A Novel Erythrocyte Binding Antigen-175 Paralogue from Plasmodium falciparum Defines a New Trypsin-resistant Receptor on Human Erythrocytes*

Tim-Wolf GilbergerDagger, Jennifer K. Thompson, Tony Triglia, Robert T. Good, Manoj T. Duraisingh, and Alan F. Cowman§

From The Walter and Eliza Hall Institute of Medical Research, Melbourne 3050, Australia

Received for publication, November 10, 2002, and in revised form, January 28, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The recognition and invasion of human erythrocytes by the most lethal malaria parasite Plasmodium falciparum is dependent on multiple ligand-receptor interactions. Members of the erythrocyte binding-like (ebl) family, including the erythrocyte binding antigen-175 (EBA-175), are responsible for high affinity binding to glycoproteins on the surface of the erythrocyte. Here we describe a paralogue of EBA-175 and show that this protein (EBA-181/JESEBL) binds in a sialic acid-dependent manner to erythrocytes. EBA-181 is expressed at the same time as EBA-175 and co-localizes with this protein in the microneme organelles of asexual stage parasites. The receptor binding specificity of EBA-181 to erythrocytes differs from other members of the ebl family and is trypsin-resistant and chymotrypsin-sensitive. Furthermore, using glycophorin B-deficient erythrocytes we show that binding of EBA-181 is not dependent on this sialoglycoprotein. The level of expression of EBA-181 differs among parasite lines, and the importance of this ligand for invasion appears to be strain-dependent as the EBA-181 gene can be disrupted in W2mef parasites, without affecting the invasion phenotype, but cannot be targeted in 3D7 parasites.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmodium falciparum is the causative agent of the most lethal form of malaria in humans and is responsible for more than 2 million deaths per year. Because of the increasing resistance of this parasite toward the commonly used antimalarial drugs there is an urgent need for the development of a vaccine (1, 2). P. falciparum invades erythrocytes and develops and expands within this host cell followed by release of invasive merozoite forms into the blood stream. The merozoite is exposed to the host immune system, and proteins involved in the invasion process are potential vaccine candidates.

The invasion of erythrocytes by merozoites is a rapid and complex process that relies on an orchestrated cascade of interactions between invading parasite and host cell (3, 4). The parasite utilizes multiple erythrocyte receptors for merozoite invasion (5-7); however, the molecular basis of this process is poorly understood. Some erythrocyte receptors have been identified using mutant red blood cells and/or defined modifications of the erythrocyte surface by enzyme treatments (8-11). The erythrocyte binding antigen-175 (EBA-175)1 binds to glycophorin A (the major glycoprotein of the erythrocyte), and this interaction mediates an invasion pathway for merozoite entry into erythrocytes (12-16). EBA-140 (BAEBL) (10, 11) a paralogue of EBA-175, has been shown to bind to glycophorin C and functions in a pathway for merozoite invasion (17).

EBA-175 and EBA-140 are members of the ebl superfamily (erythrocyte binding-like), which includes at least five members: EBA-140, EBA-165 (PEBL), EBA-175, EBA-181 (JESEBL), and EBL-1 (18, 19). The plurality of the ebl genes provides ligand diversity and potential usages of different host receptors. Members of this gene family have similar intron-exon structure and have presumably evolved from a common gene ancestor (15, 19, 20). The EBA-165 gene can be disrupted; however, no protein has been identified in the P. falciparum lines tested, suggesting it may be a pseudogene (21). The EBA-181 gene in P. falciparum has been identified from the P. falciparum genome sequence; however, the putative protein encoded by this gene has not yet been analyzed (18).

In this report, we demonstrate that EBA-181 binds to the surface of erythrocytes in a sialic acid-dependent manner to a trypsin-resistant/chymotrypsin-sensitive receptor. To evaluate the function of this protein we have disrupted the gene in P. falciparum and analyzed the effect on merozoite invasion. Our results suggest that EBA-181 is an important component of the merozoite invasion machinery but is differentially utilized in P. falciparum strains.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Parasites; Culture, Strains, and Transfection-- P. falciparum parasites were cultured in human 0+ erythrocytes according to standard procedures (35). The 3D7 parasite strain was obtained from David Walliker at Edinburgh University. W2mef is derived from the Indochina III/CDC strain. W2mef and 3D7 parasites were transfected as described previously (22-24) with 80 µg of purified plasmid DNA (Qiagen). All transfections were performed twice in independent experiments. Transfectants were selected with 10 nM WR99210 and where relevant were grown in 4 µM ganciclovir to select against transgenic parasites expressing thymidine kinase. Transfected parasites with integrated plasmids were cloned.

Nucleic Acid and Sequence Analysis-- Chromosome 1 of P. falciparum was sequenced by the Sanger Centre (www.sanger.ac.uk). Pulse-field gel electrophoresis, preparation of intact chromosomes, and isolation of genomic DNA were performed as described previously (25, 26). DNA sequencing was performed using BigDye Terminator Cycle Sequencing (PerkinElmer Life Sciences). Southern blotting was carried out using standard procedures.

The pHTKDelta 181 plasmid vector for disruption of the EBA-181 gene was constructed using pHTK (24). The 5' (~1 kb) and 3' flanks (~1.2 kb) for homologous recombination into the EBA-181 gene were amplified from genomic DNA of W2mef or 3D7 with the primer pairs 5'- GATCACTAGTATGAAAGGGAAAATGAATATGTG-3'/5'-GATCAGATCTAAGACTCCTATAGGTATAACACTC-3' (inserts a 5' SpeI and 3' BglII site) and 5'-GATCGAATTCTCGGAATCAGGTTTAAATCCAACTGATG-3'/5'-GATCCCATGGTCTAAAACAATTATATGTAGGGTC-3' (inserts a EcoRI and NcoI site), respectively. Both fragments were inserted into the appropriate restriction sites of pHTK such that they flanked the human dihydrofolate reductase gene, resulting in the vectors pHTKDelta 181W2mef and pHTKDelta 1813D7.

Antisera, Immunoblots, and Immunofluorescence-- Rabbit and mouse antisera were raised against two domains of the EBA-181 protein using the pGEX system (Amersham Biosciences). One immunogen is located between the F2 domain and the 3' cysteine-rich region (EBA181ex) and encompasses the amino acids 755-1339 of EBA-181 (Fig. 1). The coding region was amplified from genomic DNA (3D7) using the primers 5'-ACAGGATCCAGTGAGAAAAATGGAGAGGAAG-3' and 5'-AGCGCTCGAGGTCAAAATCTTTTTCATCTTTTTGTTG-3' and cloned into the BamHI/XhoI sites of pGEX-4T. The second immunogen (EBA181ct) comprises the cytosolic domain of EBA-181 encompassing amino acids 1510-1568. A 0.2-kb fragment of the 3' end of the EBA-181 gene encoding this region was amplified from cDNA using the primers 5'-GCGCGGAATTCTATAGGAAGAATTTGGATG-3' and 5'-GCGCGCTCGAGTTAAAATGTCGTTGTGTC-3' and cloned into the EcoRI/XhoI sites of pGEX-4T. The glutathione S-transferase fusion proteins were purified from Escherichia coli BL21(DE3) by standard procedures using glutathione-Sepharose 4B (Sigma). Purified proteins were injected into New Zealand White rabbits and BalbC mice. Rabbit antisera were purified on affinity columns using the recombinant protein as ligand. Other antibodies used in immuno-detection were rabbit anti-EBA-175 (7) and anti-HSP70 antibodies (27).

For immunoblots, parasite proteins were separated on 6% SDS-PAGE gels and transferred to nitrocellulose membranes (Schleicher & Schuell). EBA181ex, EBA181ct, and EBA-175 rabbit antisera were diluted 1:250, and HSP70 antisera was diluted 1:5000. The secondary antibody was sheep anti-rabbit IgG horseradish peroxidase (Silenus). The immunoblots were developed by chemiluminescence using ECL (Amersham Biosciences).

Immunofluorescence experiments were performed on synchronized W2mef, W2mefDelta 181c1, and W2mefDelta 181c2 parasites. Smears of mature schizonts were air-dried and fixed for 1 min with 100% methanol at -20 °C. Slides were incubated for 1 h with a mixture of rabbit anti-EBA-175 (1:1000) and mouse anti-EBA-181 (1:1250), washed 3 times for 10 min with 0.05% Tween 20, phosphate-buffered saline, and then incubated for 1 h with fluorescein isothiocyanate-labeled sheep anti-mouse IgG antibodies (Silenus Laboratories), rhodamine-labeled goat anti-rabbit IgG antibodies (Chemicon), and 4,6-diamidino-2-phenylindole (1:1250, Roche Molecular Biochemicals). Dual-color fluorescence images were captured using a Zeiss Axioskop 2 microscope and a digital camera (PCO sensicam).

Erythrocyte Enzyme Treatments and Erythrocyte Binding Assay-- Human erythrocytes were modified by treatment with neuraminidase and proteases as described previously (11). 500 µl of packed erythrocytes were incubated with 50 milliunits of Vibrio cholerae neuraminidase (Calbiochem), trypsin (Sigma) in two different concentrations (0.1 mg/ml or 1.5 mg/ml), or chymotrypsin (1.5 mg/ml, Sigma). Culture supernatants were collected from synchronized parasite cultures after rupture of the schizonts (>10%). 500 µl of this supernatant material was incubated with 100 µl of packed, uninfected erythrocytes for 30 min at room temperature (6, 11, 28). The cells were centrifuged through 500 µl of silicone oil (Dow Corning 550) to eliminate unbound supernatant material. Bound proteins were eluted from the erythrocyte by incubation with 20 µl of 1.5 M NaCl for 15 min at room temperature followed by centrifugation at 12,000 × g for 5 min. An equal volume of 2× SDS reducing sample buffer was added. The eluted proteins were separated by SDS-PAGE and identified by immunoblotting.

To quantify binding of EBA-181 to the surface of different erythrocytes we absorbed parasite proteins in a multistep procedure (10). Briefly, 250 µl of culture supernatant was incubated with 100 µl of packed, uninfected erythrocytes for 30 min at room temperature. The cells were centrifuged through 500 µl of silicone oil (Dow Corning 550) to eliminate unbound supernatant material. 25 µl of this depleted supernatant was mixed with 2× SDS reducing sample buffer for further SDS-PAGE analysis. Bound proteins were eluted from the erythrocyte by incubation with 20 µl of 1.5 M NaCl for 15 min at room temperature followed by centrifugation at 12,000 × g for 5 min. An equal volume of 2× SDS reducing sample buffer was added. In the next step the remaining 225 µl of depleted supernatant was used in a second round of binding with 100 µl of new packed, uninfected erythrocytes. Similar to the first cycle of binding and elution, proteins bound to the erythrocytes were eluted from the erythrocytes, and an aliquot of the further-depleted supernatant was mixed with 2× SDS reducing sample buffer. This procedure was repeated four times. The samples from each step were separated by 6% SDS-PAGE, and parasite proteins were detected by Western blotting.

Erythrocyte Invasion Assay-- Parasite erythrocyte invasion assays were performed to characterize the invasion phenotype of the W2mefDelta 181 transgenic lines (7). Surface modification of the erythrocytes was achieved by similar treatment as previously described; 500 µl of packed erythrocytes were incubated with either 50 milliunits of neuraminidase, trypsin (0.1 or 1.5 mg/ml), chymotrypsin (1.5 mg/ml), or a combination of neuraminidase (50 milliunits) and trypsin (0.1 mg/ml), neuraminidase (50 milliunits) and trypsin (1.5 mg/ml), or chymotrypsin (1.5 mg/ml) and trypsin (0.1 mg/ml). Synchronized parasites were treated with neuraminidase and trypsin to prevent re-invasion as follows. Ring stage cultures were adjusted to 2% parasitemia and treated with neuraminidase for 1 h at 37 °C (50 milliunits for 200 µl of packed, infected erythrocytes in 1 ml of RPMI-1640, HEPES, 0.2% NaHCO3). After washing twice with RPMI, HEPES, 0.2% NaHCO3, the infected cells were incubated with trypsin (1 mg/ml in RPMI-1640, HEPES, 0.2% NaHCO3) for 1 h at 37 °C followed by incubation with trypsin inhibitor (1 mg/ml in RPMI-1640, HEPES, 0.2% NaHCO3, Sigma) for 10 min at room temperature. The cultures were washed, resuspended in complete medium, and incubated overnight under standard conditions. The invasion assays were prepared the following day by mixing equal amounts of infected double-enzyme-treated cells (schizont stage) with uninfected normal or enzyme-modified erythrocytes to a final hematocrit of 3%. 100-µl aliquots of each culture were put into a 96-well microtiter plates in triplicate. The parasitemia in each well was counted by FACScan using Retic-COUNT (BD Biosciences). After 36 h of incubation, the parasitemia of each well was counted again by FACScan and compared with the starting parasitemia. Control wells with unstained culture and stained uninfected erythrocytes were included in FACScan counting to enable background correction.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Comparison of Gene Structure and Primary Sequence for EBA-181-- The EBA-181 gene has been identified from the P. falciparum genome and is composed of 4 exons and 3 introns and spans 5049 bp (Fig. 1, PFA0125c) (29). The exon/intron structure is identical to that described for other members of the ebl family including EBA-140 and EBA-175 and encodes a putative polypeptide of 1567 amino acids with a calculated molecular mass of 181 kDa (18). The predicted protein consists of an N-terminal hydrophobic signal sequence, two cysteine-rich regions in the extracellular domain (F1/F2 DBL and 3' Cys-rich domain), a transmembrane domain, and a short C-terminal cytoplasmic domain. EBA-181 has similarity to EBA-175 and EBA-140, particularly in the F1/F2 domain, with 25.3 and 24.6% identity, respectively. EBA-181 is not as closely related to EBA-175 as is EBA-140, with 37% identity between the latter two proteins within the F1/F2 domain. Nevertheless, EBA-181 is clearly a member of the ebl family due to its structural similarity and its sequence identity.


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Fig. 1.   EBA-181 is a member of the ebl superfamily and shares similar structural features with EBA-175 and EBA-140. The putative functional regions of the molecule include an N-terminal signal sequence (blue), a cysteine-rich erythrocyte binding region (F1/F2, red), a further C-terminal cysteine-rich region (purple), a single transmembrane domain (black), and a short cytoplasmic domain (green). The regions of EBA-181 to which antibodies were raised are shown by a black line for anti-EBA181ex and anti-EBA181ct. AA, amino acids.

EBA-181 Is Expressed in P. falciparum Asexual Stages-- The similarity of EBA-181 to EBA-175 and EBA-140 suggests that they may share a similar function with respect to merozoite invasion (Fig. 1). To determine whether EBA-181 is expressed in P. falciparum asexual stages we raised antibodies against a portion of the ectodomain and the cytoplasmic tail of this putative protein (Fig. 1). Synchronized 3D7 parasites were sampled every 8 h throughout the asexual life cycle and analyzed by immunoblots using the anti-EBA181ex antibodies. A major band of ~190 kDa was observed predominantly in the 24- and 32-h time points (Fig. 2A). A second minor band of ~170 kDa was also detected with the same antibody and may represent a processed product. A protein of the same size and pattern of expression was observed with the anti-EBA181ct antibodies (Fig. 2, A and B). This confirms the specificity of the anti-EBA181ex and anti-EBA181ct antibodies and the identification of EBA-181 in P. falciparum. Comparison of EBA-181 expression with that of EBA-175 through the asexual blood stage of the life cycle shows that they have identical patterns of expression (Fig. 2C).


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Fig. 2.   Stage-specific expression of EBA-181. Pellets from synchronized cultures (3D7) were harvested at 8-h intervals, and parasite proteins were separated on 6% SDS-PAGE under reducing conditions. Immunodetection of EBA-181 was achieved using two antibodies, EBA181ex (A) and EBA181ct (B) raised against different parts of the molecule. Maximal expression of EBA-181 occurs in late schizonts. C, identical blots were probed with anti-EBA-175 and anti-HSP70 antibodies.

Analysis of EBA-181 expression in two different P. falciparum lines suggests that this protein can be expressed in different amounts with respect to other proteins. Schizont pellets of 3D7 and W2mef were analyzed by immunoblots using anti-EBA181ex antibodies (Fig. 3). The signal observed for the 190-kDa protein band in 3D7 was significantly higher compared with W2mef with the same antibody. Sequence analysis of the EBA-181 gene in the region to which the antibody was raised showed no differences between 3D7 and W2mef, suggesting that the observed difference in signal was due to expression rather than altered reactivity of the antibodies (data not shown). This was confirmed by analysis of the same schizont samples in both parasite lines with antibodies to EBA-175 and HSP-70. Both of these proteins were expressed at approximately equivalent levels (Fig. 3B). These results suggest that EBA-181 is expressed in 3D7 at significantly higher levels compared with that observed in W2mef.


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Fig. 3.   The level of EBA-181 expression is strain-dependent. Proteins from synchronized W2mef and 3D7 schizonts were separated by 6% SDS-PAGE under reducing conditions. A, EBA-181 was detected with anti-EBA181ex. B, to equalize loading of parasite proteins anti-EBA175 and anti-HSP70 were used.

EBA-181 Is Localized to the Micronemes of Merozoites-- The subcellular localization of EBA-181 was determined by immunofluorescence using anti-EBA181ex rabbit antibodies and compared with EBA-175 by co-localization with anti-EBA-175 mouse antibodies. EBA-175 is localized in the microneme organelles at the apical end of the merozoite (30). Anti-EBA-181 antibodies give a punctate pattern in mature schizonts that co-localizes with EBA-175 (Fig. 4). Similarly, EBA-181 co-localizes with EBA-175 at the apical end of free merozoites, suggesting that the subcellular localization of EBA-181 is within the micronemes, consistent with it playing a role in merozoite invasion (Fig. 4).


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Fig. 4.   Co-localization of EBA-181 and EBA-175 in schizonts and free merozoites. Smears of W2mef parasites were incubated with anti-EBA181ex and anti-EBA175 followed by fluorescein isothiocyanate-labeled anti-mouse, rhodamine-labeled anti-rabbit antibodies, and 4,6-diamidino-2-phenylindole (DAPI). The top row corresponds to schizont stages, whereas the middle row is free merozoites of W2mef parasites. The bottom row is a higher magnification of a free merozoite that is attached to the surface of the erythrocyte and may be undergoing the invasion process. In the first column the parasites are stained with 4,6-diamidino-2-phenylindole (blue). In the second column the parasites have been labeled with anti-EBA-175 antibodies (red). In the third column the parasites have been labeled with anti-EBA181ex antibodies (green). In the fourth column co-localization of EBA-175 and EBA-181 (yellow) is shown by an overlay of anti-EBA175 and anti-EBA181ex micrographs. The third row is an overlay of 4,6-diamidino-2-phenylindole (blue), anti-EBA175 (red), and anti-EBA181ex (green) antibodies onto a light micrograph. m, merozoite; rbc, red blood cell.

EBA-181 Binds to Erythrocytes-- EBA-175 and EBA-140 have been shown previously to bind to erythrocytes, and to determine whether EBA-181 also binds we performed assays with normal and enzyme-treated red blood cells (10, 11, 28, 31). Incubation of parasite culture supernatants with untreated erythrocytes showed that EBA-181 binds to these cells and can be eluted from the surface using 0.3 M NaCl (Fig. 5A, first lane). The specificity of EBA-181 binding to erythrocytes was examined by modifying the surface of the red cells. Treatment with neuraminidase, which removes the sialic acid moieties from the cell surface, eliminates binding of EBA-181 to the erythrocytes (Fig. 5A, second lane), showing that the receptor for EBA-181 on the erythrocyte surface contains sialic acid. To further investigate the properties of the EBA-181 receptor, we tested binding to trypsin- and chymotrypsin-treated erythrocytes. The EBA-181 erythrocyte receptor was resistant to 1.5 mg/ml trypsin (Fig. 5A, third and fourth lanes); however, it was sensitive to chymotrypsin (Fig. 5A, fifth lane). The properties of the EBA-181 receptor are in contrast to those of glycophorin A, the receptor for EBA-175 (Fig. 5B). EBA-181 interacts with trypsin-treated but not chymotrypsin-treated erythrocytes. EBA-175 binds to chymotrypsin-treated but not trypsin-treated erythrocytes. Therefore, the major EBA-181 receptor is not glycophorin A. These results show that EBA-181 binds to erythrocytes via a sialoglycoprotein that is trypsin-resistant and chymotrypsin-sensitive.


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Fig. 5.   EBA-181 binds to the surface of erythrocytes. Immunodetection of secreted parasite proteins with anti-EBA181ex and anti-EBA-175 antibodies after binding to and elution from untreated and enzyme-treated erythrocytes. Lanes begin with proteins eluted from untreated erythrocytes (+), proteins eluted from neuraminidase (N), trypsin (T 0.1 and T 1.5 are trypsin treatments with 0.1 and 1.5 mg/ml of enzyme, respectively)- and chymotrypsin (C)-treated erythrocytes. EBA-181 can be detected in eluates from untreated (+) and trypsin (T)-treated erythrocytes, indicating that the erythrocyte receptor for EBA-181 is trypsin-resistant, neuraminidase-sensitive, and chymotrypsin-sensitive.

The physical properties of EBA-181 binding to its erythrocyte receptor are consistent with the characteristics of glycophorin B. To test if EBA-181 binds to glycophorin B we used S-s-U- erythrocytes that lack detectable levels of this protein (Fig. 6), which was verified by immunoblots with anti-glycophorin B antibodies (data not shown). Binding assays of parasite culture supernatants showed detectable levels of EBA-181 eluted from S-s-U- erythrocytes in comparable amounts to that observed with normal erythrocytes (Fig. 6A). EBA-181 binding could be depleted by both S-s-U- and normal erythrocytes after two rounds of incubation with fresh cells, and these results were similar to that observed in identical experiments with EBA-175 (Fig. 6, A and B). The ability to deplete EBA-181 from parasite culture supernatants could be ablated by pretreatment of normal erythrocytes with neuraminidase, confirming the requirement for sialic acid on the erythrocyte surface. These results suggest that EBA-181 binding to the erythrocyte surface does not occur via glycophorin B. The physical properties of the sialoglycoprotein to which EBA-181 binds suggests that it is a novel receptor, and we have termed it receptor E. 


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Fig. 6.   Binding and depletion of EBA-181 to S-s-U- erythrocytes. A, depletion of EBA-181 from parasite culture supernatants (s/n) after 1-4 rounds of binding to S-s-U- erythrocytes (glyB- erythrocytes). EBA-181 and EBA-175 eluted from these erythrocytes after each round of binding was detected by anti-EBA181ex and anti-EBA-175 antibodies. B, depletion of EBA-181 from supernatants after 1-4 rounds of binding to untreated erythrocytes. EBA-181 and EBA-175 eluted from these erythrocytes after each round of binding was detected by anti-EBA181ex and anti-EBA-175 antibodies. C, depletion of EBA-181 from supernatants after 1-4 rounds of binding to neuraminidase-treated erythrocytes (neur rbc). EBA-181 and EBA-175 eluted from these erythrocytes after each round of binding was detected by anti-EBA181ex and anti-EBA-175 antibodies. Binding to and depletion with untreated and neuraminidase-treated erythrocytes are included as a control.

Disruption of the EBA-181 Gene in P. falciparum-- To analyze the role of EBA-181 in merozoite invasion of erythrocytes by P. falciparum, we constructed a transfection plasmid to generate parasites that lack expression of this protein. The plasmid pHTKDelta 181 was derived from pHTK and constructed such that it would integrate into the EBA-181 gene via double crossover recombination (Fig. 7A) (24). The parasite lines 3D7 and W2mef were transfected with pHTKDelta 181, and transfectants were selected with WR99210. Transfected lines were obtained for both 3D7 and W2mef, and these populations maintained the plasmid primarily as episomes, which was shown by hybridization of probes to chromosomes after separation by pulsed field gel electrophoresis (Fig. 7B and data not shown). To select parasites that had integrated the plasmid into chromosome 1, where EBA-181 resides, the 3D7 transfectants were grown on ganciclovir and WR99210. The resulting parasites were analyzed to determine whether integration had occurred into the EBA-181 gene.


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Fig. 7.   Disruption of the EBA-181 gene. A, schematic representation of the double recombination crossover event of pHTKDelta 181 in the EBA-181 locus. The positive selection cassette (hDHFR), represented by the black box of the pHTK-Delta 181 vector, is flanked by two ~1-kb portions of the EBA-181 coding region. The crosses refer to the regions where recombination events took place. The EcoRV (E) restriction sites are marked, and the approximate fragment sizes upon EcoRV digestion after hybridization with the 5' EBA-181 probe are indicated. Recombination of pHTKDelta 181 as a double recombination crossover into EBA-181 leads to disruption of the gene. B, pulsed field gel electrophoresis analysis of chromosomes from W2mef to determine integration of pHTKDelta 181 into chromosome 1. The numbers on the left side of the panel indicate the identity of chromosomes under the chosen pulsed field gel electrophoresis conditions (Chrom). The first panel was probed with EBA-181, and the second panel was probed with hDHFR. The first lane in the first panel shows chromosome 1 from the parental W2mef parasites. The following lanes show chromosomes after transfection (O cycle), after the first selection (1 cycle), and after the second selection, where complete integration of pHTKDelta 181 into chromosome 1 has occurred. Lanes 1 and 2 in the second panel show integration of the hDHFR gene into chromosome 1. C, Southern blot analysis of genomic DNA from W2mef and W2mefDelta 181c1/2 parasites reveals that the plasmid has integrated via double crossover recombination into EBA-181. Sizes of the hybridizing bands are shown in kb.

The 3D7 parasites transfected with pHTKDelta 181 were analyzed by pulsed field gel electrophoresis and shown that they maintain the plasmid as an episome even after selection on WR99210 and ganciclovir (data not shown) (24). This suggested that either the plasmid was not competent for integration or the disruption of EBA-181 in 3D7 was deleterious. Further attempts to obtain 3D7 parasites that had integrated the pHTKDelta 181 plasmid or constructs containing different regions of the gene were unsuccessful. We conclude that disruption of EBA-181 in 3D7 is deleterious.

In contrast to 3D7, analysis of W2mef parasites transfected with pHTKDelta 181 suggested that the plasmid had rapidly integrated into chromosome 1 without selection on ganciclovir (24). Hybridization of an EBA-181 probe to chromosomes from W2mef transfected with the plasmid revealed a number of bands that do not correspond to chromosomes and is typical of the pattern observed for an episomal-maintained plasmid. This was confirmed by hybridization of the hDHFR probe to the same chromosome blots where the same bands were observed. After a single cycle of drug selection the hDHFR probe hybridized predominantly to chromosome 1, suggesting that the plasmid had integrated. Analysis of parasites after a second cycle of drug selection showed that no episomal plasmid remained, and all parasites appeared to have integrated this region of the plasmid into the smallest chromosome (Fig. 7B). These transfected parasites were cloned to generate W2mefDelta 181c1 and W2mefDelta 181c2

Southern hybridization with genomic DNA from W2mefDelta 181c1 and W2mefDelta 181c2 confirmed that the plasmid had integrated into the EBA-181 gene by double crossover recombination (Fig. 7C) (24). Restriction enzyme-digested genomic DNA of parental and transfected cloned parasite lines was prepared, separated, and hybridized with the EBA-181 probe. The parental W2mef parasites revealed hybridizing fragments of 11 and 3.8 kb with the EBA-181 probe corresponding to the endogenous EBA-181 gene. Integration of pHTKDelta 181 by double crossover recombination removed the endogenous 3.8-kb fragment and generated a novel 0.9-kb band. The 11-kb fragment was retained in both parental and transfected lines. The results obtained are consistent with insertion of the plasmid by double crossover recombination, resulting in loss of the plasmid backbone and TK gene as well as the central region of the EBA-181 gene including the F2 domain (Fig. 7A).

Western blot analysis and immunofluorescence microscopy were used to determine the expression of EBA-181 in W2mefDelta 181c1 and W2mefDelta 181c2 compared with parental W2mef (Fig. 8, A and B). Parasite proteins from late schizonts were separated on SDS-PAGE, blotted onto nitrocellulose membrane, and incubated with antibodies raised against EBA-181. As expected a 190-kDa protein band was observed in W2mef; however, no reactivity was seen in either W2mefDelta 181c1 or W2mefDelta 181c2, confirming that they do not express the EBA-181 protein. Immunofluorescence assays were performed to visualize EBA-181 expression in schizonts from W2mef, W2mefDelta 181c1, and W2mefDelta 181c2. EBA-175 was detected in W2mefDelta 181c1/2 parasites, and the localization was typical of that seen previously. However, no reactivity with anti-EBA-181 antibodies was observed in the transfected lines, consistent with the lack of expression as observed in immunoblots. In contrast, typical EBA-175 and EBA-181 expression and co-localization was observed in W2mef (Fig. 8B).


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Fig. 8.   EBA-181 is not expressed in W2mefDelta 181 parasites. A, Western blot analysis of EBA-181 expression in W2mefDelta 181 (W2mefDelta 181c1 and W2mefDelta 181c2 showed identical results with no EBA-181 protein detectable). Proteins from synchronized schizonts were separated by 6% SDS-PAGE under reducing conditions and immunodetected using anti-EBA181-ex (first panel). As a loading control anti-EBA-175 and anti-HSP70 antibodies were used (second and third panel). B, immunofluorescence assays with anti-EBA-175 and anti-EBA181ct on synchronized W2mef, W2mefDelta 181. EBA-181 is not expressed in W2mefDelta 181 parasites. DAPI, 4,6-diamidino-2-phenylindole.

Lack of Expression of EBA-181 in W2mef Does Not Affect Merozoite Invasion-- Previously, it has been shown that loss of EBA-175 or EBA-140 expression can have an effect on the ability of merozoites to invade enzyme-treated erythrocytes (7, 17). To determine whether loss of EBA-181 expression in W2mefDelta 181c1/2 resulted in alterations in the pattern of merozoite invasion into human erythrocytes we compared the efficiency of invasion of these transfected lines with W2mef into neuraminidase-, trypsin-, and chymotrypsin-treated erythrocytes. We could not detect any significant differences between W2mef and W2mefDelta 181 parasites in their capacity to invade these different erythrocytes (data not shown).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Invasion of human red blood cells by P. falciparum merozoites involves attachment, reorientation, and penetration of the host cell, and this is a complex process involving multiple ligand-receptor interactions (3, 4). EBA-175 was the first ligand identified in P. falciparum that functions in merozoite invasion by binding to the host receptor glycophorin A. The complexity of the invasion process and the ability of the parasite to utilize alternative invasion pathways that involves some redundancy suggests that there are other parasite proteins with similar function to EBA-175 (5, 7). The identification of EBA-175 homologues in the genome sequence of P. falciparum provides candidate ligands that may function in different alternate invasion pathways (29). We have shown that EBA-181 is a paralogue of EBA-175 and binds to the human erythrocyte.

P. falciparum is known to utilize a number of receptors on the erythrocyte surface for merozoite invasion including glycophorin A, B, and C and an unknown receptor X (6). EBA-175 is the parasite ligand for glycophorin A (14), whereas EBA-140 utilizes glycophorin C for merozoite invasion (17). The parasite ligand that binds to glycophorin B has not been identified, and receptor E has similar properties because ligand binding to both proteins is neuraminidase-sensitive, trypsin-resistant, and chymotrypsin-sensitive (6). We have ruled out the possibility that EBA-181 interacts with glycophorin B because this ligand binds efficiently to S-s-U- erythrocytes that lack this receptor. Similarly, receptor E does not correspond to glycophorin A or C because these sialoglycoproteins are trypsin-sensitive and chymotrypsin-resistant (11). Merozoite invasion via receptor X is neuraminidase-resistant (6, 12, 13) and does not correspond to receptor E. This suggests that EBA-181 binds to a novel sialoglycoprotein on the surface of human erythrocytes.

EBA-181 is a paralogue of EBA-175 and EBA-140 because it shares important features with these proteins and all bind to sialoglycoproteins on the human erythrocyte surface. Although the binding of these parasite ligands to their cognate receptor is sialic acid-dependent, they show distinctive binding specificities to erythrocyte receptors (12, 17). This suggests that the specificity of binding for each ligand is defined by the protein backbone of the receptor, and this has been shown directly for EBA-175 and glycophorin A (32). The binding of EBA-181 to erythrocytes is chymotrypsin-sensitive, suggesting that the specificity of receptor binding is narrow and likely to be a single receptor.

The demonstration that EBA-181 binds erythrocytes suggests that it plays a role in merozoite invasion and it is likely to function in an analogous manner to EBA-175 and EBA-140 as an alternative parasite ligand. Disruption of EBA-181 in W2mef did not affect the pattern or efficiency of merozoite invasion, suggesting that it is redundant in this parasite line. This was in contrast to our inability to disrupt EBA-181 in 3D7, suggesting that the protein may play a more important role in this genetic background. This would be analogous to the differential function of EBA-140 that has been observed in 3D7 and W2mef (17). It is likely that the differences in expression may reflect the relative role and importance of EBA-181 in these two strains. It was shown that EBA-140 function is significantly increased in 3D7 compared with W2mef, and this appears to be related to polymorphisms in the F1 domain of W2mef that decrease affinity of binding to glycophorin C (17).

EBA-181 and the other members of the ebl gene family, EBA-175, EBA-140, EBA-165, EBL-1, MAEBL, are all located in the subtelomeric region of different chromosomes (Fig. 9) (29) in the P. falciparum genome, and this provides a mechanism for expansion from an ancestral progenitor. The subtelomeric region of chromosomes has a conserved structure and contains a number of gene families and repetitive sequences that would favor pairing of heterologous chromosomes and ectopic recombination. This recombination may drive the generation of diversity in the subtelomeric variety gene family that is responsible for antigenic variation in P. falciparum. Ectopic recombination would provide a mechanism for duplication and expansion of gene families that are located in the subtelomeric region of the chromosomes (25, 33, 34). This suggests that the ebl gene family has arisen from a single precursor by recombination between heterologous chromosomes and then diverged to provide parasite ligands with different host receptor specificities.


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Fig. 9.   Chromosomal localization of the ebl gene family in P. falciparum. Chromosomes 1-14 are shown, and on the right are the approximate sizes in kilobase pairs. All members of the gene family including EBA-181 are located in the subtelomeric region of different chromosomes. The subtelomeric region consists of a number of gene families and conserved repetitive regions favoring pairing between heterologous chromosomes and ectopic recombination.

The P. falciparum ligands EBA-175, EBA-140, and EBA-181 are a family of functionally equivalent proteins in merozoite invasion of human erythrocytes. Binding of these ligands to their cognate receptor requires sialic acid, and they, therefore, function in sialic acid-dependent invasion pathways. The three ligands show some redundancy in function as the genes for all can be disrupted; however, this can be dependent on the genetic background (7, 17). For example, EBA-181 can be disrupted in W2mef but not 3D7, whereas EBA-175 can be disrupted in W2mef and 3D7 but not D10. The D10 parasite line already lacks the EBA-140 gene, and it is likely that a minimum complement of the parasite ligands are required for sialic acid-dependent invasion processes.

Why does P. falciparum have multiple ligands with equivalent functions that can be redundant in some parasite lines for merozoite invasion? The molecules on the surface of the host erythrocyte are highly polymorphic within the population and also during red blood cell development (1). Additionally, immune responses in an infected individual to a subset of the ligands involved in merozoite invasion could block particular invasion pathways. It would be highly advantageous for the parasite to have multiple ligands with different receptor specificities to circumvent blockage of a subset of invasion pathways. An important point in considering proteins such as EBA-181, EBA-175, and EBA-140 as vaccine candidates is that they can be in some cases functionally redundant and strain-dependent, and if they are to move forward as vaccine candidates, it will be important that they be used in combination with other proteins involved in different invasion pathways.

    ACKNOWLEDGEMENTS

We thank the Red Cross Blood Service (Melbourne, Australia) for supply of erythrocytes and serum. We thank the international Malaria Genome Project for making sequence data from the genome of P. falciparum public before publication of the completed sequence.

    FOOTNOTES

* This work was supported by the National Health and Medical Research Council of Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Funded by a Deutsche Forschunggemeinschaft Emmy Noether Fellowship.

§ A Howard Hughes International Scholar. To whom correspondence should be addressed: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Melbourne, 3050, Australia. Tel.: 61-9345-2555; E-mail: cowman@wehi.edu.au.

Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M211446200

    ABBREVIATIONS

The abbreviations used are: EBA, erythrocyte binding antigen; kb, kilobase(s).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Miller, L. H., Baruch, D. I., Marsh, K., and Doumbo, O. K. (2002) Nature 415, 673-679[CrossRef][Medline] [Order article via Infotrieve]
2. Butler, D. (2002) Nature 419, 426-428[CrossRef][Medline] [Order article via Infotrieve]
3. Barnwell, J. W., and Galinski, M. R. (1998) in Malaria: Parasite Biology, Pathogenesis and Protection (Sherman, I. W., ed) , pp. 93-120, American Society for Microbiology, Washington, D. C.
4. Chitnis, C. E., and Blackman, M. J. (2000) Parasitol. Today 16, 411-415[CrossRef][Medline] [Order article via Infotrieve]
5. Dolan, S. A., Miller, L. H., and Wellems, T. E. (1990) J. Clin. Invest. 86, 618-624[Medline] [Order article via Infotrieve]
6. Dolan, S. A., Proctor, J. L., Alling, D. W., Okubo, Y., Wellems, T. E., and Miller, L. H. (1994) Mol. Biochem. Parasitol. 64, 55-63[CrossRef][Medline] [Order article via Infotrieve]
7. Reed, M. B., Caruana, S. R., Batchelor, A. H., Thompson, J. K., Crabb, B. S., and Cowman, A. F. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 7509-7514[Abstract/Free Full Text]
8. Hadley, T. J., Klotz, F. W., Pasvol, G., Haynes, J. D., and McGinniss, M. H. (1987) J. Clin. Invest. 80, 1190-1193[Medline] [Order article via Infotrieve]
9. Haynes, J. D., Dalton, J. P., Klotz, F. W., McGinniss, M. H., Hadley, T. J., Hudson, D. E., and Miller, L. H. (1988) J. Exp. Med. 167, 1873-1881[Abstract]
10. Mayer, D. C., Kaneko, O., Hudson-Taylor, D. E., Reid, M. E., and Miller, L. H. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 5222-5227[Abstract/Free Full Text]
11. Thompson, J. K., Triglia, T., Reed, M. B., and Cowman, A. F. (2001) Mol. Microbiol. 41, 47-58[CrossRef][Medline] [Order article via Infotrieve]
12. Camus, D., and Hadley, T. J. (1985) Science 230, 553-556[Medline] [Order article via Infotrieve]
13. Sim, B. (1990) Mol. Biochem. Parasitol. 41, 293-296[Medline] [Order article via Infotrieve]
14. Orlandi, P. A., Klotz, F. W., and Haynes, J. D. (1992) J. Cell Biol. 116, 901-909[Abstract]
15. Adams, J. H., Sim, B. K. L., Dolan, S. A., Fang, X., Kaslow, D. C., and Miller, L. H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7085-7089[Abstract]
16. Sim, B. K., Carter, J. M., Deal, C. D., Holland, C., Haynes, J. D., and Gross, M. (1994) Exp. Parasitol. 78, 259-268[CrossRef][Medline] [Order article via Infotrieve]
17. Maier, A. G., Duraisingh, M. D., Reeder, J. C., Patel, S. S., Kazura, J. W., Zimmerman, P. W., and Cowman, A. F. (2003) Nat. Med. 9, 87-92[CrossRef][Medline] [Order article via Infotrieve]
18. Adams, J. H., Blair, P. L., Kaneko, O., and Peterson, D. S. (2001) Trends Parasitol. 17, 297-299[CrossRef][Medline] [Order article via Infotrieve]
19. Michon, P., Stevens, J. R., Kaneko, O., and Adams, J. H. (2002) Mol. Biol. Evol. 19, 1128-1142[Abstract/Free Full Text]
20. Cowman, A. F., and Crabb, B. S. (2002) Science 298, 126-128[Abstract/Free Full Text]
21. Triglia, T., Thompson, J. K., and Cowman, A. F. (2001) Mol. Biochem. Parasitol. 116, 55-63[CrossRef][Medline] [Order article via Infotrieve]
22. Crabb, B. S., and Cowman, A. F. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7289-7294[Abstract/Free Full Text]
23. Wu, Y., Kirkman, L. A., and Wellems, T. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1130-1134[Abstract/Free Full Text]
24. Duraisingh, M. T., Triglia, T., and Cowman, A. F. (2002) Int. J. Parasitol. 32, 81-89[CrossRef][Medline] [Order article via Infotrieve]
25. Rubio, J. P., Thompson, J. K., and Cowman, A. F. (1996) EMBO J. 15, 4069-4077[Abstract]
26. Triglia, T., Wang, P., Sims, P. F. G., Hyde, J. E., and Cowman, A. F. (1998) EMBO J. 17, 3807-3815[Abstract/Free Full Text]
27. Bianco, A. E., Favaloro, J. M., Burkot, T. R., Culvenor, J. G., Crewther, P. E., Brown, G. V., Anders, R. F., Coppel, R. L., and Kemp, D. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8713-8717[Abstract]
28. Galinski, M. R., Medina, C. C., Ingravallo, P., and Barnwell, J. W. (1992) Cell 69, 1213-1226[Medline] [Order article via Infotrieve]
29. Gardner, M. J., Hall, N., Fung, E., White, O., Berriman, M., Hyman, R. W., Carlton, J. M., Pain, A., Nelson, K. E., Bowman, S., Paulsen, I. T., James, K., Eisen, J. A., Rutherford, K., Salzberg, S. L., Craig, A., Kyes, S., Chan, M. S., Nene, V., Shallom, S. J., Suh, B., Peterson, J., Angiuoli, S., Pertea, M., Allen, J., Selengut, J., Haft, D., Mather, M. W., Vaidya, A. B., Martin, D. M., Fairlamb, A. H., Fraunholz, M. J., Roos, D. S., Ralph, S. A., McFadden, G. I., Cummings, L. M., Subramanian, G. M., Mungall, C., Venter, J. C., Carucci, D. J., Hoffman, S. L., Newbold, C., Davis, R. W., Fraser, C. M., and Barrell, B. (2002) Nature 419, 498-511[CrossRef][Medline] [Order article via Infotrieve]
30. Sim, B., Toyoshima, T., Haynes, J., and Aikawa, M. (1992) Mol. Biochem. Parasitol. 51, 157-159[CrossRef][Medline] [Order article via Infotrieve]
31. Narum, D. L., Fuhrmann, S. R., Luu, T., and Sim, B. K. (2002) Mol. Biochem. Parasitol. 119, 159-168[CrossRef][Medline] [Order article via Infotrieve]
32. Kain, K. C., Orlandi, P. A., Haynes, J. D., Sim, B. K. L., and Lanar, D. E. (1993) J. Exp. Med. 178, 1497-1505[Abstract]
33. Corcoran, L. M., Thompson, J. K., Walliker, D., and Kemp, D. J. (1988) Cell 53, 807-813[Medline] [Order article via Infotrieve]
34. Freitas-Junior, L. H., Bottius, E., Pirrit, L. A., Deitsch, K. W., Scheidig, C., Guinet, F., Nehrbass, U., Wellems, T. E., and Scherf, A. (2000) Nature 407, 1018-1022[CrossRef][Medline] [Order article via Infotrieve]
35. Trager, W., and Jansen, J. B. (1976) Science 193, 673-675[Medline] [Order article via Infotrieve]


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