Cell, Vol. 104, 153–164, January 12, 2001, Copyright 2001 by Cell Press
A Central Role for P48/45 in Malaria
Parasite Male Gamete Fertility
midgut to establish the parasitic infection of the vector.
Sex is, therefore, an obligatory part of the life cycle of
the malaria parasite.
The central role of zygote formation in the life cycle
and transmission of the parasite makes gametes and
zygotes attractive targets for interruption strategies to
approach to block transmission of malaria parasites is
immunization of the human host with parasite proteins
that will generate a transmission-blocking immune re-
ous surface proteins of the gametes and zygotes have
been proposed as candidate antigens for the develop-
against P. falciparum, for example, Pfs48/45, Pfs230,
and Pfs25/28 (Kaslow et al., 1988; Kocken et al.,1993;
have successfully targeted events during sexual devel-
opment of the parasite, resulting in reduction of trans-
mission capacity (Kaslow, 1996; Carter et al., 2000).
Antibodies against Pfs48/45 cause a significant re-
duction of transmission due to the inhibition of zygote
development and mosquito infection (Vermeulen et al.,
1985; Carter et al., 1990; Targett et al., 1990; Roeffen et
al., 1996). Pfs48/45 is specifically expressed in gameto-
cytes and gametes and is thought to be anchored at
the parasite surface by a glycosylphosphatidylinositol
linkage (Vermeulen et al., 1986; Kocken et al., 1993). It
contains two copies of a six-cysteine (6-cys) structural
domain, unique to Plasmodium, and is a member of a
conserved structure based upon these 6-cys domains
(Carter et al., 1995; Templeton and Kaslow, 1999). Blast
analysis of sequences present in the P. falciparum ge-
nome sequencing project has identified nine proteins in
this family to date, including Pf12, Pf47, and Pfs230 and
its paralog (J. T., unpublished data; Elliott et al., 1990;
Templeton and Kaslow,1999). Pfs230, like Pfs48/45, is
present in gametocytes and gametes and appears to
physically interact with Pfs48/45 (Kumar, 1987). The ex-
pression pattern and precise function of other members
of the P48/45 superfamily is currently unknown. Deter-
mination of the essential nature, function, and interac-
cycle is crucial for a rational approach to improve and
develop effective transmission-blocking components
for a malaria vaccine (Kaslow, 1996).
Genetic modification of malaria parasites is now an
established approach to examine the function of pro-
genes expressed in the mosquito stages of parasite
development (Dessens et al., 1999; Wengelnik et al.,
1999; Me ´nard, 2000). In this study, we disrupted the
pfs48/45 of P. falciparum and isolated and disrupted
son of the phenotype of knockout (ko) parasites of both
species demonstrated a central and conserved role for
P48/45 in male gamete fertility.
Melissa R. van Dijk,*?Chris J. Janse,*?
Joanne Thompson,* Andrew P. Waters,*§
Joanna A. M. Braks,†Huub J. Dodemont,†
Henk G. Stunnenberg,†Geert-Jan van Gemert,‡
Robert W. Sauerwein,‡and Wijnand Eling‡
*Laboratory for Parasitology
Leiden University Medical Center
P.O. Box 9600
2300 RC Leiden
†Department of Molecular Biology
University of Nijmegen
6525 ED Nijmegen
‡Department of Medical Microbiology
University Medical Centre St. Radboud
P.O. Box 9101
6500 HB Nijmegen
Fertilization and zygote development are obligate fea-
tein PFS48/45 is expressed by male and female ga-
metes of Plasmodium falciparum and PFS48/45 anti-
bodies preventzygote developmentand transmission.
Here, gene disruption was used to show that Pfs48/
45 and the ortholog Pbs48/45 from a rodent malaria
parasite P. berghei play a conserved and important
role in fertilization. p48/45?parasites had a reduced
capacity to produce oocysts in mosquitoes due to
greatly reduced zygote formation. Unexpectedly, only
male gamete fertility of p48/45?parasites was af-
fected, failing to penetrate otherwise fertile female
gametes. P48/45 is shown to be a surface protein of
malaria parasites with a demonstrable role in fertil-
The malaria parasite (Plasmodium spp.) must infect its
Anopheline mosquito vector to undergo transmission
between vertebrate hosts. A developmental pathway
is initiated when the mosquito ingests the specialized
sexualprecursor cells(gametocytes)ofthe parasitethat
circulate in the blood of an infected host. Within the
mosquito, these gametocytes rapidly differentiate to
zygotes. The zygote develops withinthe blood meal into
a motile form, the ookinete, which traverses the midgut
wall forming an oocyst on the hemolymph side of the
§To whom correspondence should be addressed (e-mail: waters@
?These authors contributed equally to the work.
Figure 1. Comparison of the P48/45 Proteins
and Organization of Their Genetic Loci
(A) Comparison of the predicted amino acid
sequence of the P48/45 protein of P. berghei,
P. falciparum, and the nonhuman primate
parasite P. reichenowi. The different do-
mains, including the two six-cysteine do-
mains, are shaded and schematically shown.
Arrowheads indicate the conserved cysteine
residues. *, identical; :, conserved; ., semi-
conserved substitution. S, secretory signal
sequence; 6C, six-cysteine domain; IR, in-
tervening region; A, anchor domain.
(B) Schematic diagram ofthe P48/45 and P47
genome organization in P. falciparum and P.
berghei. Overlapping P. berghei genomic
clones, 47.1 and pBL1, and the position of
oligonucleotide primers used to amplify the
intergenic regions are shown.
P48/45 and the P47 gene in the genome of
P. falciparum (Pf) and P. berghei (Pb).
pfs48/45. Western analysis of gametocytes using spe-
cific polyclonal antiserum revealed a (broad) band of
Pbs48/45 of 53 kDa, most probably consisting of a pro-
tein doublet that under reducing conditions migrated as
a single well-resolved 55 kDa protein comparable to
Pfs48/45 (Figure 2D).
Identification of a Pfs48/45 Ortholog in P. berghei
pbs48/45 was isolated from a P. berghei genomic DNA
library. Pfs48/45 and Pbs48/45 are 71% and 54% identi-
cal at the DNA and protein level, respectively (Figure
1A). All cysteine residues are positionally conserved
apart from a single cysteine residue that is also not
zee parasite closely related to P. falciparum (Figure 1A).
pbs48/45 is located on one of the two largest chromo-
somes of P. berghei (chromosome 13/14; Figure 2B).
Transcription of pbs48/45 was in young (24 hr) and ma-
ture (30 hr) gametocytes (Figure 2C) and comparable to
P. berghei also Contains an Ortholog of pf47,
Linked to pbs48/45 in the Genome
BLAST analysis of the Pfs48/45 protein sequence
against the P. falciparum genome sequence database
uously and most closely with Pf47, a gene described by
Templeton and Kaslow (1999) (identity 26%, similarity
P48/45 Is Central to Plasmodium Gamete Fertility
Figure 2. Generation of Pbs48/45ko Parasites of P. berghei and Analysis of the Genotype and Expression of Pbs48/45
(A) Schematic diagram of the pbs48/45-locus and the replacement vector p54 used to disrupt the Pbs48/45 gene. This vector contains the
TgDHFR/TS selection cassette flanked by pbs48/45 targeting sequences for replacement. Correct integration of the BamHI/KpnI fragment of
p54 results in the disrupted gene as shown. Open box, pbs48/45 untranslated regions; black box, pbs48/45 coding region; hatched box, T.
gondii DHFR/TS selection cassette; HIII, HindIII.
(B) Chromosome analysis of P. berghei wt and Pbs48/45ko parasites. Chromosomes were hybridized to a pbs48/45-specific probe (48/45;
first panel) and to a probe specific for the P. berghei dhfr/ts 3? UTR region (DT-3?; second and third panel). This latter probe hybridizes to the
dhfr/ts locus on chromosome 7 and the pbs45/48 locus in the Pbs48/45ko parasites as a result of integration of the selection cassette.
(C) Transcription of the Pbs48/45 and Pbs47 genes during blood stage development in wt and Pbs48/45ko parasites. The transcription pattern
of the Pbs48/45 and Pbs47 genes in the wt parasite clone is shown in the left hand panel. Total RNA isolated from old trophozoites/young
schizonts (16, 19, and 22 hr post invasion (hpi)) and from purified young (24 hpi) and mature (30 hpi) gametocytes (gam.) was subsequently
hybridized to a pbs48/45 locus- (48/45) and a pbs47 locus- (47) specific probe. In the right hand panel, transcription of the Pbs48/45 and
Pbs47 genes in young gametocytes (24 hpi) of wt and Pbs48/45ko parasite clones is shown.
(D) Western blot analysis of expression of Pbs48/45 in gametocytes of wt and ko parasites. Proteins were reacted with a polyclonal antiserum
raised against a recombinant Pbs48/45 protein. A polyclonal antiserum against P. falciparum ?-tubulin was used as a control for the amount
of protein loaded. NR, nonreduced; R, reduced.
(E) Southern blot analysis of HindIII-digested genomic DNA from wt and ko parasites demonstrates the expected disruption of the Pbs48/45
gene. DNA was hybridized to the probe DT-48/45, which detects the dhfr/ts locus as well as the pbs48/45 locus. In wt parasites, a 5 kb
fragment containing the dhfr/ts locus and a 10 kb fragment derived from the pbs48/45 locus hybridized. In ko parasites, the 10 kb fragment
increased in size to 13 kb and an additional fragment of 0.7 kb derived from the pbs48/45 locus was obtained as a result of the integration
event. The lanes KoB2m and KoB3m contain DNA collected from blood stages after mosquito (m) transmission of the Pbs48/45ko parasites
KoB2 and KoB3.
(F) PCR analysis of genomic DNA of wt and ko parasites demonstrates correct disruption of pbs48/45. Using primers #450/#451 that specifically
amplify the wt Pbs48/45 gene showed a PCR fragment of 1.5 kb in wt parasites (upper panel). Integration-specific PCR primers #450/#313
amplified the expected fragment of 1.3 kb only in the Pbs48/45ko parasites. The lanes KoB1–4m contain fragments amplified from DNA
collected from blood stages after mosquito (m) transmission of the ko parasites.
43%). It has been found before that two genes ex-
pressed during sexual development, pfs230 and pfs25,
are closely linked to paralogous genes in the Plasmo-
dium genome (Duffy and Kaslow, 1997; Gardner et al.,
1998). We therefore examined the possible linkage of
the Pfs48/45 and Pf47 genes. Assembly of sequence
data of pfs48/45 and pf47 obtained from early release of
sequences from the P. falciparum genome sequencing
projects indicated that pf47 is closely linked to pfs48/
45(Figure 1B)andlies only1.5kb immediatelyupstream
of pfs48/45 (Figure 1C).
We identified a potential ortholog of pf47, pb47, as a
sequence tag in the P. berghei gene sequence tag proj-
ect (clone UFL_258PbD10, see Experimental Proce-
dures) and used it as a probe to isolate the complete
and with Pfs48/45 of only 27%. All the cysteine residues
within the 6-cys domains of Pb47 were conserved. pb47
lies approximately 1.5 kb upstream of pbs48/45 (Figure
1C) and is also transcribed in gametocytes (Figure 2C).
Therefore, the genomic organization of the P48/45 and
P47 genes is conserved in P. falciparum and P. berghei
and they are a third paralogous gene pair expressed
during sexual development.
these parasites contain pI48 correctly integrated at the
pfs48/45-locus (some in a concatemeric arrangement),
but also episomally maintained plasmids (Figures 3B
No Pfs48/45 protein could be detected in most of the
Pfs48/45ko clones by immunofluorescence assay (IFA)
and Western blot analysis of proteins from purified ga-
metocytes (Figures 3D and 3E) whereas in wt parasites,
all mature (male and female) gametocytes reacted
strongly by IFA (Figure 3D). The level of transcription of
ble in Pfs48/45ko and in wt parasites as determined by
45ko clones, however, we could detect by PCR low
levels ofparasites with thewt genotype, evenin multiply
cloned lines. Some pfs48/45ko gametocytes of the
clones (f 10?4–10?6) reacted strongly with anti-Pfs48/45
antibodies by IFA. Phenotype analysis of Pfs48/45ko
parasites may, therefore, be complicated by low fre-
quency reversion to the wild type. Pfs230 has been
shown to form a complex with Pfs48/45 at the gameto-
cyte/gamete surface (Kumar, 1987). Western blot analy-
sis using mAb 18F25 specific for Pfs230 demonstrated
sites (Figure 3E and data not shown).
Generation of P48/45ko Parasites
To study the function of the P48/45 proteins, P48/45ko
parasites of P. berghei and P. falciparum were gener-
ated. The P. berghei Pbs48/45 gene was disrupted by
ure 2A). Four Pbs48/45ko clones from two independent
experiments, KoB1–4, were analyzed further by PCR
and Southern blot analyses of separated chromosomes
and restriction fragments (Figures 2B, 2E, and 2F) to
confirm the expected disruption of the wild-type (wt)
Pbs48/45ko parasites produced normal numbers of
female and male gametocytes (see below). Northern
analysis of total RNA and Western analysis of proteins
obtained from purified gametocytes of the Pbs48/45ko
parasites demonstrated that the disrupted Pbs48/45
gene was not transcribed and the protein was absent
(Figures 2C and 2D). The level of transcription of the
paralog pbs47 in Pbs48/45ko parasites was unaffected
(Figure 2C). Therefore, phenotypic effects observed in
Pbs48/45ko parasites are solely due to a lack of Pbs48/
45 in this mutant.
The P. falciparum Pfs48/45 gene was disrupted using
plasmid pI48 that generates two nonfunctional copies
of pfs48/45 in the P. falciparum genome following site-
specific integration of pI48 (Figure 3A). In contrast to the
of p54 in P. berghei, the single crossover mechanism
of integration used in P. falciparum is potentially a re-
of the wt gene in parasites as has been described for P.
berghei (Me ´nard and Janse, 1997). In two independent
and contained a disrupted Pfs48/45 gene. Two clones
from each experiment, KoF1–4, were chosen for further
analysis. PCR,Southern blot,and plasmidrescue analy-
ses of Pfs48/45ko parasite DNA demonstrated that
P48/45ko Parasites Produce Normal Numbers
of Gametocytes and Gametes
As expected, disruption of the P48/45 gene had no ob-
servable effect on blood stage asexual development. In
P. berghei, the multiplication rate of asexual parasites
Pbs48/45ko and wt parasites. In P. falciparum, Pfs48/
45ko parasites showed a normal growth and multiplica-
tion of P48/45 did not affect the production or ratio of
male and female gametocytes in either P. berghei or P.
falciparum (Tables 1 and 2). Furthermore, every charac-
teristic of the development of P48/45ko gametocytes
into gametes was comparable to wt; conversion rate,
morphology,male gametemotility,and theirattachment
to uninfected erythrocytes, resulting in the presence
of characteristic exflagellation centers, female gamete
emergence, and their expression of the gamete/zygote-
specific protein Pbs21 or Pfs25 on their surfaces, re-
spectively (Table 2; Figure 4B).
Zygote Formation and Transmission Capacity
Is Strongly Impaired in P48/45ko Parasites
Although disruption of the P48/45 gene did not have an
observable effect on gametocyte and gamete develop-
ment, there was a dramatic effect on development of
both P. falciparum and P. berghei zygotes and ooki-
netes. In vitro, 52%–69% of the wt P. berghei female
gametes compared with only 0.001%–0.03% of Pbs48/
45ko female gametes developed into mature ookinetes
(Table 2). In P. falciparum, ookinetes were readily ob-
served in midguts of mosquitoes infected with wt para-
sites whereas no ookinetes were found in mosquitoes
infected with Pfs48/45ko parasites (Table 1).
To test whether the observed inhibition of ookinete
P48/45 Is Central to Plasmodium Gamete Fertility
Figure 3. Generation of Pfs48/45ko Parasites (clone KoF1-KoF4) of P. falciparum and Analysis of the Genotype and Expression of Pfs48/45
(A) Schematic diagram of the pfs48/45-locus and insertion vector pI48 used to disrupt the Pfs48/45 gene. This vector contains a TgDHFR/TS
selection cassette and a truncated copy of the Pfs48/45 gene, which serves as the site for homologous recombination. Correct site-specific
integration of this plasmid results in the disrupted gene as shown. Open box, genomic DNA; black box, pfs48/45 ORF; hatched box, T. gondii
DHFR/TS selection cassette; dotted line, plasmid sequences; HcII, HincII.
(B) Southern blot analysis of HincII-digested genomic DNA from wt and Pfs48/45ko parasites demonstrates the expected disruption of Pfs48/
45 gene. DNA was hybridized to the pfs48/45-specific probe. In wt parasites, this probe hybridized to a single fragment of 8 kb, whereas it
hybridizes to three fragments in Pfs48/45ko parasites. Two fragments of 4 kb and 12 kb arise as a result from correct integration of the
insertion plasmid in the Pfs48/45 gene, whereas the third band of 7.3 kb corresponds to either the presence of episomally maintained plasmids
or the existence of multiple copies of the insertion plasmid in the genome. In the Pfs48/45ko clones, no hybridization was obtained with the
8 kb fragment of the wt gene. The presence of episomes was confirmed by performing plasmid rescue experiments (data not shown). KoP,
parental Pfs48/45ko parasite population.
(C) PCR analysis ofgenomic DNA of wt and Pfs48/45koparasites demonstrates correct disruption of Pfs45/48 genein the Pfs48/45ko parasites.
Primers #428/#429 specifically amplify a 1.2 kb fragment of the wt Pfs48/45 gene (upper panel). Integration-specific primers #428 and #430
amplified the recombinant 1.3 kb fragment only in the Pfs48/45ko parasites (lower panel).
(D) Expression of Pfs48/45 in gametocytes of wt and Pfs48/45ko parasites as shown in an IFA using FITC-conjugated mAb 32F3. Pfs48/45
is present in both male and female wt gametocytes and absent in gametocytes of the Pfs48/45ko (KoF3) clone.
(E) Western blot analysis of protein expression in gametocytes of wt and Pfs48/45ko parasites. Proteins were reacted with the following
polyclonal antisera: anti-Pfs48/45 (K96); anti-Pfs16, a P. falciparum gametocyte protein; P. falciparum ?-tubulin and mAb 18F25 specific for
Pfs230. R, reduced; NR, nonreduced.
formation in the P48/45ko parasites correlated with a
reduction in transmission capacity, as defined by the
rate of oocyst production, mosquitoes were fed with
Pfs48/45ko and Pbs48/45ko gametocytes. P48/45ko
oocyst production was strongly reduced compared to
oocyst production in wild-type parasites (Tables 1 and
2). In P. falciparum, a maximum of 2.5% of the mosqui-
pared to an average wt infection rate of 93% (max. 180
oocysts per mosquito). It is possible that these oocysts
resulted from fertilization of gametes that have reverted
Pbs48/45ko oocyst numbers are 27–63 times lower
compared to wt parasites (Table 2). In the mosquito
midgut, an average of 0.09% of Pbs48/45ko female ga-
metes developed into ookinetes, which is significantly
than the average in vitro conversion rate of 0.02%.
Therefore, Pbs48/45ko parasites were able to produce
oocysts at a significantly higher rate than expected on
oocysts had normalmorphology under light microscopy
and produced sporozoites 10–12 days after mosquito
infections. Pbs48/45ko sporozoites were able to infect
naı ¨ve mice as effectively as wt P. berghei sporozoites
(data not shown). Transmitted Pbs48/45ko parasites
Table 1. Gamete Formation in Pfs48/45ko Parasites Is Normal but Ookinete and Oocyst Production Is Strongly Inhibited
? : ?
Range; n ? 8
Range; n ? 12
Range; n ? 2
Range; n ? 5
aGametocyte production is the number of mature gametocytes per 102erythrocytes at day 14 after start of the cultures and the pfs48/45ko
parasites (KoF1–3) are comparable to wild type.
bOokinete production is the mean number of ookinetes per mosquito midgut 21 hr after feeding.
cOocyst production is the mean number of oocysts at day 7 after feeding mosquitoes.
and the observed reduction in the rate of ookinete and
oocyst production of Pbs48/45ko parasites (data not
To test the fertility of Pbs48/45ko male gametes, the
determined after mixing Pbs48/45ko male and female
gametes with wt female gametes. Formation of wt male
gametes was blocked by the DNA polymerase I inhibitor
Aphidicolin. Three independent crosses produced no
ookinetes (Table 3; cross B), indicating that Pbs48/45ko
male gametes were unable to fertilize wt female ga-
metes. To prove the viability of the wt female gametes
in cross B after exposure to Aphidicolin, wt female ga-
metes were mixed with male and female gametes of the
Pbs21ko clone. This clone produces normal numbers
of gametes and ookinetes that lack the Pbs21 protein
on the surface (A. Tomas et al., submitted). The conver-
mined by counting the number of ookinetes expressing
Pbs21 visualized with a specific mAb. Large numbers
of ookinetes were Pbs21 positive (Table 3; cross A),
demonstrating that wt female gametes could be fertil-
ized by the Pbs21ko male gametes. Pbs21-negative
ookinetes were also present resulting from normal self-
fertilization of the Pbs21ko parasites. Therefore, wt fe-
male gamete fertility is unaffected after Aphidicolin
treatment (cross A) and male Pbs48/45ko gametes are
infertile (cross B).
Since the P48/45 protein is abundantly present on the
to contribute to the lack of self-fertilization of P48/45ko
parasites. To investigate the fertility of the Pbs48/45ko
female gametes, we again mixed gametes of the Pbs48/
45ko parasites with gametes of the Pbs21ko clone and
counted Pbs21-positive and -negative ookinetes (Table
3; cross C). If Pbs48/45ko females were infertile, only
Pbs21-negative ookinetes from the self-fertilization of
Pbs21ko mutant gametes would be observed. Unex-
pectedly, in five independent repetitions of this assay,
we observed ookinetes that reacted strongly with the
tion of Pbs48/45ko female gametes by Pbs21ko males
(Table 3, cross C; Figure 4B). The conversion rate of the
rable to the conversion rates obtained in standard fertil-
ization assays of wt parasites (Table 3, cross C). There-
fore, disruption of Pbs48/45 does not affect fertility of
female gametes or further development of zygotes.
Pbs48/45ko Male Gametes Have an Impaired Ability
to Attach to and Penetrate Female Gametes
The reduced capacity of P48/45ko gametes to develop
tion or subsequent zygote development. The role of
ization assays. The behavior of gametes was examined
in vitro after induction of gamete development. In six
independent preparations of wt parasites, motile male
female gametes. Individual fertilization events involving
male penetration of a female gamete were readily ob-
served (a range of 6–18 fertilization events per prepara-
tion). In contrast, in ten independent preparations of
Pbs48/45ko parasites (clone KoB2 and KoB3), no single
fertilization event was detected. Motile Pbs48/45ko
male gametes attached only to erythrocytes and not to
female gametes. The observed fertilization defect was
confirmed by examination of Giemsa-stained parasites
prepared 1 hr after induction of gamete formation. In
two independent preparations of wt parasites, 46% and
54% of the female gametes had been fertilized and ex-
hibited the characteristic features of a zygote (Janse
et al., 1985a; 1986); 15% and 21% of these zygotes
contained the two, still separated, nuclei of the female
and male gametes. These fertilization features were ab-
sent from equivalent preparations of Pbs48/45ko para-
sites. Only large clusters of single nucleated female ga-
metes were found (Figure 4A). Therefore, fertilization is
strongly impaired in Pbs48/45ko parasites.
Gamete Fertility Is Strongly Impaired in Pbs48/45ko
Male but Not Female Gametes
could result, therefore, from a loss of either male or
periments between gametes of different P. berghei wt
and Pbs48/45ko clones.
P48/45 Is Central to Plasmodium Gamete Fertility
Figure 4. Female Gametes and Ookinetes of
Pbs48/45ko Parasites Produced during In
Vitro Fertilization Assays
(A) Large clusters of unfertilized female ga-
metes are present in Pbs48/45ko parasites
16 hr after induction of gamete formation,
whereas in wt parasites characteristic clus-
ters of ookinetes are observed. Parasites
shown are stained with Giemsa and photo-
graphed at 1000? magnification. One of the
few ookinetes found in the in vitro cultures of
Pbs48/45ko parasites is shown in the insert.
(B) Unfertilized female gametes of Pbs48/
45ko parasites express the Pbs21 surface
protein specific forfemale gametes and ooki-
netes. Parasites are stained with mAb 13.1
and photographed at 1000? magnification.
(C) Ookinetes of Pbs48/45ko parasites, re-
sulting from cross-fertilization of the Pbs21-
sites by males from the Pbs21ko parasite
clone 602. Parasites visualized as in (B).
Round forms are unfertilized female gametes
and arrows indicate mature ookinetes.
tion, adhesion, or presentation of accessory molecules
such as P230. In P. falciparum, male gametes attach to
erythrocytes in a sialic acid and glycophorin-dependent
manner (Templeton et al., 1998). Disruption of pbs48/
45 does not affect this interaction, however, suggesting
that P48/45 is not the gamete receptor for erythrocyte
Ko parasites defective in male gamete fertility will be
useful tools for the identification and study of additional
genes involved in the fertilization process of malaria
parasites. Other mutants of Plasmodium have been de-
scribed that show aberrant sexual development that
remains undescribed at the molecular level (Guinet et
al., 1996). Genetic studies have now started to define
P48/45 Is Necessary for Male Gamete
Fertility in Plasmodium
Disruption of P48/45 strongly reduces zygote formation
in both species resulting from a dramatic diminution in
male gamete fertility. In addition, although P48/45 is
(Kaushal and Carter, 1984; Vermeulen et al., 1985), it
has no apparent effect on the ability of female gametes
to be formed or fertilized. The inability of P48/45ko male
gametes to adhere to and penetrate female gametes
indicates that P48/45 plays a direct role in fertilization
mediated by male gametes, which may include recogni-
Table 2. Gamete Formation in Pbs48/45ko Parasites Is Normal but Ookinete and Oocyst Production Is Strongly Inhibited
CR (vitro) CR
? : ?
n ? 6
n ? 2
n ? 2
n ? 2
n ? 5
n ? 2
n ? 2
n ? 2
n ? 6
n ? 2
n ? 2
n ? 2
n ? 6
n ? 2
n ? 2
n ? 2
n ? 6
n ? 2
n ? 2
n ? 8
n ? 8
n ? 2
n ? 2
n ? 5
n ? 8
n ? 2
n ? 2
n ? 5
n ? 2
aConversion (CR) rate of asexual parasites into gametocytes is the percentage of ring forms that develop into gametocytes in synchronized
infections in mice and wild-type (wt) and Pbs84/54ko (KoB2–4) parasites are comparable.
bConversion rate of gametocytes into ? gametes is the percentage of ? gametocytes that escape from the host cell and subsequently express
the Pbs21 protein on their surface in in vitro cultures.
cConversion rate of ? gametocytes into ? gametes is the percentage of ? gametocytes that underwent exflagellation in in vitro cultures.
dConversion rate of female gametes into ookinetes in vitro is the percentage of female gametes that develop within 18 hr into ookinetes in
in vitro cultures.
eConversion rate of female gametes into ookinetes in vivo is the percentage of Pbs21-positive ookinetes of the total of Pbs21-positive cells
(gametes and ookinetes) present in the midguts of mosquitoes 21 hr after feeding.
fOocyst production is the mean number of oocysts at day 7 after feeding mosquitoes on infected mice.
Table 3. Cross Fertilization Studies Reveal that Male Gametes of Pbs48/45ko Parasites Are Infertile yet Female Gametes Can Be
aCross A: Male gametes of Pbs21ko parasites were able to fertilize female gametes (Pbs21-positive) of wt parasites.
bCross B: Male gametes of Pbs45/48ko (KoB4) parasites were unable to fertilize female gametes of wt parasites.
cCross C: Pbs21-positive female gametes of Pbs48/45ko (KoB4) parasites were fertilized by male gametes of Pbs21ko parasites. The outcome
of this cross was unaffected by prior treatment of Pbs48/45ko (KoB4) gametes with 5 ? 10?4M Aphidicolin.
dMale gamete formation in the wt parasites was blocked by 5 ? 10?4M Aphidicolin (Aphi).
eThe numbers of gametocytes and ookinetes are expressed per 105erythrocytes. The number of male gametocytes is determined by counting
the number of exflagellations. Male/female ratio in infected blood of the different clones was approximately 1 (i.e., the number of female
gametocytes equals the number of males).
fIn the crosses, we mixed infected blood in amounts based on the numbers of gametocytes present in the infected blood: Cross A: 4 ?l wt
and: 8 ?l Pbs21ko; Cross B: 6 ?l wt and 6 ?l Pbs48/45ko; Cross C: 8 ?l Pbs21ko and 4 ?l Pbs48/45ko. Inhibition of male gamete formation
in wt parasites was achieved by adding Aphidicolin to the culture medium at a concentration of 5 ? 10?4M during the first 10 min, after which
Aphidicolin was removed by washing. Gametes of the different clones were then mixed in fresh culture medium and incubated for 18 hr.
Comparable conversion rates for Pbs48/45ko (KoB4) female gametes into ookinetes were observed in the absence (26%) of presence (27%)
of drug. Pbs21ko female gametes and ookinetes can be distinguished from the gametes/ookinetes of wt and Pbs48/45ko parasites by the
lack of staining with mAb 13.1 (Pbs21-positive parasites).
the role of sex-specific proteins. Disruption of the gene
encoding Pfg27 through genetic modification resulted
in the arrest of early gametocyte development (Lobo et
al., 1999), and a genetic cross of P. falciparum demon-
mosome 12 plays a role in male fertility (Vaidya et al.,
1995). Identification of proteins involved in the sexual
development and determination of their function might
facilitate design of improved strategies for blocking
transmission of malaria parasites.
Most members of the Pfs48/45 superfamily are con-
served in the genome of other human malarias (P. vivax)
and the rodent parasites, P. berghei and P. chabaudi.
A further 6-cys superfamily member, P47, is also ex-
pressed during sexual development in both P. falci-
parum and P. berghei and is predicted to be a gameto-
cyte/gamete-surface antigen. All members of the 6-cys
and are gametocyte/gamete-specific suggesting that
this family has evolved to play a role in the sexual cycle
of Plasmodium. Whether all these proteins mediate ga-
mete interactions, as exemplified by P48/45, remains to
be determined. Interestingly, it has been shown that
Pfs48/45 physically interacts with another member of
the protein family, Pfs230, and antibodies to Pfs230 can
also inhibit the development of zygotes (Kumar, 1987;
Williamson et al., 1993; Kaslow, 1996).
Linkage of p48/45 and its paralog, p47, is conserved
in the genome of P. berghei and P. falciparum. Tight
scribed in gametocytes and encode surface proteins
appears to be a recurrent theme in malaria parasites.
The genes encoding Pfs230 and P25, transcribed dur-
ing the sexual stages, form tightly linked gene pairs
with their paralogs, separated by approximately 2 kb
(Gardner et al., 1998; A. Tomas et al., submitted). Gene
duplication resulting in paralogous gene pairs appears,
therefore, to be an ancient and coordinated event in
Plasmodium. We have recently demonstrated a high
level of redundancy between the P25 gene pair (A. To-
Members of the 6-Cys Domain Gene Superfamily
Are Unique and Conserved within the Plasmodium
Genus and Expressed during Sexual Development
In general, the mutual recognition by male and female
gametes required for fertilization is mediated by struc-
receptor proteins. These proteins are typically rapidly
evolving, highly structured members of families of pro-
interactions leading to and ensuring sex specificity of
fertilization (Vacquier, 1998). P48/45 belongs to a family
of proteins that share a conserved structure defined by
apparently species-specific 6-cys domains (Carter et
al., 1995; Templeton and Kaslow, 1999). Database
searches do not reveal any Pfs48/45 or other 6-cys do-
main family member homologs outside the genus Plas-
modium, including the related Apicomplexan Toxo-
plasma gondii. Although full genome sequences are
necessary, current indications are that the 6-cys motif
is unique to and distributed throughout Plasmodium.
P48/45 Is Central to Plasmodium Gamete Fertility
those proteins does not result in a significant inhibition
of zygote and oocyst development, whereas the devel-
opment of zygotes into oocysts is blocked when both
genes are disrupted. In contrast, this study shows that
P47 cannot compensate in the same way for P48/45
disruption. Furthermore, the few P48/45ko parasites
that can be transmitted by mosquitoes still exhibit the
defective male gamete phenotype after mosquito trans-
mission. The possibility is, therefore, excluded that a
proportion of the parasites stably switch to the expres-
sion of alternative genes that are able to complement
the defect of disruption of P48/45. Stable switching of
genotype and phenotype has been shown before in
Plasmodium in relation to switching antigenic types (al-
of erythrocyte invasion (Dolan et al., 1990; Reed et al.,
that immune responses against these proteins will be
boosted by natural infection. Antibodies against Pfs48/
45 and Pfs230 are present in naturally infected persons
and serum levels of Pfs48/45 antibodies correlate with
fen et al., 1996; Mulder et al., 1999). Characterization of
the pattern of expression and possible role of the other
ization is, therefore, desirable. This and an evaluation
of the immunological properties of the 6-cys domain
gene superfamily members will assist in a rational ap-
proach to transmission-blocking vaccine design.
P. berghei: Clone 15cy1A (ANKA), a gametocyte-producer clone,
producer clone (Janse et al., 1989); Clone 602, a knockout mutant
parasite lacking the Pbs21 surface protein of gametes/zygotes (A.
Tomas et al., submitted). P. falciparum: Cloned line NF54.
P. falciparum parasites were cultured using a semi-automated cul-
ture system. Asexual parasites were removed by treatment of the
cultures with 50 mM N-acetyl-glucosamine between days 8 and 12
(Gupta et al., 1985). Mature gametocytes were isolated and concen-
trated as described by Staalsoe et al., 1999.
Implications for Vaccine Development
This study demonstrates the important role of Pfs48/
45 in malaria parasite transmission and emphasizes its
value as a leading candidate antigen for the develop-
ment of a transmission-blocking vaccine. However, dis-
ruption of the P48/45 gene did not result in complete
blockage of transmission. Malaria transmission is re-
markably efficient even at low gamete densities (Janse
et al., 1985b; Boudin et al., 1993), and it is likely that
factors present in the mosquito midgut can enhance
fertilization (Billker et al., 1998). These observations are
reinforced by the demonstration here that, although
tectable in vitro, P48/45ko parasites do undergo low-
have serious implications for development of transmis-
additional molecules for the formulation of a multivalent
against the selection of escape mutants. We also found
that reduction in zygote formation in vivo is less marked
in Pbs48/45ko parasites than in Pfs48/45ko parasites.
The assays used to quantify zygote formation differ be-
tween the two parasites and these distinctions may ex-
plain the apparently more efficient zygote formation in
ko P. berghei parasites. Transmission of in vitro culti-
vated gametocytes to mosquitoes via membrane feed-
ing is significantly less efficient than direct mosquito
tions). It is not possible to perform human transmission
studies of P. falciparum ko parasites and so P. berghei
is a useful and more sensitive model system to test
the essential nature of transmission-blocking candidate
Members of the 6-cys domain gene superfamily pos-
sess important features that make them attractive tar-
gets for a transmission-blocking vaccine: (1) There is
remarkable conservation of the different p48/45 super-
family members within Plasmodium; (2) this conserva-
tion and genus-wide distribution indicates that the mo-
of this family of proteins are also highly conserved; and
(3) most importantly, it appears that a number of these
proteins are already expressed on the surface of the
gametocytes in the blood, and therefore, it is possible
Isolation and Characterization of the Pbs48/45
and Pb47 Genes
A ?ZAP P. berghei genomic DNA library was screened using 1 kb
of the Pfs48/45 gene as a probe, amplified by PCR using primers
#343 and #344 (oligonucleotides used in this study are available
at http://www.cell.com/cgi/content/full/104/1/153/DC1) using stan-
dard conditions (Birago et al., 1996). A clone, pBL1, containing the
Pbs48/45 coding region, 358 bp of upstream and 1.5 kb of down-
stream region, was further characterized and sequenced. The Pb47
gene was cloned by gene walking upstream of Pbs48/45 and
through use of clone from the University of Florida P. berghei ge-
nome sequencing tag project (http://parasite.vetmed.ufl.edu). A ge-
nomic clone, 47.1, containing the Pbs47 coding region, 500 bp of
upstream and 1.3 kb of downstream region, was isolated and se-
Analysis of p48/45 and p47 Linkage in the Genome
A contig ofthe Pfs48/45 and Pf47 locus wasassembled from BLAST
analysis of sequence data for P. falciparum chromosome 13 (http://
www.sanger.ac.uk/Projects/P_falciparum/). Sequencing of P. falci-
parum chromosome 13 was accomplished as part of the Malaria
Genome Project with support by The Wellcome Trust. Preliminary
sequence data for P. falciparum chromosomes 10 and 11 were
obtained from The Institute for Genomic Research (www.tigr.org).
For P. falciparum, primer pair irR (nt 14–25) and irF (nt 1206–1227)
of pfs48/45 and pf47, respectively, and for P. berghei, primer pairs
#486 and #651 corresponding to nt 1210–1233 of pbs47 were used
to amplify the intergenic region by PCR.
Generation of P. falciparum and P. berghei
P. berghei: The Pbs48/45 gene of P. berghei was disrupted with
replacement vector p54, a derivative of the previously described
construct pDB.DT?H.DB(Wengelnik et al., 1999). The 5? pbs48/45 tar-
geting sequence (0.7 kb) was amplified from clone pBL1 using
primer #406(200 nt upstreamof the predicted startcodon) introduc-
ing HindIII and BamHI sites, respectively, and primer #407 (nt 527–
47) introducing an HindIII site, and cloned into pDB.DT?H.DBgiving
rise to vector p53. The 3? pbs48/45 targeting sequence (1.5 kb) was
amplified using primer #408 (nt 1219–39) introducing an EcoRV site
in combination with the pBS-SK forward primer and cloned into p53
and KpnI, the DNA fragment gel purified and used for transfection.
P. berghei parasites were transfected and cloned as described
(Me ´nard and Janse, 1997).
P. falciparum: The Pfs48/45 gene of P. falciparum was disrupted
with insertion vector pI48, a derivative of the previously described
pDT.Tg23 (Wu et al., 1996). pI48 was constructed by cloning 1088
bp ofthe codingsequence of pfs48/45into pDT.Tg23.This fragment
was obtained by PCR amplification using primers #343/#344 con-
taining BglII and SpeI sites, respectively, and cloned into pDT.Tg23.
selection, and cloning of recombinant parasites were as described
by Fidock and Wellems,1997. To select pfs48/45ko clones, the ab-
sence of expression of the Pfs48/45 in mature gametocytes was
assayed by immunofluorescence assay (IFA).
assay (Janse et al., 1985a, 1985b). The fertility of male and female
ko gametes was determinedby cross-fertilization of different clones
of P. berghei in the fertilization assays. Cross-fertilization between
gametes of two clones (A and B) was obtained as follows: infected
blood from a mouse infected with clone A and infected blood from
a mouse infected with clone B were separately transferred into
ookinete culture medium to induce gamete formation. 10 min after
induction, equal numbers of gametes of both clones were mixed
and 18 hr later, the number of ookinetes and unfertilized gametes
were counted. To specifically block male gamete formation in one
of the two clones, Aphidicolin at a concentration of 5 ? 10?4M was
added to the cultures during the first 10 min of gamete induction
(Janse et al., 1986). Further detail on the precise methods used
in the phenotype analysis is available at http://www.cell.com/cgi/
Genotype Analysis of P48/45ko Parasites
Disruption of the Pbs48/45 and Pfs48/45 gene in the ko parasites
was analyzed by PCR, DNA, and RNA blot analyses (Me ´nard and
Janse, 1997). In P. berghei parasites, homologous integration at the
target gene was detected by PCR amplification using a primer pair
#450/#313 and #428/#430 for P. falciparum. Control PCR amplifica-
tions to detect the wt gene were performed using primer pairs, P.
berghei, #450/#451; P. falciparum, #428/#429. P. berghei chromo-
somes were separated by Field Inversion Gel Electrophoresis (FIGE)
as described (Birago et al., 1996) and hybridized to a pbs48/45 ORF
probe or a probe of the 3?UTR of the P. berghei dhfr/ts locus (DT-3?)
(Wengelnik et al., 1999). P. berghei genomic DNA, digested with
HindIII, was hybridized to a hybrid probe (DT-48/45, amplified using
primers #450/#313) that hybridizes to the 5?UTR of the P. berghei
DHFR/TS gene and the pbs48/45 locus (see Figure 2A). Genomic
DNA from P. falciparum, digested with HincII, was hybridized to a
Pfs48/45 ORF-specific probe.
We would like to thank Jai Ramesar, Auke van Wigcheren, Marga
van der Vegte-Bolmer, Ton Lensen, and Jo Hooghof for excellent
technical assistance; Annemarie van der Wel and Clemens Kocken
for great help with the fertilization assays; and Prof. R. E. Sinden
for the gift of antibodies. Financial support was from the QLRT-KA2
program of the DGXII Department of the European Commission
(Contract Number QLK2-CT-1999-00753), the Medical Sciences
Foundation (901-15-079), which is subsidized by the Netherlands
Organization for Scientific Research, and DGIS/SO grant N1002701.
The Sanger Centre website at http://www.sanger.ac.uk/Projects/
P_falciparum/. Sequencing of P. falciparum chromosome 13 was
accomplished as part of the Malaria Genome Project with support
by The Wellcome Trust. Preliminary sequence data for P. falciparum
chromosomes 10 and 11 were obtained from The Institute for Geno-
mic Research website (www.tigr.org). Sequencing of chromosomes
10 and 11 was part of the International Malaria Genome Sequencing
Project and was supported by an award from the National Institute
of Allergy and Infectious Diseases, National Institutes of Health.
Sequence data for P. berghei were obtained from the University of
Florida Gene Sequence Tag Project Website at: http://parasite.
vetmed.ufl.edu. Funding was provided by the National Institute of
Allergy and Infectious Diseases.
Generation of Pbs48/45-Specific Antiserum
Using Recombinant Pbs48/45
A fragment of the Pbs48/45 gene (encoding amino acids 62–408)
was amplified by PCR using primers pBEXP1and pBEXP2 and
cloned into the expression vector pET-15b (Novagen) providing an
N-terminal 6-Histidine tag under the control of the T7lac promoter
(pET-pbs48/45). Recombinant Pbs48/45 were purified by affinity
chromatography on Ni-NTA super flow beads (Qiagen) under dena-
clonal antiserum was raised in a New Zealand rabbit by injection of
200 ?g of gel-purified recombinant protein. Boosting was carried
outsubcutaneouslywith 3-weekintervalsusing200 ?grecombinant
protein in incomplete Freund’s adjuvant. Serum (Pc48/45) obtained
2 weeks after the third boost was immunopurified on immobilized,
purified recombinant Pbs48/45.
Received August 16, 2000; revised November 17, 2000.
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GenBank Accession Number
The GenBank accession number for the entire nucleotide sequence
of the ORFs of pbs47 and pbs48/45 and their intergenic regions is