene by PCR, are shown. SS, signal sequence; TM, transmembrane domain; CYT, cytoplasmic domain.
B. Transfection construct used for deletion of P. knowlesiα gene. DNA sequences from the 5′ and 3′ flanking regions of P. knowlesiα gene (5′Pkα and 3′Pkα) were cloned to flank the selection cassette containing the gene encoding pyrimethamine-resistant Tg dhfr-ts in the transfection plasmid b2DDDPKA. Plasmid b2DDDPKA was linearized by digestion with restriction enzymes ClaI and BamHI and used to transfect P. knowlesi H strain by electroporation. Integration of the Tg dhfr-ts cassette by homologous recombination results in deletion of the P. knowlesiα gene. Positions of primers P1, P2, P3 and P4 used for PCR to confirm integration of the Tg dhfr-ts casette and loss of the P. knowlesiα gene are shown. A DNA probe (dotted line) based on sequence of region II of P. knowlesiα gene (PkaRII) was used for Southern blot hybridization. Restriction sites BamHI, B; ClaI, C; EcoRI, E and HindIII, H are shown.

Results

Disruption of the P. knowlesiα gene by homologous recombination

A plasmid construct, b2DDDPKA, containing a mutagenized Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene (Tg dhfr-ts), which confers resistance to pyrimethamine, cloned between 5′ and 3′ flanking sequences of the P. knowlesiα gene, was linearized and used for transfection to disrupt the P. knowlesiα gene by homologous recombination as previously described (van der Wel et al., 1997; Kocken et al., 2002) (Fig. 1). Pyrimethamine-resistant P. knowlesi clones BA and AE were derived by limiting dilution and analysed for chromosomal integration of the Tg dhfr-ts gene and loss of P. knowlesiα gene by polymerase chain reaction (PCR) amplication and Southern blot hybridization.

Polymerase chain reaction using primers P1 and P2, which are based on the sequence of the 5′ upstream region of the P. knowlesiα gene and Tg dhfr-ts sequence, respectively (Fig. 1), yields a product of the expected size when genomic DNA preparations from clones BA and AE are used as template (Fig. 2, data for AE not shown) confirming integration of Tg dhfr-ts in the P. knowlesi genome at the α gene locus. As expected, PCR using primers P1 and P2 with genomic DNA from wild-type P. knowlesi parasites (PkWT) does not yield any product (Fig. 2). PCR using primers P3 and P4, which are based on the sequence of the P. knowlesiα gene (Fig. 1), yields a product of expected size with genomic DNA from PkWT parasites as template but does not yield any product when genomic DNA from clones BA or AE are used as template confirming deletion of the P. knowlesiα gene in these clones (Fig. 2, data for AE not shown).

Figure 2.

Confirmation of the deletion of P. knowlesiα gene in PkαKO parasites.
A. PCR analysis. Genomic DNA isolated from PkαKO parasite line BA (lanes 1 and 3) and P. knowlesi WT parasites (lanes 2 and 4) was used for PCR either with primers P1 and P2, which are based on 5′ flanking sequence of P. knowlesiα gene and sequence of Tg dhfr-ts, respectively (lanes 1 and 2) or primers P3 and P4, which are based on the P. knowlesiα gene (lanes 3 and 4).
B. Southern blot hybridization. Genomic DNA isolated both from PkWT or cloned PkαKO lines BA and AE was digested with enzymes HaeIII and TaqI and probed with a ∼1.0 kb probe based on region II of P. knowlesiα gene (PkaRII). Arrows indicate the fragments expected from P. knowlesiα gene that hybridize with the probe based on PkαRII. Molecular weight markers (M) are shown in kilobase pairs (Kb).
C. Transcriptional analysis of P. knowlesi α, β and γ genes in PkWT and PkαKO parasites. Transcription of P. knowlesiα, β and γ genes was analysed in PkWT and PkαKO parasites. RNA was isolated from PkWT and PkαKO schizont stage parasites and reverse-transcribed (+RT) using random hexamers. Mock reverse-transcribed RNA (–RT) was used as control. cDNA products were used as templates for PCR using primers specific for P. knowlesiα, β and γ genes. Molecular weight markers (M) are shown in base pairs.
D. Detection of PkDBP in PkWT and PkαKO parasites by immunoprecipitation. Metabolically labelled supernatants (Sup.) of PkWT and PkαKO parasites were used for immunoprecipitation with rabbit sera raised against recombinant P. knowlesiα region II (PkαRII) either before (None) or after pre-absorption with Duffy-positive human erythrocytes (Fy(a+b+)), Duffy-negative human erythrocytes (Fy(ab)) or rhesus erythrocytes (Rh). Rabbit sera raised against PkαRII immunoprecipitate 135 kDa and 138 kDa proteins from culture supernatant of PkWT parasites. The 135 kDa protein is pre-absorbed with Duffy-positive human erythrocytes but not Duffy-negative human erythrocytes identifying it as PkDBP. The 135 kDa PkDBP is not detected in supernatants of PkαKO parasites by immunoprecipitation with sera raised against PkαRII.

Genomic DNA preparations from PkWT parasites and P. knowlesiα gene knockout (PkαKO) clones BA and AE were digested with HaeIII or TaqI, separated by agarose gel electrophoresis and tested for presence of P. knowlesiα gene by Southern blot hybridization using a 32P-labelled DNA probe based on P. knowlesiα region II (PkαRII) (Fig. 2). Digestion of P. knowlesiα gene with HaeIII results in two fragments of 1.13 kb and 0.55 kb that hybridize with the probe. These fragments are not seen with HaeIII digests of genomic DNA from PkαKO clones BA and AE indicating that the P. knowlesiα gene has been deleted in these parasites. Digestion of the P. knowlesiγ gene with HaeIII yields a 1.67 kb DNA fragment that contains sequences from P. knowlesiγ region II (PkγRII) that cross-hybridize with the PkαRII-based probe. The 1.67 kb fragment is observed in Southern blot hybridization experiments with genomic DNA from PkWT as well as PkαKO clones BA and AE confirming that the P. knowlesiγ gene is not disrupted in the knockout parasites. Digestion of P. knowlesiα gene with TaqI results in a fragment of 1.7 kb that hybridizes with the PkαRII-based probe. This fragment is only seen with genomic DNA from PkWT parasites and not with genomic DNA from PkαKO clones BA and AE. Digestion with TaqI also yields fragments of 1.9 kb, 0.33 kb and 0.22 kb, which contain PkγRII sequences that cross-hybridize with the PkαRII-based probe. These fragments are seen in PkWT as well as PkαKO clones BA and AE. PCR and Southern blot hybridization confirm that the Tg dhfr-ts cassette has integrated at the P. knowlesiα gene locus in clones BA and AE resulting in the deletion of the P. knowlesiα gene. The PkαKO clone BA was used for further analysis.

Expression of P. knowlesiα gene in PkWT and PkαKO parasites

Transcription of the P. knowlesiα, β and γ genes was analysed in PkWT and PkαKO parasites. RNA isolated from schizont stage PkWT and PkαKO parasites was reverse-transcribed (RT) using random hexamers and the resulting cDNA was used as template for PCR using primers specific for the P. knowlesiα, β and γ genes. RT-PCR products of expected sizes were observed for P. knowlesiβ and γ genes in both PkWT and PkαKO parasites (Fig. 2). However, RT-PCR with primers based on the P. knowlesiα gene yields a product only with RNA isolated from PkWT parasites (Fig. 2). Transcripts corresponding to the P. knowlesiα gene are not detected in RNA isolated from PkαKO parasites (Fig. 2).

Rabbit serum raised against recombinant PkαRII (Singh et al., 2002), the binding domain of PkDBP, was used to detect the 135 kDa PkDBP in metabolically labelled culture supernatants of PkWT and PkαKO parasites. Immunoprecipitation of PkαRII from PkWT culture supernatants using anti-PkαRII serum yields a doublet of closely migrating proteins with sizes of ∼135 kDa and ∼138 kDa (Fig. 2) as observed previously (Adams et al., 1990). The 135 kDa protein is not detectable if immunoprecipitation is performed after pre-absorption of the PkWT culture supernatant with Duffy-positive human erythrocytes but is detected if pre-absorption is performed with Duffy-negative human erythrocytes that lack the Duffy antigen (Fig. 2). This confirms the identity of the 135 kDa protein as PkDBP. The 135 kDa PkDBP is not detected in supernatants of PkαKO parasites (Fig. 2). The 138 kDa protein, which is also immunoprecipitated by anti-PkαRII sera, is detected in supernatants of both PkWT and PkαKO parasites and is pre-absorbed by rhesus erythrocytes but not by human erythrocytes. The 138 kDa erythrocyte binding protein is probably encoded by one of the P. knowlesiα gene paralogues such as P. knowlesiβ or γ genes, which encode parasite ligands that bind receptors other than Duffy antigen on rhesus erythrocytes (Adams et al., 1990; Ranjan and Chitnis, 1999).

Invasion phenotype of PkαKO parasites

The ability of PkαKO parasites to invade human and rhesus erythrocytes was tested using in vitro invasion assays. PkWT and PkαKO schizonts were purified and incubated with fluorescein isothiocyanate (FITC)-labelled target erythrocytes to allow invasion. Parasites were detected in newly formed rings following invasion by staining with the fluorescent DNA intercalating dye 4′,6-diamidino-2-phenyindole (DAPI). The invasion efficiency was determined by scoring the percentage of FITC-labelled target erythrocytes that contain DAPI-stained parasites (Table 1). Both PkWT and PkαKO parasites invade rhesus erythrocytes as well as chymotrypsin-treated rhesus erythrocytes (Table 1). Deletion of the P. knowlesiα gene has no measurable effect on invasion of rhesus erythrocytes by P. knowlesi. However, deletion of the P. knowlesiα gene has a significant effect on invasion of human erythrocytes. PkαKO parasites are unable to invade Duffy-positive human erythrocytes (Table 1). The presence of a functional P. knowlesiα gene is thus absolutely necessary for invasion of human erythrocytes by P. knowlesi. The ability of PkWT and PkαKO parasites to invade rhesus erythrocytes treated with a variety of other enzymes was also tested (Table 1). Both PkWT and PkαKO parasites invade rhesus erythrocytes treated with neuraminidase, trypsin, proteinase K and combination of neuraminidase and trypsin. Deletion of the P. knowlesiα gene does not affect invasion of rhesus erythrocytes by pathways other than the Duffy receptor pathway.

Table 1.  Invasion of human and rhesus erythrocytes by wild-type P. knowlesi (PkWT) and P. knowlesiα gene knockout (PkαKO) parasites.
ErythrocytesTreatmentaPkWTb Invasion rate
(%) (Avg ± SD)
PkαKoc Invasion rate
(%) (Avg ± SD)
P-valued
  • a

    . Erythrocytes were either used after treatment with chymotrypsin (Chy), trypsin (Try), neuraminidase (Neu) or Proteinase K (Prot K).

  • b

    . Invasion rates (average ± standard deviation) of PkWT parasites into human and enzyme-treated rhesus erythrocytes are expressed as percentage of invasion rate of PkWT parasites into normal rhesus erythrocytes.

  • c

    . Invasion rates (average ± standard deviation) of PkαKO parasites into human and enzyme-treated rhesus erythrocytes are expressed as percentage of invasion rate of PkαKO parasites into normal rhesus erythrocytes.

  • d

    .P-values were calculated using paired, two-tailed t-test method. P-values < 0.05 indicate that differences between paired values are significant whereas P-values > 0.05 indicate that differences between paired values are not significant.

  • e

    . Representative data from one of three similar experiments is presented. Invasion assays was performed in triplicate in each experiment. Invasion rates of PkWT and PkαKO parasites in normal rhesus erythrocytes were 14.48 ± 1.03% and 6.02 ± 0.65% respectively.

  • f

    . Invasion rates represent average (± standard deviation) from two independent experiments. Each invasion assay was performed in triplicate in each experiment. Invasion rates of PkWT and PkαKO parasites into normal rhesus erythrocytes were 15.07 ± 1.96% and 16.90 ± 1.16% respectively.

Experiment 1e
 RhesusNone100100NA
 RhesusChy 87.78 ± 13.26106.98 ± 19.440.3057
 Human Fy(a+b+)None 74.24 ± 7.44  00.0001
 Human Fy(ab)None  0  0NA
Experiment 2f
 RhesusNone100100NA
 RhesusNeu 82.65 ± 9.40 95.89 ± 0.560.1479
 RhesusTry 74.90 ± 4.90 78.00 ± 15.200.6541
 RhesusProt K 73.09 ± 14.17 81.67 ± 8.330.1256
 RhesusNeu + Try 64.15 ± 14.15 68.43 ± 16.780.1061

PkαKO parasites fail to form a junction with Duffy-positive human erythrocytes

Electron microscopy was used to study the interaction of PkαKO parasites with Duffy-positive human erythrocytes to identify the step at which invasion is arrested. Purified PkαKO late-stage schizonts were ruptured by the method of syringe release and free merozoites were isolated by centrifugation (Ward et al., 1993). The released merozoites were treated with cytochalasin B and allowed to interact with rhesus erythrocytes and Duffy-positive human erythrocytes. Cytochalasin treatment blocks polymerization of actin and arrests invasion at the step of junction formation (Miller et al., 1979). Following fixation, sections were examined by electron microscopy. A total of 25 merozoites interacting with rhesus erythrocytes were observed. Of these 19 were in close apposition with rhesus erythrocytes and the formation of a junction between PkαKO merozoites and rhesus erythrocytes was observed (Fig. 3). In the case of PkαKO merozoites interacting with Duffy-positive human erythrocytes, 63 merozoites were examined. Of these, 59 were oriented with their apical end facing the red cell membrane. In each case, the interaction was not close and no junction was visible (Fig. 3). These studies provide direct evidence to demonstrate that the interaction of PkDBP with the Duffy antigen is necessary for the critical step of junction formation during invasion.

Figure 3.

Electron micrographs of PkαKO merozoites interacting with human and rhesus erythrocytes. Cytochalasin B treated PkαKO merozoites were allowed to interact with rhesus erythrocytes (A) and Duffy-positive human erythrocytes (B). The interaction was analysed by electron microscopy. PkαKO merozoites (M) interact closely with rhesus erythrocytes (RBC) and a junction is visible at the point of contact with the erythrocyte (see arrows, A). In case of PkαKO merozoites (M) interacting with Duffy-positive human erythrocytes (RBC), although the PkαKO merozoite is oriented with the apical end facing the red cell membrane, the interaction is not close and a junction does not form (see arrows, B).

Discussion

The invasion of red cells by Plasmodium merozoites requires specific molecular interactions between host receptors and parasite ligands (Chitnis, 2001). The EBP family of Plasmodium binds erythrocyte receptors to mediate red cell invasion. Genetic approaches have been used to analyse the roles of EBPs in invasion. Deletion of EBA-175 and its paralogues has allowed the analysis of invasion receptors and pathways used by P. falciparum for red cell invasion (Kaneko et al., 2000; Reed et al., 2000; Duraisingh et al., 2003; Gilberger et al., 2003; Maier et al., 2003). However, the precise functional roles of EBPs in the multi-step invasion process are still not clearly understood. The use of P. falciparum for mechanistic studies on invasion is limited by the unavailability of methods to isolate viable P. falciparum merozoites that retain their invasive ability. In contrast, it is possible to isolate viable P. knowlesi merozoites that invade red cells in vitro (Ward et al., 1993). Moreover, the availability of culture-adapted P. knowlesi has eliminated the need for access to rhesus macaques making P. knowlesi more accessible for investigation (Wel et al., 2004). We have used the advantages offered by P. knowlesi to directly address the role of EBPs in red cell invasion.

The P. knowlesiα gene, which encodes PkDBP, was deleted by insertion of the Tg dhfr-ts cassette at the P. knowlesiα gene locus by homologous recombination. Integration of the Tg dhfr-ts cassette at the P. knowlesiα gene locus and deletion of the P. knowlesiα gene in PkαKO parasites was confirmed by PCR and Southern blot hybridization. Transcripts for P. knowlesiα gene were not detectable by RT-PCR and the encoded PkDBP was not detectable by immunoprecipitation in PkαKO parasites. The invasion phenotype of PkαKO parasites was analysed using in vitro erythrocyte invasion assays. PkαKO parasites invade rhesus erythrocytes normally but are unable to invade human erythrocytes. Deletion of the P. knowlesiα gene results in the complete loss of ability to invade Duffy-positive human erythrocytes. This provides the first direct evidence for the role of PkDBP in invasion of human erythrocytes by the Duffy pathway. The observation that PkαKO parasites are incapable of invading Duffy-positive human erythrocytes indicates that P. knowlesi is completely dependent on PkDBP for interaction with the Duffy antigen and invasion of human erythrocytes. P. knowlesi lacks redundancy in parasite ligands that can interact either with the Duffy antigen or with alternative receptors on human erythrocytes to mediate invasion. Deletion of EBA-175 in P. falciparum strain W2mef, which cannot invade neuraminidase-treated erythrocytes, results in a switch in invasion phenotype to sialic acid-independent pathways (Reed et al., 2000; Duraisingh et al., 2003). In an effort to identify similar changes in receptors used by PkαKO parasites, we tested the invasion of PkWT and PkαKO parasites into rhesus erythrocytes treated with a variety of enzymes. Rates of invasion by PkWT and PkαKO parasites into normal and enzyme-treated rhesus erythrocytes were comparable indicating that deletion of the P. knowlesiα gene does not affect invasion of rhesus erythrocytes by Duffy-independent pathways.

Erythrocyte invasion by malaria parasites is a complex multi-step process. Components of apical organelles such as micronemes play a vital role in these steps during invasion. Recently, a microneme protein, apical merozoite antigen-1 (AMA-1) has been implicated in apical reorientation during red cell invasion (Mitchell et al., 2003). Here, we have investigated the functional role of another micronemal protein, PkDBP, in the invasion process. It has been previously demonstrated that when P. knowlesi merozoites interact with Duffy-negative human erythrocytes, initial interaction and apical orientation take place normally but a junction does not develop and invasion is aborted (Aikawa et al., 1978). These studies indicated that interaction of P. knowlesi merozoites with the Duffy antigen is critical for junction formation during red cell invasion. Here, we have deleted the P. knowlesiα gene to directly assess the role of PkDBP in junction formation during invasion. When PkαKO merozoites interact with rhesus erythrocytes a junction forms (Fig. 3) and invasion proceeds to completion. However, when PkαKO merozoites interact with Duffy-positive human erythrocytes, initial attachment and apical reorientation take place normally, but a junction does not develop (Fig. 3) and invasion is aborted at this step. These studies provide direct evidence for the essential role of PkDBP in junction formation during invasion of human erythrocytes by P. knowlesi. The observation that PkαKO parasites form a normal junction with rhesus erythrocytes indicates that P. knowlesi merozoites can use alternative receptors on rhesus erythrocytes to form a junction and invade by Duffy-independent pathways. PkDBP paralogues such as P. knowlesiβ and γ, which bind Duffy-independent receptors on rhesus erythrocytes (Chitnis and Miller, 1994; Ranjan and Chitnis, 1999), may mediate junction formation and invasion of rhesus erythrocytes by such Duffy-independent pathways.

Like P. knowlesi, the human malaria parasite, P. vivax, is also dependent on interaction with the Duffy antigen for invasion of human erythrocytes (Miller et al., 1976). Duffy-negative human erythrocytes are refractory to invasion by P. vivax and Duffy-negative individuals are resistant to P. vivax malaria (Miller et al., 1976). A single copy gene encodes the P. vivax Duffy binding protein (PvDBP), which shares close homology with PkDBP (Fang et al., 1991; Adams et al., 1992). As in case of P. knowlesi, P. vivax is also likely to be completely dependent on PvDBP for interaction with the Duffy antigen to mediate junction formation during invasion of human erythrocytes. Absolute dependence on interaction of PvDBP with Duffy antigen for invasion identifies this receptor–ligand interaction as an attractive target for receptor-blocking strategies to block invasion by P. vivax. Indeed, inhibition of the PkDBP–Duffy antigen interaction with either chemokines or antibodies raised against the binding domain, PkαRII, has been shown to inhibit P. knowlesi invasion into human erythrocytes efficiently (Horuk et al., 1993; Singh et al., 2002). Antibodies directed against the homologous receptor-binding domain, region II of PvDBP, should also block erythrocyte invasion by P. vivax. By analogy, the P. falciparum orthologues of PkDBP, namely, EBA-175, EBA-140 and EBA-181, also probably mediate junction formation during red cell invasion. Inhibition of these interactions should block erythrocyte invasion by P. falciparum. Indeed, antibodies to EBA-175 have been shown to block red cell invasion even by P. falciparum strains that use multiple invasion pathways (Pandey et al., 2002; Narum et al., 2000). These observations support the development of therapeutic or prophylactic strategies that target the interaction of EBPs with erythrocyte receptors to block junction formation and inhibit red cell invasion by Plasmodium merozoites to provide protection against malaria.

Experimental procedures

Plasmid construct used for deletion of P. knowlesiα gene by homologous recombination

The transfection vector, b2DDD, has a selection cassette, which contains mutagenized Tg dhfr-ts that can confer resistance to pyrimethamine flanked by P. berghei dhfr-ts flanking sequences (van der Wel et al., 1997; Kocken et al., 2002). DNA fragments containing 5′ and 3′ flanking regions of P. knowlesiα gene were amplified by PCR and cloned at the 5′ and 3′ ends of the Tg dhfr-ts casette in the transfection vector b2DDD (van der Wel et al., 1997; Kocken et al., 2002) to yield plasmid b2DDDPKA as shown in Fig. 1. Primers P1cgF2 (5′-AC TGC AAG CTT TGT GTG CTC ATG GTA CAG-3′) and P1cgR (5′-GAT CGC AAG CTT TAG CTA GGT GTT CTA CA-3′) were used with plasmid p1cg, which contains the 5′ end of the P. knowlesiα gene, as template to amplify a 1.1 kb fragment from the 5′ flanking region of P. knowlesiα gene. Both primers contain HindIII restriction sites (shown in bold). The 1.1 kb PCR product was digested with HindIII and cloned in the HindIII site at the 5′ end of Tgdhfr-ts in transfection vector b2DDD to yield plasmid b2DDD-PKA5. Orientation of the 5′ flanking fragment in plasmid b2DDD-PKA5 was confirmed by restriction digest. Primer P6DF (5′-GAT CGA GAA TTC AAT TGT GAT AGT ACA GCT-3′), which contains an EcoRI site (shown in bold), and primer P6DR (5′-CTA GTC GGA TCC AGG TTA AAA TTT TGT CCA AT-3′), which contains a BamHI site (shown in bold), were used for PCR with plasmid p6D, which contains the 3′ end of P. knowlesiα gene, as template to amplify a 1.0 kb DNA fragment from the 3′ flanking region of P. knowlesiα gene. The PCR product was digested with EcoRI and BamHI and cloned at the 3′ end of Tgdhfr-ts gene cassette in plasmid b2DDD-PKA5 to yield plasmid b2DDDPKA (Fig. 1). Plasmid b2DDDPKA was linearized by digestion with ClaI and BamHI (Fig. 1), precipitated with ethanol, resuspended in Cytomix buffer (van den Hoff et al., 1992) and used for transfection as previously described (van der Wel et al., 1997; Kocken et al., 2002).

Parasite culture and transfection

Plasmodium knowlesi H strain was cultured in vitro and transfected as described previously (Kocken et al., 2002). Briefly, P. knowlesi culture [0.5 ml packed cell volume (PCV)] with ∼10% parasitemia containing ∼70% schizonts was washed once with RPMI 1640 and resuspended in Cytomix buffer in a total volume of 0.7 ml. Linearized plasmid b2DDDPKA (25 µg) was resuspended in 100 µl of Cytomix, added to 0.7 ml of P. knowlesi schizonts resuspended in Cytomix, and electroporated using a Bio-Rad electroporator with the following conditions: voltage, 2.5 kV; capacitance, 25 µF; resistance, 200 ohms. After electroporation, the parasites were chilled on ice for 5 min, mixed with 1 ml of rhesus red cells at 50% hematocrit and 20 ml complete RPMI medium containing 20% fetal calf serum (FCS) and allowed to grow at 37°C in a T75 flask flushed with mixed gas (5% CO2, 5% O2, 90% N2). The following day, the culture was washed once with incomplete RPMI 1640 medium and resuspended in complete RPMI 1640 medium containing pyrimethamine (25 ng ml−1). Transfected P. knowlesi parasites were cultured in complete RPMI 1640 medium containing pyrimethamine for 2 weeks with daily changes of growth medium. Ring stage pyrimethamine-resistant parasites were cryo-preserved to create master stocks at this stage. Two pyrimethamine resistant P. knowlesiα gene knockout (PkαKO) clones, BA and AE, obtained by limiting dilution were selected for further study.

PCR and Southern analysis of pyrimethamine-resistant P. knowlesi transfectants

Primer P1 (5′-GCA CTA TTG TGT GTT CAT GG-3′) based on 5′ flanking sequence of P. knowlesiα gene and primer P2 (5′-GTG TCT ATA TTA CCA ACT C3′) based on sequence of the Tg dhfr-ts gene cassette were used for PCR with genomic DNA from PkαKO clones, BA and AE, to test for integration of the Tg dhfr-ts gene cassette at the P. knowlesiα gene locus. The size of the expected PCR product using primers P1 and P2 with PkαKO genomic DNA as template is 1.5 kb. Primers P3 (5′-TGC GAA TTC TGT AAG GAT ATA AGA TG-3′) and P4 (5′-ATA GTT TAG CGG CCG CTC AGA GAT GAT GAT GAT GAT GTT CAG TTA TCG GAT TAG A-3′) based on the P. knowlesiα gene were used for PCR with genomic DNA derived from PkαKO clones, BA and AE, and PkWT parasites. The size of the expected PCR product using primers P3 and P4 with PkWT genomic DNA as template is 0.74 kb.

Genomic DNA (5.0 µg) from PkWT and PkαKO clones BA and AE was digested with 20 units of either TaqI or HaeIII for 2 h, separated by gel electrophoresis on a 1% agarose gel transferred to nylon membrane by capillary transfer and probed with a 32P-labelled 1.0 kb probe based on PkαRII using standard methods for Southern blot hybridization. The 32P-labelled probe was prepared using a random primer labelling kit (Roche, Germany) and a DNA fragment encoding PkαRII as template.

Detection of transcripts of P. knowlesiα gene by RT-PCR

RNA was isolated from PkαKO and PkWT cultures when majority of parasites (>70%) were in the schizont stage using TRIZOLTM (Invitrogen, USA) as described by the manufacturer. RNA (10 µg) derived from both PkαKO and PkWT parasites was reverse-transcribed using SuperScript II RNase H reverse transcriptase (Invitrogen, USA) and random hexamers (Invitrogen, USA). Mock reverse transcriptase reactions were performed in which no reverse transcriptase was added. The products of reverse transcriptase and mock reverse transcriptase reactions were used as templates for PCR with specific primers based on sequences of P. knowlesiα, β and γ genes. Primers P3 and P4 described above were used to detect transcripts for P. knowlesiα gene by RT-PCR. Primers BF1630 (5′-TAA CAA CGT ATA TAG CGA AGA C3′) and BR2057 (5′-CAT TGC ATG GAG ACA TTG TAC A3′) based on P. knowlesiβ gene and primers GF1564 (5′-CTG CAT GGA ATT GTA AAG AAG ATG3′) and GR1781 (5′-TCC TGT TAT CCA TTC ATC ATA TGA3′) based on P. knowlesiγ gene were used to detect transcripts of the P. knowlesiβ and γ genes respectively. RT-PCR products were separated by gel electrophoresis on 1.2% agarose gels and visualized after staining with ethidium bromide.

Detection of PkDBP in metabolically labelled P. knowlesi culture supernatants by immunoprecipitation

PkWT and PkαKO parasites were cultured in vitro and metabolically labelled as follows. Mature schizonts purified from PkWT and PkαKO cultures by centrifugation on 45% Percoll. Purified schizonts (3 × 107 ml−1) were cultured for 1 h in RPMI 1640 medium lacking cysteine and methionine [cysmet RPMI 1640 (Sigma, USA)]. Parasites were metabolically labelled by adding fresh cysmet RPMI 1640 containing 1% human serum, 2 mM glutamine, and 77 µCi Promix (Amersham, UK) per ml, and allowing parasites to grow for a further 8 h. Culture supernatants were harvested by centrifugation and stored at −70°C.

Rabbit serum raised against recombinant PkαRII (Singh et al., 2002) was used to detect PkDBP in P. knowlesi culture supernatants by immunoprecipitation. Metabolically labelled PkWT and PkαKO culture supernatants were used for immunoprecipitation either directly or after pre-absorption three times with 500 µl of packed Duffy-positive human erythrocytes, Duffy-negative human erythrocytes or rhesus erythrocytes. Parasite culture supernatants were incubated for 2 h on ice with 1:50 dilution of anti-PkαRII rabbit serum and with 100 µl of Protein A Sepharose beads at room temperature for 1 h. The beads were collected by centrifugation, washed once with 0.5% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 50 mM Tris pH 7.4 (NETT) containing 0.5% bovine serum albumin (BSA) and twice with NETT buffer without BSA. Bound proteins were eluted by boiling, separated on 7.5% sodium dodecyl sulphate polyacrylamide gels (SDS-PAGE) and detected by autoradiography.

Analysis of the invasion phenotype of PkαKO parasites

Rhesus and human erythrocytes (100 µl PCV) were washed three times with RPMI 1640 and incubated with 1 ml FITC (Sigma, USA), 4.0 mg ml−1 in phosphate-buffered saline (PBS) for 1 h with gentle rocking at room temperature. Cells were washed three times with 50 volumes of RPMI 1640, resuspended in RPMI 1640 containing 10% human serum and left overnight at 4°C. Cells were washed two more times in RPMI 1640 and used for invasion assays within 3 days of labelling. Invasion assays were performed in triplicate in 100 µl volume in 96-well flat bottom sterile plates. Percoll-purified PkWT or PkαKO schizonts (2 × 106) were incubated with FITC-labelled erythrocytes (2 × 107) for 12 h at 37°C in presence of mixed gas (5% CO2, 5% O2, 90% N2) to allow invasion. Smears were made in duplicate, fixed with methanol and stained with DAPI (Sigma, USA), 1 µg ml−1 in PBS. The percentage of FITC-stained erythrocytes containing DAPI-stained parasites was determined using a NIKON TE300 fluorescence microscope to estimate erythrocyte invasion rates. At least 2000 FITC-labelled erythrocytes were scored for presence of DAPI-stained parasites. Rhesus erythrocytes were treated with trypsin, proteinase K, chymotrypsin and neuraminidase as previously described (Thompson et al., 2001) prior to use in invasion assays.

Electron microscopy

Invasive P. knowlesi merozoites were obtained from P. knowlesi schizonts as previously described (Ward et al., 1993). Briefly, P. knowlesi schizonts purified from in vitro culture were allowed to grow in presence of leupeptin and chymostatin (50 µg ml−1 each) to reversibly arrest development at late schizont stage. Free merozoites were released from mature schizonts by forcing the schizonts through a gauge 25 needle (Becton Dickinson, USA) 10–15 times. Debris and unlysed cells were removed by centrifugation at 1600 r.p.m. for 5 min at room temperature in Sorvall RT7 centrifuge with a swinging bucket rotor. Merozoites were collected from the supernatant by centrifugation at 7000 r.p.m. for 3 min at room temperature. Merozoites in the pellet were resuspended in RPMI 1640 containing 20% FCS, treated with cytochalasin B (10 µg ml−1) for 3 min at 37°C, and incubated with 2 × 107 human or rhesus erythrocytes for 30 min at 37°C to allow interaction. Erythrocytes with attached merozoites were collected by centrifugation, resuspended in RPMI 1640 containing 10% FCS and fixed overnight in chilled 2% glutaraldehyde in 100 mM phosphate buffer containing 160 mM sucrose. Samples were then treated with 1% osmium tetraoxide at 4°C for 2–4 h and washed extensively with excess of triple-distilled water. Samples were dehydrated gradually by addition of increasing concentration of ethanol (10% increment in ethanol concentration per step up to 100% ethanol in final step). Samples were infiltrated at 37°C for 6–12 h with embedding medium (Dr Spurr medium, medium viscosity) diluted in ethanol at v/v ratios of 1:3, 1:1 and 3:1. Sample blocks were made in undiluted embedding media and allowed to polymerize at 60°C for 48 h. Thin sections were prepared, stained with uranyl acetate and lead citrate, and observed in a Philips CM-10 electron microscope.

Acknowledgements

We thank Dr Tapas K. Das, All India Institute of Medical Sciences (AIIMS), New Delhi, the staff of Electron Microscope Facility, AIIMS, New Delhi and Dr Nirupam Roychowdhary, ICGEB, New Delhi for help with electron microscopy, A. M. van der Wel, BPRC, Rijswijk for technical assistance with P. knowlesi transfection and Amit Sharma, ICGEB, New Delhi for comments on the manuscript. This work was supported with grants from the European Commission to S.K.P., A.T. and C.E.C. (IC18CT980369) and Howard Hughes Medical Institute, USA to C.E.C. C.E.C. is an International Research Scholar of the Howard Hughes Medical Institute (HHMI), USA and International Senior Research Fellow of The Wellcome Trust, UK.

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