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Variation in Plasmodium falciparum Erythrocyte Invasion Phenotypes and
Merozoite Ligand Gene Expression across Different Populations in Areas of
Malaria Endemicity
Article  in  Infection and immunity · April 2015
DOI: 10.1128/IAI.03009-14 · Source: PubMed
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Variation in Plasmodium falciparum Erythrocyte Invasion Phenotypes
and Merozoite Ligand Gene Expression across Different Populations
in Areas of Malaria Endemicity
Paul W. Bowyer,a* Lindsay B. Stewart,a Harvey Aspeling-Jones,a Henrietta E. Mensah-Brown,b Ambroise D. Ahouidi,c
Alfred Amambua-Ngwa,d Gordon A. Awandare,b David J. Conwaya
Pathogen Molecular Biology Department, London School of Hygiene and Tropical Medicine, London, United Kingdoma; Department of Biochemistry, Cell and Molecular
Biology, University of Ghana, Legon, Ghanab; Le Dantec Hospital, Universite Cheikh Anta Diop, Dakar, Senegalc; Medical Research Council Unit, Fajara, Banjul, The Gambiad
Plasmodium falciparum merozoites use diverse alternative erythrocyte receptors for invasion and variably express cognate li-
gands encoded by the erythrocyte binding antigen (eba) and reticulocyte binding-like homologue (Rh) gene families. Previous
analyses conducted on parasites from single populations in areas of endemicity revealed a wide spectrum of invasion phenotypes
and expression profiles, although comparisons across studies have been limited by the use of different protocols. For direct com-
parisons within and among populations, clinical isolates from three different West African sites of endemicity (in Ghana,
Guinea, and Senegal) were cryopreserved and cultured ex vivo after thawing in a single laboratory to assay invasion of target
erythrocytes pretreated with enzymes affecting receptor subsets. Complete invasion assay data from 67 isolates showed no dif-
ferences among the populations in the broad range of phenotypes measured by neuraminidase treatment (overall mean, 40.6%
inhibition) or trypsin treatment (overall mean, 83.3% inhibition). The effects of chymotrypsin treatment (overall mean, 79.2%
inhibition) showed heterogeneity across populations (Kruskall-Wallis P  0.023), although the full phenotypic range was seen in
each. Schizont-stage transcript data for a panel of 8 invasion ligand genes (eba175, eba140, eba181, Rh1, Rh2a, Rh2b, Rh4, and
Rh5) were obtained for 37 isolates, showing similar ranges of variation in each population except that eba175 levels tended to be
higher in parasites from Ghana than in those from Senegal (whereas levels of eba181 and Rh2b were lower in parasites from
Ghana). The broad diversity in invasion phenotypes and gene expression seen within each local population, with minimal differ-
ences among them, is consistent with a hypothesis of immune selection maintaining parasite variation.
The major human malaria parasite Plasmodium falciparum uses these (7), and some components may be best considered in com-diverse ligand-receptor interactions in merozoite invasion of binations (16).
erythrocytes (1). Parasite ligands include proteins belonging to If acquired immune responses to parasites can inhibit different
the erythrocyte binding antigen (EBA) and reticulocyte binding ligand-receptor interactions, a diversity of enzyme-sensitive
protein-like homologue (Rh) families, including EBA175, EBL-1, erythrocyte invasion phenotypes is expected to be maintained
EBA140, and Rh4, which bind, respectively, to glycophorin A within each endemic population by frequency-dependent im-
(GpA), GpB, GpC, and complement receptor 1 (CR1) on eryth- mune selection. This may be influenced by levels of endemicity,
rocytes (1–6). These ligand-receptor interactions are variably used such that a low incidence of infection and minimal acquired im-
by different P. falciparum lines, while interaction between mero- munity may allow many parasites to use a favored primary path-
zoite Rh5 and erythrocyte surface basigin is apparently used by all way, while a broader range of alternative invasion phenotypes may
lines (7, 8). Other members of these protein families expressed in
P. falciparum merozoites for which cognate erythrocyte receptors
have not yet been identified are EBA181 (9) and Rh1 (10, 11) and Received 11 December 2014 Returned for modification 8 February 2015
the closely related proteins Rh2a and Rh2b (12). Variation in in- Accepted 4 April 2015
vasion phenotypes has been widely characterized by assessing the Accepted manuscript posted online 13 April 2015
ability of parasites to invade erythrocytes treated with enzymes to Citation Bowyer PW, Stewart LB, Aspeling-Jones H, Mensah-Brown HE, Ahouidi
selectively remove parts of the receptor repertoire, following pio- AD, Amambua-Ngwa A, Awandare GA, Conway DJ. 2015. Variation in Plasmodium
neering studies in the 1980s (13, 14). In particular, neuraminidase falciparum erythrocyte invasion phenotypes and merozoite ligand gene
expression across different populations in areas of malaria endemicity. Infect
treatment removes sialic acids from glycophorins and other eryth- Immun 83:2575–2582. doi:10.1128/IAI.03009-14.
rocyte receptors, whereas trypsin treatment cleaves the peptide Editor: J. H. Adams
backbone of several receptors (including GpA, GpC, and CR1), Address correspondence to David J. Conway, david.conway@lshtm.ac.uk.
and chymotrypsin cleaves others (including GpB and CR1) (1, 2) * Present address: Paul W. Bowyer, National Institute of Biological Standards and
(see Table S1 in the supplemental material). Polymorphism and Control, South Mimms, Potters Bar, Hertfordshire, United Kingdom.
plasticity of invasion phenotypes may be adaptive for the parasite P.W.B. and L.B.S. contributed equally to this article.
due to selection by acquired immunity to individual merozoite Supplemental material for this article may be found at http://dx.doi.org/10.1128
ligands (7, 15) or possibly due to diversity in the structure and /IAI.03009-14.
abundance of receptors on erythrocytes. This is of applicable im- Copyright © 2015, American Society for Microbiology. All Rights Reserved.
portance, as particular ligands are being developed as vaccine an- doi:10.1128/IAI.03009-14
tigens, with EBA175 and Rh5 being the lead candidates among
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Bowyer et al.
FIG 1 Locations of the three sites in West Africa from which P. falciparum clinical isolates were sampled for assays of erythrocyte invasion and gene expression.
The map on the left shows the locations of Pikine in Senegal (44 isolates), Faranah in Guinea (23 isolates), and Kintampo in Ghana (63 isolates) and utilizes
graphics from d-maps.com (http://d-maps.com/carte.php?num_car753&langen). Of the 130 isolates tested, 67 yielded invasion phenotype data. On the
right, the area is shown within a map of Africa which indicates the estimated community prevalence of P. falciparum slide positivity in children aged between 2
and 10 years in 2010 (reprinted from the Malaria Journal [40]).
be selected for in areas of greater endemicity. Studies on clinical SD, 10.1 years). Patients attending Pikine health center in Dakar, Senegal,
isolates have indicated a high diversity of invasion phenotypes in were recruited in 2013; 44 of the patients with P. falciparum parasitemia of
India (17), Brazil (18, 19), Peru (19), Colombia (19), The Gambia 0.5% were included here (mean patient age, 8.9 years; SD, 3.8 years).
(20, 21), Kenya (22, 23), Tanzania (24), and Senegal (25, 26). Written informed consent was obtained from patients or their parents or
However, apart from a small number of isolates from Peru and guardians, and additional assent was received from children who were 10
Colombia that were cultured together in a single laboratory (19), years of age or older. Antimalarial treatment was provided according to
the relevant national and local guidelines. Immediately prior to treatment,
all other samples from each country were analyzed in different
venous blood samples of up to 5 ml in volume were collected into hepa-
laboratories at different times, using a variety of assay protocols. rinized Vacutainer tubes in Ghana and Guinea and into EDTA anticoag-
Therefore, these data do not enable a standardized analysis of ulant tubes in Senegal. At each site, blood samples were centrifuged,
variation within and between populations. plasma and leukocyte buffy coats were removed, and erythrocytes were
We report the first comparative analysis of erythrocyte inva- cryopreserved in glycerolyte and stored frozen at 80°C or in liquid ni-
sion and merozoite ligand gene expression by population samples trogen until shipment on dry ice to the London School of Hygiene and
of malaria parasites from different countries in regions of Tropical Medicine for assay. Approval of the study was granted by the
endemicity, assayed in the first parasite cycle ex vivo in a single Ethics Committees of the Ghana Health Service, the Kintampo Health
laboratory. Clinical isolates of P. falciparum from three different Research Centre, and the Noguchi Memorial Institute for Medical Re-
countries in West Africa were sampled and cryopreserved, prior to search (University of Ghana), the National Ethics Committee for Health
assaying the isolates together with identical protocols in a blind Research in the Republic of Guinea, the Ethics Committee of the Ministry
manner. Virtually all the diversity in invasion phenotypes and of Health in Senegal, and the Ethics Committee of the London School of
Hygiene and Tropical Medicine.
gene expression was seen within each local population, as ex- Parasite culture and erythrocyte invasion assays. A total of 130 iso-
pected from a hypothesis of immune selection maintaining para- lates that each had parasitemia represented by at least 0.5% infected eryth-
site variation at all of these sites of endemicity. rocytes were thawed and given serial codes for culture by one laboratory
investigator at the London School of Hygiene and Tropical Medicine.
MATERIALS AND METHODS Prior to parasite culture being initiated by the same investigator, erythro-
Plasmodium falciparum clinical isolates from three populations in ar- cytes from each sample were washed 4 times in total to ensure that no
eas of endemicity. Malaria patients attending local health facilities at anticoagulant or plasma components from the samples were included.
three different sites in West Africa (Fig. 1) who tested positive for P. The schedule typically involved processing between 6 and 10 isolates from
falciparum malaria by immunochromatic rapid diagnostic testing and a mixture of the different source population samples each week, and blind
who had reported not taking antimalarial drugs during the preceding 3 processing was ensured, as the subsequent invasion assays were conducted
days were invited to participate in the study. Patients attending Kintampo by a different laboratory investigator who had no role in the sample col-
Municipal Hospital in the Brong-Ahafo region of central Ghana were lection, storage, thawing, or culture initiation procedures. Parasites were
recruited in 2011 and 2012; 63 patients with P. falciparum parasitemia of cultured at 2% hematocrit in RPMI 1640 medium containing 2% human
0.5% infected erythrocytes were included in this study, with a mean AB serum (GE Healthcare, Amersham, United Kingdom) and 0.3%
patient age of 5.3 years (standard deviation [SD], 3.5 years). Patients at- Albumax II (Life Technologies, Paisley, United Kingdom) under an at-
tending the Tiro and Banian local health centers near Faranah in the mosphere of 1% O2, 3% CO2, and 96% N2.
Republic of Guinea were recruited in 2012; 23 patients with P. falciparum Erythrocytes (blood group A, rhesus positive) for each invasion assay
parasitemia of 0.5% were included here (mean patient age, 12.9 years; were labeled by incubation at 4% hematocrit in 5 M 7-hydroxy-9H-(1,3-
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Plasmodium falciparum Merozoite Variation
dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE) ume of TRIzol reagent (Life Technologies). Aliquots were stored at 80°C
fluorescent dye (Life Technologies, Paisley, United Kingdom)–RPMI for subsequent RNA extraction using an RNeasy Micro kit (Qiagen). The
1640 with rotation in the dark for 2 h at 37°C. The erythrocytes were then extracted RNA was quantified using Qubit fluorometric quantitation
washed twice with RPMI 1640 and suspended at 4% hematocrit into sep- (Life Technologies), and samples below the detection limit for this assay
arate tubes for alternative enzyme treatments. Neuraminidase (Vibrio were not carried forward for transcript analysis. The mRNA was reverse
cholerae; Sigma, United Kingdom) was used at a final concentration of 100 transcribed (RT) with oligo(dT) using TaqMan reagents (Life Technolo-
mU/ml, trypsin (TPCK [tosylsulfonyl phenylalanyl chloromethyl ketone] gies), and cDNA was quantified in a fluorogenic 5=-nuclease assay on a
treated, bovine) (Sigma, United Kingdom) at 104 U/ml, and chymotryp- Rotor-Gene 3000 system (Corbett Life Sciences/Qiagen). Gene-specific
sin (TLCK [N-p-tosyl-L-lysine chloromethyl ketone] treated) (Wor- TaqMan primer and probe sets for each gene were exactly as described
thington, USA) at 45 U/ml. All enzyme treatments were performed at previously for eba140, eba175, and eba181 in reference 28, for Rh1, Rh2a,
37°C for 1 h, following which erythrocytes were pelleted, washed 3 times Rh2b, and Rh4 in reference 29, and for Rh5 in reference 21. All primers and
in RPMI 1640, and suspended at 4% hematocrit. To check that the en- probes were designed to match unique sequences that had no polymor-
zyme treatments were effective, 20 l of each of the cell preparations was phisms in P. falciparum, and this was rechecked with all additional se-
added to 20 l of anti-M monoclonal antibody (MAb) (successful trypsin quence data available at www.plasmodb.org on 1 May 2013, revealing no
digestion of GpA leads to loss of agglutination), anti-S MAb (successful polymorphisms in any of the matching sequences except for a single nu-
chymotrypsin digestion of GpB leads to loss of agglutination), or peanut cleotide polymorphism (SNP) in 1 of 71 isolates in the sequence corre-
lectin (successful neuraminidase digestion of sialic acids leads to gain of sponding to the reverse primer for eba181. These protocols were used to
agglutination). The efficacy of enzyme treatment was also confirmed in allow direct comparisons with results from a previous study conducted in
one batch of erythrocytes by flow cytometric analysis, showing the ex- The Gambia, and as in that study, the ebl-1 gene which contains a stop
pected loss of GpA (CD235a), GpC (CD236), and CR-1 (CD35), as de- codon in many isolates was not analyzed here. Real-time PCRs were per-
tected by monoclonal antibodies (anti-CD235a MAb clone E4 [diluted formed using a Kapa Probe Fast quantitative PCR (qPCR) kit (Kapa Bio-
1/100], anti-CD236 MAb clone E5 [diluted 1/1,000], and anti-CD35 MAb systems) in 10-l volumes with 250 nM concentrations of each primer
clone J3D3 [diluted 1/100]; all antibodies from Santa Cruz Biotechnology and 250 nM concentrations of each probe at 95°C for 3 min followed by 40
Inc.). Effects of these enzyme treatments on different erythrocyte recep- cycles of 95°C for 3 s and 60°C for 30 s. Each run included controls and
tors as defined by previous studies are listed in Table S1 in the supplemen- 3D7 genomic DNA standards, with each sample assayed in duplicate and
tal material. standard curves generated during each run. The threshold fluorescence
Invasion assays were performed using a protocol based on a previously value was determined automatically by Rotor-Gene 3000 software for
described method (27), with each cell preparation tested in triplicate wells each run. The relative transcript level for each individual gene determined
of round-bottom 96-well plates, in a culture volume of 200 l per well at by RT-qPCR was normalized as the proportion of the sum of the tran-
1% hematocrit. Plates were gassed with 1% O2, 3% CO2, and 96% N2 in a script levels for the 8 genes assayed in each isolate.
hypoxia chamber (Billups Rothberg Inc., USA) and incubated at 37°C. Statistical analysis. Nonparametric statistics were used to analyze the
Assay plates were removed from the incubator after 48 h and centrifuged distribution of culture phenotypes. The Kruskal-Wallis test was applied to
at 450  g for 1 min to pellet the cells, and the supernatant was removed. test for statistical significance with respect to heterogeneity across the
To each assay well, 200 l of phosphate-buffered saline (PBS) containing three sampled populations. Where this was significant, Mann-Whitney
2 SYBR green I was added. The plates were incubated for 1 h at 37°C and tests were applied to investigate pairwise differences between different
then removed from the incubator and centrifuged as before, and the su- populations, with Bonferroni correction for open testing of multiple pair-
pernatant was removed. The cells were washed a further 3 times in PBS wise interpopulation comparisons. Correlations between the distribu-
before being suspended in PBS at a final hematocrit of 0.15% for flow tions of different enzyme inhibition phenotypes were tested by Spear-
cytometry on a FACSCalibur cell analyzer (BD Biosciences, CA, USA). man’s 	 (rho).
Erythrocytes were gated using forward-scatter and side-scatter character-
istics, and DDAO-SE-positive cells were determined by emission at 645 to RESULTS
677 nm following excitation at 635 nm, while levels of SYBR green
Erythrocyte invasion phenotype variation. The first ex vivo cycle
I-positive cells were determined by emission at 500 to 560 nm following
excitation at 488 nm. Invasion of enzyme-treated erythrocytes was deter- invasion phenotypes for all of the different enzyme pretreatments
mined by analysis of the proportion of 50,000 counted DDAO-SE-posi- were successfully obtained for 67 (52%) of the 130 clinical parasite
tive cells that were also SYBR green I positive, and data were analyzed isolates that had been thawed for short-term culture, including 34
using FlowJo (Tree Star Inc.). Percent inhibition of invasion of each type (54%) of 63 isolates from Ghana, 12 (52%) of 23 isolates from
of each treated cell preparation (TC) was calculated in comparison with Guinea, and 21 (48%) of 44 isolates from Senegal. Those that did
negative-control cells (NC [no enzyme treatment]) and positive-control not yield reliable results had either poor parasite growth or fewer
cells (PC [treated with all the enzymes, i.e., trypsin, chymotrypsin, and than 100 postinvasion parasitized erythrocyte counts that differed
neuraminidase]) as follows: between the untreated and triple-enzyme-treated erythrocytes.
   The proportions from each of the three populations that had suc-% TC infected  % NC infected% inhibition  1   100 cessful growth were not significantly different from those ob-% PC infected  % NC infected tained in previous assays of fresh West African clinical isolates
An isolate was determined to have passed quality control if the differ- without any cryopreservation step, including the largest study
ence between the mean percentages of infected cells (from triplicate well which yielded results for 163 (63%) of 263 isolates assayed in The
counts) in positive controls (PC) and negative controls (NC) was 0.2, Gambia (21).
corresponding to a difference of more than 100 parasites for 50,000 There was wide variability among the isolates in the ability to
counted erythrocytes from each well.
invade erythrocytes that had been treated with each of the en-
RNA extraction and quantitative transcript analysis. For parasite
RNA preparation, the equivalent of at least 100 l of packed erythrocytes zymes compared to the control erythrocytes (Fig. 2). The overall
from each isolate was cultured in complete RPMI 1640 at 2% hematocrit mean level of invasion inhibition by neuraminidase treatment was
and 37°C for up to 48 h in separate flasks until most parasites were at the 40.6% (range, 0 to 78.1%), and the mean levels were similar across
schizont stage of parasite development, following which erythrocytes were samples from the three different countries (Kruskal-Wallis test,
resuspended at 50% hematocrit and immediately mixed with a 4 vol- P  0.441) (Fig. 2A). Trypsin treatment of erythrocytes led to
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Bowyer et al.
greatly reduced invasion by most isolates, with an overall mean
inhibition level of 83.3% (range, 3.7% to 100%), and the mean
levels were similar across the different countries (Kruskal-Wallis
test, P  0.338) (Fig. 2B). Chymotrypsin treatment of erythrocytes
caused an overall mean invasion inhibition level of 79.2% (range,
13.9% to 100%), with significant heterogeneity across the samples
from different countries (Kruskal-Wallis test, P  0.023) (Fig.
2C). Isolates from Guinea were inhibited more by chymotrypsin
treatment (mean inhibition of 90.8%) than isolates from Ghana
(mean of 78.3%; Mann-Whitney uncorrected P  0.033, P cor-
rected for multiple pairwise comparisons  0.10) or Senegal
(mean of 74.1%; Mann-Whitney uncorrected P  0.004, P cor-
rected for multiple pairwise comparisons  0.012).
Across all isolates, the inhibition by neuraminidase treatment
of erythrocytes was positively correlated with inhibition by trypsin
treatment (Spearman’s rho  0.319, P  0.009) (Fig. 3A) but not
with inhibition by chymotrypsin treatment (Spearman’s rho 
0.016, P  0.9) (Fig. 3B). Invasion inhibition by chymotrypsin
was positively correlated with inhibition by trypsin (Spearman’s
rho  0.447, P 
 0.001) (Fig. 3C).
Analysis of transcript levels. In parallel with the invasion assay
cultures, ex vivo parasites were cultured for up to 48 h until they
reached stages consisting predominantly of schizonts, which
yielded sufficient RNA to allow relative transcript levels of 8 eba
and Rh genes to be successfully assayed for 37 isolates of the 67 that
had had their invasion phenotypes determined (26 from Ghana, 3
from Guinea, and 8 from Senegal). The remaining 30 isolates had
too low a yield of overall RNA for reliable quantification after
extraction from the limited amount of schizont-stage culture
available.
Overall, the eba gene transcripts were more abundant than the
Rh gene transcripts, with eba175 being most abundant (mean of
28% of the total for the 8 genes in each isolate) followed by eba140
and eba181 (means of 21% and 20%, respectively) (Fig. 4). The
profiles were broadly similar across the samples from different
countries. However, the relative levels of eba175 transcripts
tended to be lower in isolates from Senegal than in those from
Ghana (respective means of 17% and 32% of the total transcripts
across all 8 genes; Mann-Whitney uncorrected P  0.0002, Bon-
ferroni corrected P  0.005); conversely, the relative levels of
eba181 were higher in isolates from Senegal than in those from
Ghana (respective means of 26% and 18%; uncorrected P 
0.0012, P corrected for multiple comparisons  0.03). The relative
levels of Rh2b transcripts were also higher in isolates from Senegal
than in those from Ghana, although the differences were not sta-
tistically significant after correction for multiple comparisons (re-
spective means of 9% and 6%; uncorrected P  0.008, P corrected
FIG 2 Invasion phenotypes of P. falciparum isolates from Ghana (n  34),
Guinea (n  12), and Senegal (n  21), determined by the reduction in inva-
sion of erythrocytes treated with enzymes compared to untreated control
erythrocytes. Horizontal bars show the mean values within each group. (A)
Percentages of inhibition for all countries following neuraminidase treatment.
(B) Percentages of inhibition for all countries following trypsin treatment. (C)
Percentages of inhibition following chymotrypsin treatment. Pairwise Mann-
Whitney tests for differences between population samples in the chymotrypsin
treatment phenotypes showed two differences: for Ghana versus Guinea, un-
corrected P  0.033, P corrected for multiple pairwise comparisons  0.10; for
Guinea versus Senegal, uncorrected P  0.004, P corrected for multiple pair-
wise comparisons  0.012. Invasion data values for all individual isolates are
listed in Dataset S1 in the supplemental material.
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Plasmodium falciparum Merozoite Variation
FIG 3 Pairwise correlations of invasion phenotypes across isolates as defined by different enzyme pretreatments of erythrocytes. Sources of individual isolates
are identified by different shaded symbols as follows: Ghana, black; Guinea, white; and Senegal, gray. (A) Significant positive correlation between neuraminidase
treatment and trypsin treatment (Spearman’s rho  0.319, P  0.009). (B) No significant correlation between neuraminidase treatment and chymotrypsin
treatment (Spearman’s rho  0.016, P  0.9). (C) Significant positive correlation between trypsin treatment and chymotrypsin treatment (Spearman’s rho 
0.497, P 
 0.001).
for multiple comparisons  0.19) (Fig. 4). Profiling across the 8 P  0.047), although these were not significant after Bonferroni
transcripts for individual isolates showed that most had higher correction.
levels of eba175 transcripts than of transcripts of any of the other
genes, although a proportion of isolates (including 6 of the 8 Sen- DISCUSSION
egalese isolates) had higher levels of eba181 transcripts (Fig. 5). This first direct comparison of data reflecting erythrocyte invasion
Among the isolates from Ghana, tests were performed for corre- by clinical P. falciparum isolates from three different countries in
lations between the 8 individual gene transcript levels and the 3 regions of endemicity shows that almost all of the overall pheno-
different enzyme inhibition measurements. Of the 24 correlations typic diversity is contained within each local population. There
tested, two were marginally significant without correction for were no significant differences among the populations in the in-
multiple comparisons (neuraminidase inhibition versus eba140 vasion inhibition of clinical isolates by neuraminidase or trypsin
relative levels, r  0.47, uncorrected P  0.017; chymotrypsin treatment of target erythrocytes. A slightly greater effect of chy-
inhibition versus eba181 relative levels, r  0.39, uncorrected motrypsin treatment on the invasion of parasites from Guinea
FIG 4 Relative transcript levels of 3 eba genes and 5 Rh genes in ex vivo schizont-stage cultures of P. falciparum clinical isolates. (A) Isolates from Ghana (n  26).
(B) Isolates from Guinea (n  3). (C) Isolates from Senegal (n  8). Each dot shows the percentage of transcript for each gene in a single clinical isolate as a
proportion of the total for all 8 genes in the isolate. Horizontal lines denote the mean for each transcript across all isolates sampled in each population. Levels of
eba175 were higher in isolates from Ghana than in isolates from Senegal (Mann-Whitney uncorrected P  0.0002, P corrected for multiple comparisons  0.005);
conversely, the levels of eba181 were higher in isolates from Senegal than in isolates from Ghana (Mann-Whitney uncorrected P  0.0012, P corrected for
multiple comparisons  0.03), as were the relative levels of Rh2b (Mann-Whitney uncorrected P  0.008), although these data were not statistically significant
after correction for multiple comparisons (P  0.19). The results of all other pairwise tests comparing transcript levels between countries were not statistically
significant. Transcript data values for all individual isolates are listed in Dataset S1 in the supplemental material.
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Bowyer et al.
FIG 5 Individual isolate expression profiles for 8 eba and Rh genes in ex vivo schizont-stage cultures. (A) Isolates from Ghana (n  26 [two panels]). (B) Isolates
from Guinea (n  3). (C) Isolates from Senegal (n  8). The percentage of each transcript is normalized as a proportion of the sum of all 8 gene transcripts. To
assist visual clarity, the plotted order of the 8 genes for each isolate is the same as the order of the genes in the legend.
than on those from Ghana and Senegal is not likely to reflect involved with invasion is tightly regulated as part of the parasite
differences in endemicity, as the disease is more endemic in the developmental cycle (30, 31), and variation between parasites is
sampled sites in both Guinea and Ghana than in the site in Sene- known to be under epigenetic regulation (32–34), but the selective
gal. The correlation between the levels of inhibition by trypsin and determinants of transcript profiles seen in vivo are not well under-
chymotrypsin seen here was not seen in previous studies on West stood. A diversity of eba and Rh gene expression profiles has been
African parasites, conducted in Senegal or The Gambia (21, 25). previously seen in separate studies of clinical isolates from indi-
This might reflect differences in protocols among laboratories or vidual populations, but epidemiological correlations with func-
variations in the activities of commercial enzyme batches or re- tion or host immunity remain to be established (21, 24, 25, 29). In
ceptor properties of target erythrocytes used for the assays, illus- a large study of Gambian children, there were no differences in
trating the value of conducting comparisons within a single labo- expression of these genes between isolates from patients with se-
ratory where possible. vere malaria and those from patients with mild malaria, although
Expression of genes encoding P. falciparum merozoite ligands expression of eba175 correlated negatively with patient age,
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Plasmodium falciparum Merozoite Variation
whereas expression of eba181 correlated positively (21). It was based on known parasite ligands involved in invasion would need
suggested that use of EBA175 might be favored by parasites in to incorporate a broad range of alternative EBA and Rh proteins in
nonimmune hosts but that acquired immune responses to order to be effective for use in any local population, unless an
EBA175 may select for parasites using alternative receptors such as effective vaccine can be based on a ligand such as Rh5 which ap-
EBA181 in older individuals. In the present study, isolates from pears to be consistently required for invasion (7).
Ghana had slightly higher levels of eba175 gene expression, and
concomitantly lower levels of eba181, than isolates from Senegal. ACKNOWLEDGMENTS
Although the Ghanaian patients were slightly younger on average We are grateful to all the malaria patients and clinical staff for their par-
than those in Senegal, the endemicity of malaria was lower overall ticipation in the study. The sample collection was facilitated by staff at
at the study site in Senegal, so this observed difference in expres- Kintampo Health Research Centre, at the National Institute for Public
sion does not correlate with a simple prediction based on likely Health in Guinea, and at Pikine Health Centre in Senegal. We appreciate
differences in immunity. The expression profiles seen for each of the support of many colleagues at the Medical Research Council Unit in
the samples here fall within the range of those reported in the The Gambia, the Laboratory of Bacteriology and Virology, Le Dantec
previous large study from The Gambia (21). Hospital in Senegal, Noguchi Memorial Institute for Medical Research
The phenotypes and expression profiles described here that and the University of Ghana, and the London School of Hygiene and
correspond to the first replicative cycle ex vivo necessarily refer to Tropical Medicine in enabling this work. We thank Amy Bei, Michel
Theron, and Julian Rayner for helpful advice and discussion on invasion
total parasite populations sampled from each infected patient assay protocols.
rather than to isolated individual parasite types. Most clinical P. This study was funded by an ERC Advanced Award (grant AdG-2011-
falciparum infections in these areas of West Africa contain a mix- 294428 to D.J.C.) and a Leverhulme-Royal Society Africa Award (grant
ture of different parasite genotypes (35, 36), so analysis of individ- AA110050 to G.A.A. and D.J.C.).
ual genotypes and replication of assays on parasite lines would
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