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
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ABSTRACT |
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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.
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.
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 pHTK 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,
W2mef 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
W2mef 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.
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).
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.
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).
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.
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 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 pHTK
The 3D7 parasites transfected with pHTK
In contrast to 3D7, analysis of W2mef parasites transfected with
pHTK
Southern hybridization with genomic DNA from W2mef
Western blot analysis and immunofluorescence microscopy were used to
determine the expression of EBA-181 in W2mef 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 W2mef 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 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 pHTK
181W2mef
and pHTK
1813D7.
181c1, and W2mef
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).
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
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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.
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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.
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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.
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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.
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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.
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.
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 pHTK
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 pHTK 181 in the EBA-181
locus. The positive selection cassette (hDHFR), represented
by the black box of the pHTK-
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 pHTK
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 pHTK
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 pHTK
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 W2mef
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.
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
pHTK
181 plasmid or constructs containing different regions of the
gene were unsuccessful. We conclude that disruption of
EBA-181 in 3D7 is deleterious.
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 W2mef
181c1 and
W2mef
181c2
181c1 and
W2mef
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 pHTK
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).
181c1 and W2mef
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 W2mef
181c1 or W2mef
181c2, confirming
that they do not express the EBA-181 protein. Immunofluorescence assays
were performed to visualize EBA-181 expression in schizonts from W2mef,
W2mef
181c1, and W2mef
181c2. EBA-175 was detected in
W2mef
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
W2mef 181 parasites. A, Western
blot analysis of EBA-181 expression in W2mef
181 (W2mef
181c1 and
W2mef
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, W2mef
181.
EBA-181 is not expressed in W2mef
181 parasites. DAPI,
4,6-diamidino-2-phenylindole.
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
W2mef
181 parasites in their capacity to invade these different
erythrocytes (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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The abbreviations used are: EBA, erythrocyte binding antigen; kb, kilobase(s).
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