Cell-Passage Activity Is Required for the Malarial Parasite to Cross the Liver Sinusoidal Cell Layer

Abstract

Liver infection is an obligatory step in malarial transmission, but it remains unclear how the sporozoites gain access to the hepatocytes, which are separated from the circulatory system by the liver sinusoidal cell layer. We found that a novel microneme protein, named sporozoite microneme protein essential for cell traversal (SPECT), is produced by the liver-infective sporozoite of the rodent malaria parasite, Plasmodium berghei. Targeted disruption of the spect gene greatly reduced sporozoite infectivity to the liver. In vitro cell invasion assays revealed that these disruptants can infect hepatocytes normally but completely lack their cell passage ability. Their apparent liver infectivity was, however, restored by depletion of Kupffer cells, hepatic macrophages included in the sinusoidal cell layer. These results show that malarial sporozoites access hepatocytes through the liver sinusoidal cell layer by cell traversal motility mediated by SPECT and strongly suggest that Kupffer cells are main routes for this passage. Our findings may open the way for novel malaria transmission-blocking strategies that target molecules involved in sporozoite migration to the hepatocyte.

Full text

Cell-Passage Activity Is Required
for the Malarial Parasite
to Cross the Liver Sinusoidal Cell Layer
Tomoko Ishino
1,2
, Kazuhiko Yano
1
, Yasuo Chinzei
1,2
, Masao Yuda
1,2
*
1 Mie University School of Medicine, Mie, Japan, 2 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi,
Saitama, Japan
Liver infection is an obligatory step in malarial transmission, but it remains unclear how the sporozoites gain access to
the hepatocytes, which are separated from the circulatory system by the liver sinusoidal cell layer. We found that a
novel microneme protein, named sporozoite microneme protein essential for cell traversal (SPECT), is produced by the
liver-infective sporozoite of the rodent malaria parasite, Plasmodium berghei. Targeted disruption of the spect gene
greatly reduced sporozoite infectivity to the liver. In vitro cell invasion assays revealed that these disruptants can
infect hepatocytes normally but completely lack their cell passage ability. Their apparent liver infectivity was, however,
restored by depletion of Kupffer cells, hepatic macrophages included in the sinusoidal cell layer. These results show
that malarial sporozoites access hepatocytes through the liver sinusoidal cell layer by cell traversal motility mediated
by SPECT and strongly suggest that Kupffer cells are main routes for this passage. Our findings may open the way for
novel malaria transmission-blocking strategies that target molecules involved in sporozoite migration to the
hepatocyte.
Introduction
Malaria is one of the most devastating infectious diseases in
the world, killing more than 1 million people per year.
Malaria is transmitted by bites of infected mosquitoes that
inject sporozoites under the skin. The first obligatory step for
these parasites to establish infection in humans is migration
to hepatocytes, where they proliferate and develop into the
erythrocyte-invasive form (Sinnis 1996). This liver-invasive
stage has been demonstrated as a promising target for
antimalarial strategies that aim to establish sterile immunity
against the malarial parasite (Nu ssenzweig et al . 1967;
Hoffman et al. 1996). However, the mechanisms underlying
the parasite’s liver infection are largely unknown. I n
particular, it has been controversial how sporozoites reach
the hepatocytes that are separated from blood circulation by
the liver sinusoidal layer. The routes the sporozoites use to
cross this layer, the modes of motility on w hich their
migration is based, and the molecules of the parasite involved
in this process are poorly understood.
Malarial parasites develop into sporozoites in the mosquito
midgut and then invade the salivary gland, where they wait to
be transferred to the mammalian host (Menard 2000). Once
injected by mosquito bites under the skin, sporozoites enter
the blood circulation and are carried to the liver by the
bloodstream (Sinnis and Nussenzweig 1996; Menard 2000;
Mota and Rodriguez 2002). In the liver, they are thought to be
arrested on the inner surface of the liver sinusoidal vein and
then leave the vein and infect the hepatocytes by crossing the
sinusoidal wall (Sinnis and Nussenzweig 1996). This wall is a
single-cell layer mainly composed of sinusoidal endothelial
cells and Kupffer cells, which are hepatic macrophages.
Several models have been proposed to explain how spor-
ozoites cross th is layer. Some authors proposed that
sporozoites infect hepatocytes after crossing the liver
endothelial cell through fenestrations in this cell (Vanderberg
and Stewart 1990), but these openings are too small for
sporozoites to freely pass through (Mota and Rodriguez 2002).
Other authors have suggested that Kupffer cells are gates for
sporozoites to access hepatocytes, based on the ultrastruc-
tural observation that sporozoites were found inside Kupffer
cells shortly after intravenous inoculation (Mota and Rodri-
guez 2002). This Kupffer cell hypothesis, however, has not
been convincingly demonstrated, because other tools for
probing into this event were lacking. Furthermore, the
observation that the sporozoites in Kupffer cells sometimes
have a vacuole around them makes the conclusion uncertain.
Some authors have proposed that sporozoites are passively
engulfed by Kupffer cells and then carried to the hepatocyte
(Meis et al. 1983), and some have proposed that this migration
is based on active motility accompanied by vacuole formation
(Pradel and Frevert 2001).
The malarial parasite has no locomotory organelles such as
flagella or cilia. Motility of the host-invasive stages of the
malarial parasite, including the sporozoite, is dependent on
secretion of micronemes that are organelles occupying the
cytoplasm of the parasite (Sultan 1999; Menard 2 001).
Micronemal components, which may include several attach-
Received September 12, 2003; Accepted October 28, 2003; Published January
20, 2004
DOI: 10.1371/journal.pbio.0020004
Copyright: Ó2004 Yuda et al. This is an open-access article distributed under
the terms of the Creative Commons Attributio n License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
Abbreviations: Cl
2
MDP, dichloromethylene diphosphonate; CS, circum sporozoite
protein; DAPI, 49,6- diamidino -2-phenylindole; EEF, exoerythrocytic form; EST,
expressed sequence tag; FITC, fluorescein isothiocyanate; GST, glutathione S-
transferase; SPECT, sporozoite microneme protein essential for cell traversal
Academic Editor: Gary Ward, University of Vermont
* To whom correspondence should be addressed. E-mail: m-yuda@doc.medic.
mie-u.ac.jp
PLoS Biology | http://biology.plosjournals.org January 2004 | Volume 2 | Issue 1 | Page 0077
P
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BIOLOGY

ment proteins, are secreted from the apical pore during
parasite movement and are translocated backwards along the
parasite cell surface by actomyosin motors of the parasite.
This surface movement is believed to generate traction for
parasite-invasive motility.
Salivary gland sporozoites display three modes of motility
in vitro dependent on secretion of micronemes (Mota and
Rodriguez 2002). One mode is gliding motility on a solid
surface, which can be observed as circular movement on a
glass slide, probably representing gliding motility on the cell
surface. The other two are cell-invasi ve motilities: cell-
infection and cell-traversal motility (Mota et al. 2001; Kappe
et al. 2003). Cell-infection motility is accompanied by vacuole
formation and is followed by parasite development into
exoerythrocytic forms (EEFs). Cell-traversal motility, on the
other hand, involves plasma-membrane disruption and is
followed by migration through the cytoplasm and eventual
escape from the cell. Recently, Mota et al. (2002) revealed that
this type of cell-invasion motil ity can be i dentified by
conventional cell-wound assay. According to the observation
that passage through some hepatocytes by this motility
precedes hepatocyte infection, they proposed the hypothesis
that this motility is necessary for sporozoites to be activated
for hepatocyte infection (Mota et al. 2002). However, the role
of this motility in liver infection remains unclear.
Aiming at identification of molecules involved in spor-
ozoite infection, we screened an expressed sequence tag (EST)
database of the salivary gland sporozoite of a rodent malarial
parasite, Plasmodium berghei. In this paper, we report a novel
microneme protein, named SPECT (sporozoite microneme
protein essential for cell traversal), which is specifically
produced by the liver-infective sporozoite and is essential
for the sporozoite’s cell-passage ability. By using spect-
disrupted parasites, we show that cell-passage ability of the
sporozoite plays a critical role in malarial transmission to the
vertebrate host and is required for sporozoites to access
hepatocytes by traversal of the liver sinusoidal cell layer. In
addition, we provide a model of sporozoite liver infection,
which suggests an answer to the question of how sporozoites
reach the hepatocytes.
Results
Identification of cDNA Encoding SPECT from P. berghei
Salivary Gland Sporozoite EST Database
Sporozoites acquire the ability to infect the mammalian
liver after infection of the mosquito salivary glands (Sultan et
al. 1997), indicating that novel protein synthesis for liver
infection begins in this stage (Matuschewski et al. 2002). To
search for malarial genes involved in liver infection, we
screened an EST database of P. berghei salivary gland
sporozoites. We assembled 3,825 ESTs, obtained 502 contigs,
and screened them for genes encoding secretory proteins or
membrane-associated proteins, which may participate in
host–parasite interactions. This screening was started from
the contigs containing a high number of ESTs, since the
number of ESTs may correlate with the expression levels of
the respective genes. In this process, we identified a contig
composed of ten ESTs, encoding a putative secretory protein
of 241 amino acids (Figure 1A). The expected molecular mass
for the N-terminal signal sequence-processed form of this
protein was 25 kDa. We named this protein SPECT
(sporozoite microneme protein essential for cell traversal),
since it is essential for sporozoite passage through a host cell,
as described later.
Southern blot analysis showed that the spect gene is a single-
copy gene (data not shown). Sequence analysis of the spect
gene identified four introns (data not shown). A computer
search of Plasmodium genome databases (Carlton et al. 2002;
Gardner et al. 2002) revealed that this gene is conserved
through several Plasmodium species. The orthologous protein
in P. falciparum, the clinically most important human malaria
parasite, shared 45.6% sequence identity with P. berghei
SPECT (Figure 1B).
SPECT Is Produced Specifically by Salivary Gland Spor-
ozoites and Localized in Micronemes
The expression profile of this gene in the malarial life cycle
was investigated. Immunofluorescent analysis in all host-
invasive stages showed that SPECT production was restricted
to sporozoites in the salivary gland (Figure 2A). It is
noteworthy that SPECT is not detected in sporozoites in
the midgut, because this expression profile strongly suggests
Figure 1. Sequence Analysis of spect cDNA
(A) Nucleotide sequence of spect cDNA (top lane) and the deduced
amino acid sequence (bottom lane) are shown. The predicted N-
terminal signal sequence is under lined. The numbers indicate
positions of the nucleotides starting from the 59 end. The asterisks
indicate the termination codon.
(B) A comparison of deduced amino acid sequences of P. berghei spect
(top) and P. falciparum spect (bottom). Gaps are introduced to obtain
optical matching by using GENETIX-MAC software. Asterisks or dots
show conserved or similar residues, respectively. The amino acid
numbers from the first Met residue are shown on the left of each line.
DOI: 10.1371/journal.pbio.0020004.g001
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Role of SPECT in Malarial Transmission

that SPECT is specifically involved in liver infection. Western
blot analysis revealed SPECT as a 22 kDa protein in salivary
gland sporozoites, but not in midgut sporozoites (Figure 2B),
confirming that SPECT is produced after invasion into the
salivary gland. Immunoelectron microscopy showed that
SPECT is localized in the sporozoite to micronemes that are
secretory organelles occupying the cytoplasm (Figure 2C).
Micronemes are common to motile stages of Plasmodium
parasites and play a central role in host-invasive motility
(Sultan 1999; Menard 2001). Taken together, these results
indicate that SPECT plays a role in the liver-invasive motility
of the sporozoite.
SPECT Plays an Important Role in Sporozoite Infection of
the Host Liver
To investigate the function of SPECT protein, we gener-
ated spect-disrupted parasites by homologous recombination
(Figure 3A). The spect disruptants were selected by the
antimalarial drug pyrimethamine and were separated from
wild-type parasites by limiting dilution. Disruption of the
spect locus was confirmed by Southern blot analysis (Figure
3B). To exclude the possibility that the spect-disrupted
populations obtained were derived from a single clone, two
independently obtained spect-disrupted populations (spect()1
and spect()2) were used in the following experiments.
In the intra-erythrocytic stage, SPECT gene disruption did
not affect parasite proliferation, as the growth rates in rat
blood were almost the same in the spect-disrupted and wild-
type populations (data not shown). Furthermore, disruption
of the gene did not affect parasite development in the
mosquito vector, as numbers of sporozoites residing in the
midgut and in the salivary glands were similar in the spect-
disrupted and wild-type populations (Table 1).
Next, the liver infectivity of the spect-disrupted sporozoites
was examined. Rats were intra venously inoculated with
sporozoites, and the progress of parasitemia, the percentage
Figure 2. SPECT Is a Microneme Protein Specifically Produced in the
Liver-Infective Sporozoite Stage
(A) Indirect immunofluorescence microscopy of all four invasive
forms of the malarial parasite (indicated over the panel). Parasites
were stained with primary antibodies against SPECT, followed by
FITC-conjugated secondary antibodies. SPECT was detected only in
the salivary gland sporozoite, the liver-infective stage. The corre-
sponding phase-contrast or DAPI-stained image (Phase or DAPI) is
shown under each image. Scale bars, 5 lm
(B) Western blot analysis of SPECT production in the midgut
sporozoite (M) and the salivary gland sporozoite (S). Lysate of
500,000 sporozoites was loaded onto each lane and detected with the
same antibody used in (A). SPECT was detected as a single band of 22
kDa (arrowhead) only in the salivary gland sporozoite.
(C) Immunoelectron microscopy of sporozoites in the salivary gland.
Ultrathin sections of a mosquito salivary gland infected with
sporozoites were incubated with the same antibody used in (A)
followed by secondary antibodies conjugated with gold particles (15
nm). Particles were localized to micronemes (Mn) but not to
rhoptories (Rh). Axial (inset) and vertical images are shown. Scale
bars, 0.5 lm.
DOI: 10.1371/journal.pbio.0020004.g002
Figure 3. Targeted Disruption of the spect Gene
(A) Schematic representation of targeted disruption of the spect gene.
The targeting vector (top) containing a selectable marker gene is
integrated into the spect gene locus (middle) by double crossover. This
recombination event resulted in the disruption of the spect gene and
confers pyrimethamine resistance to disruptants (bottom).
(B) Genomic Southern blot hybridization of wild-type (WT) and
spect() populations. Genomic DNA isolated from the respective
parasite populations was digested with EcoT22I and hybridized with
the probe indicated in (A) by a solid bar. By integration of the
targeting construct, the size of detected fragments was decreased
from 1.9 kbp to 1.2 kbp. The result is shown for two independently
prepared populations, spect()1 and spect()2.
(C) Immunofluorescence microscopy of the wild-type (WT) and
spect() parasite. Sporozoites were collected from the salivary gland
and stained with primary antibody against SPECT followed by FITC-
conjugated secondary antibodies. The apical end of each sporozoite is
indicated by an arrowhead.
DOI: 10.1371/journal.pbio.0020004.g003
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Role of SPECT in Malarial Transmission

of infected erythrocytes, was measured in the exponential
growth period (from 3.5 d to 5 d after inoculation). It is
thought that the parasitemias reflect the liver infectivity of
the respective parasite populations, since the growth rates of
their in traerythrocytic stages ar e similar (shown by the
parallel slopes of the increase in parasitemia in Figure 4).
Based on the average parasitemia at 3.5 d after inoculation of
30,000 sporozoites, the liver infectivities of the two disruptant
strains were estimated to be 15- and 28-fold lower,
respectively, than that of the wild-type. These results are
consistent with the observation that the parasitemias after
injection of 30,000 disruptant sporozoites were lower than
that from 3,000 wild-type sporozoites.
The liver infectivity was also evaluated by the number of
early EEFs. Frozen sections of the rat liver was prepared 24 h
after sporozoite injection and EEFs w ere counted by
immunofluorescence microscopy. As shown in Figure 4B,
EEFs were approximately 30-fold decreased by spect gene
disruption. This reduction rate agrees well with that
estimated by parasitemia. These results indicate that SPECT
plays a role in the process of sporozoite invasion into the
liver.
SPECT Is Essential for Sporozoite Cell-Passage Ability
Localization of SPECT in micronemes indicates its involve-
ment in the invasive motility of the sporozoite. The motility
of spect-disrupted sporozoites was investigated by three in
vitro assays corresponding to three modes of motility of the
sporozoite. First, we checked gliding motility on a solid
surface, which is essential for sporozoite infectivity. Most
disruptants displayed a typical circular movement, and the
proportion of motile sporozoites was almost identical in
disruptant and wild-type parasites (63.6% and 67.5% ,
respectively), showing that their gliding moti lity is not
affected by SPECT gene disruption. Second, we examined
the ability of the sporozoites to infect hepatocytes. This was
assayed by formation of EEFs in a human hepatoma cell line,
HepG2 (Hollingdale et al. 1981). As shown in Figure 5A, the
disruptants formed EEFs in similar numbers to the wild-type,
indicating that they retain normal infectivity to the hepato-
cyte. Third, we examined cell-traversal ability that takes place
prior to hepatocyte infection. This was estimated by the
number of membrane-wounded cultured cells that were
labeled by uptake of flu orescein isothiocyanate (FITC )-
conjugated dextran from the medium (Mota et al. 2001). As
shown in Figure 5B, the cell-wound assay using HeLa cells
showed that the disruptants lost their cell-passage activity
completely. The same results were obtained in HepG2 cells
(data not shown). These results revealed that SPECT is
specifically involved in cell-traversal ability and suggest that
lack of this ability reduced liver infectivity of the disruptants.
Cell Passage Ability Is Necessary for Sporozoites to
Traverse the Sinusoidal Layer Cells and to Access
Hepatocytes
To access the hepatocytes, sporozoites must cross the
sinusoidal layer, which separates them from the circulation.
We assumed that SPECT was necessary for this process. Since
Kupffer cells are major components of this layer and have
been reported as the main gates for sporozoite access to the
hepatocyte, we prepared Kupffer cell-depleted rats by intra-
venous injection of liposome-encapsulated dichloromethy-
lene diphosphon ate (Cl
2
MDP) (Vreden et al. 1993; van
Rooijen and Sanders 1994) and tested them for infection by
disruptant and wild-type sporozoites. As shown in Figure 6A,
infectivities of spect-disruptants assessed by parasitemia were
increased by 22- and 53-fold by Kupffer cell depletion and, as
a result, became equal to that of the wild-type. The numbers
of early EEFs detected in the liver sections were also almost
identical in wild-type and spect-disrupted parasites (Figure
6B). These results show that the disruptants’ loss of infectivity
is localized at the sinusoidal cell layer and that the cell-
passage ability of the sporozoite is necessary to cross this
layer and, specifically, the Kupffer cells.
Figure 4. Targeted Disruption of spect Results in Reduction of Sporozoite
Infectivity to the Liver
(A) The salivary gland sporozoites of each parasite population were
injected intravenously into five rats. The parasitemia of each rat was
checked by a Giemsa-stained blood smear after inoculation on the
days indicated. The average parasitemia after inoculation of 30,000
sporozoites was significantly low in disruptant populations, whereas
their growth rates in the blood were essentially the same as the wild-
type. The numbers of parasites inoculated were as follows: 30,000
spect()1 (open circles), 30,000 spect()2 (open triangles), 30,000 wild-
type (filled circles), and 3,000 wild-type (filled squares). Values shown
represent the mean parasitemia (6 SEM) of five rats.
(B) The salivary gland sporozoites (500,000) of wild-type or spect-
disrupted parasites were inoculated intravenously into 3-wk-old rats.
After 24 h, the livers were fixed with paraformaldehyde and frozen.
The number of EEFs on each cryostat sections was estimated by
indirect immunofluorescence anal ysis using anti-CS antiserum.
Values shown represent the mean number of EEFs per square
millimeter (6 SEM) of at least three rats.
DOI: 10.1371/journal.pbio.0020004.g004
Table 1. SPECT Disrupted Parasites Develop Normally into
Sporozoites and Invade the Salivary Gland in the Mosquito
Vector
Parasites
Population
Number of
Midgut
Sporozoites
per Mosquito
Number of
Salivary Gland
Sporozoites
per Mosquito
spect()1 141,367 6 36,205 14,136 6 1,778
spect()2 69,800 6 9,825 16,233 6 4,138
Wild-type 86,674 6 8,454 19,396 6 3,33
Mosquitoes were fed on mice infected with spect() parasite populations or wild-
type polyclonal populations. Sporozoites were collected separately from the
midgut and the salivary glands of mosquitoes 24–28 d after feeding and then
counted. Each value is the mean of the number with its standard error from three
independent experiments.
DOI: 10.1371/journal.pbio.0020004.t001
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Role of SPECT in Malarial Transmission

Discussion
It has been reported that the Plasmodium sporozoite has the
ability to traverse cultured cells rapidly (Mota et al. 2001), but
the role of this process in liver infection has remained
unclear. On the other hand, it is poorly understood how the
sporozoite migrates from the ci rculatory system to the
hepatocyte. In this paper, we address these issues using a
gene-targeting technique. We have shown that the cell-
traversal activity of the sporozoite is necessary for it to leave
the circulatory system by crossing the liver sinusoidal cell
layer. These results are the first to reveal the role of cell-
traversal activity in malarial transmission.
In vitro cell invasion assays showed that spect-disrupted
sporozoites completely lose cell passage activity, but preserve
normal infectivity to the hepatocyte (see Figure 5). These
results clearly demonstrated that these two cell-invasion
activities are independent of each other. This conclusion
contradicts the hypothesis proposed by Mota et al. (2002) that
cell passage activates the sporozoite for hepatocyte infection.
They assumed that sporozoites traverse some hepatocytes
before infecting a hepatocyte and that this passage alters
their mode of cell invasion from passage to infection (Mota et
al. 2002). Our results, however, demonstrated that lack of
previous cell passage has no influence on the infectivity to
hepatocytes. This independence was confirmed in vivo by the
result that disruptants and wild-type showed the same liver
infectivities in Kupffer cell-depleted rats (see Figure 6).
Therefore, sporozoites may change their mode of invasive
motility according to other factors, which remain to be
elucidated. We suppose that secretion of the micronemal
contents during gliding on the cell surface might be one such
factor, since this motility may precede hepatocyte infection
as discussed below.
Our results indicate that the liver sinusoidal barrier is not
perfect, since a small proportion of the spect-disr upted
sporozoites can infect the liver (see Figure 4). It is supposed
that this layer may have a few openings and the disruptants
can migrate through them by gliding along the epithelial cell
surface. In Kupffer cell-depleted rats, on the other hand, both
disruptants and wild-type may migrate through the numerous
gaps created among the endothelial cells, resulting in
elimination of the phenotypic difference. Since Kupffer cells
constitute approximately 30% of the sinusoidal cells (Bou-
wens et al. 1986), their depletion from this layer may leave
Figure 6. Restoration of spect() Sporozoite Infectivity in Kupffer Cell-
Depleted Rats
(A) Liposome-encapsulated Cl
2
MDP (filled points) or PBS (open) was
injected intravenously into rats. After 48 h, 30,000 sporozoites of
spect()1 (circles), spect()2 (triangles), or wild-type (squares) popula-
tions were inoculated intravenously. Parasitemia of each rat was
checked by Giemsa-stained blood smears after inoculation on the
days indicated. Values shown represent the mean parasitemia (6
SEM) of five rats.
(B) Salivary gland sporozoites (500,000) of each parasite population
were inoculated intravenously into Kupffer cell-depleted rats. After
24 h, the livers were fixed with paraformaldehyde and frozen. The
number of EEFs on each cryostat section was estimated by indirect
immunofluorescence analysis using anti-CS antiserum. Values shown
represent the mean number of EEFs per square millimeter (6 SEM)
of at least three rats.
DOI: 10.1371/journal.pbio.0020004.g006
Figure 5. spect Disruption Results in Loss
of Cell-Passage Activity of the Sporozoite
(A) spect disruption does not affect
sporozoite ability to infect hepatocytes.
(Top panel) Comparison of EEF numbers
between disruptants (spect()) and wild-
type (WT) parasites. Salivary gland spor-
ozoites were added to HepG2 cells and
cultured for 48 h. EEFs formed were
counted after immunofluorescence
staining with an antiserum against CS
protein. (Bottom panels) Representative
fluorescence stained images.
(B) Sporozoites lacking SPECT cannot
traverse HeLa cells. (Top) Comparison of
cell-passage activity between disruptants
and wild-type parasites. Salivary gland
sporozoites were added to HeLa cells
and incubated for 1 h with FITC-con-
jugated dextran (1 mg/ml). Cell-passage
activity was estimated by the number of
cells wounded by sporozoite passage,
which were identified by cytosolic label-
ing with FITC-conjugated dextran. (Bot-
tom) Representative fluorescence
stained images. All data are mean num-
bers of EEFs or FITC-positive cells in a
one-fifth area of an 8-well chamber slide
with standard errors for at least three
independent experiments.
DOI: 10.1371/journal.pbio.0020004.g005
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Role of SPECT in Malarial Transmission

many gaps that cannot readily be repaired. Supposedly,
sporozoites cross these gaps in the same way as they migrate
through the few gaps in normal rats.
Experiments using Kupffer cell-depleted rats indicate that
Kupffer cells are not involved in sporozoites targeting the
liver, because the depletion did not reduce the susceptibility
of rats to sporozoite infection. Thus, sporozoites seem to be
first arrested on the endothelial cell surface or on the
glycosaminoglycans extending through endothelial fenestra-
tions and then migrate to Kupffer cells (Cerami et al. 1992;
Pradel et al. 2002). If so, gliding motility on the cell surface
would be necessary for the sporozoite to migrate from initial
attachment sites to Kupffer cells (or to gaps) along the inner
surface of the sinusoidal layer as well as for the sporozoite to
migrate through gaps. These assumptions imply that after
Kupffer cell depletion, sporozoites can arrive at the hep-
atocyte by gliding motility alone, in accord with the
observation that the disruptants can infect Kupffer cell-
depleted rats with the same infectivity as the wild-type.
Our results strongly suggest that Kupffer cells are main
gates for sporozoites to access hepatocytes. Previous electron
microscopic st udies have reported that sporozoi tes a re
observed in Kupffer cells after intravenous inoculation, and
some of them are found within vacuoles (Meis et al. 1983;
Pradel and Frevert 2001). Based on this observation, it has
been speculated that sporozoites invade the Kupffer cell by a
motility distinct from passage that does not involve para-
sitophorous vacuole formation. Our results, on the contrary,
indicate that sporozoites cross the layer by the same cell-
passage motility as observed in vitro. We think this discrep-
ancy indicates the following two possibilities. One is that the
vacuole formed in the Kupffer cell after rupture of its cell
membrane is different from the parasitophorous vacuole
formed in the hepatocyte, although their differences cannot
be distinguished by electron microscopy. Another possibility
is that the parasites seen in vacuoles were phagocytosed ones
and not in the process of invasion. In fact, if their invasion
mode is cell-traversal motility, as we believe, this event may be
rapidly completed and difficult to catch by electron micro-
scopy. Therefore, many phagocytosed parasites could be
included among those seen. Taking the evidence together, we
propose that the sporozoites access the hepatocyte through
Kupffer cells by the same cell-traversal motility that has been
identified in vitro, and we propose a model for sporozoite
liver infection in Figure 7.
In this study we have established the significance of cell-
passage ability of the sporozoite in malaria transmission and
have demonstrated that this ability is necessary for breaking
through the liver sinusoidal barrier. Cell-traversal activity
plays an important role in other invasive stages of the
malarial parasite, including the ookinete, which migrates
through the epithelial cells of the mosquito midgut, and the
sporozoite in the oocyst, which is released from the mature
oocyst and then migrates through the salivary gland cell. Our
study revealed that another cellular barrier is present in the
malarial life cycle and sporozoites must break through this
barrier by cell-traversal activity.
Our recent work has identified two other genes that are
involved in the cell passage activity of the sporozoite. Like
SPECT, the products of these genes have a secretory protein-
like structure and are localized in the micronemes. Further-
more, sporozoites disrupted for these genes have similar
phenotypic charac ter t o spect-disrupted ones, including
impaired cell-passage ability, decreased liver infectivity with
similar reduction rate, complete restoration of the infectivity
in Kupffer cell-depleted rats, normal gliding motility, and
normal hepa tocyte infectivity (unpublished data). This
suggests that the cell-traversal ability of the sporozoite is
realized by cooperation of several microneme proteins. We
suggest that these molecules could be targets for antimalarial
strategies, since success in crossing this layer is critical for the
malarial parasite to establish infection in humans. Elucida-
tion of the molecular mechanisms of passage may lead to
novel malaria transmission-blocking strategies that prevent
sporozoites from gaining access to the hepatocyte.
Materials and Methods
Parasite preparations. Female 6–10-wk-old BALB/c mice (Japan
SLC, Inc., Hamamatsu, Japan) infected with the P. berghei ANKA strain
were prepared by peritoneal injection of infected blood that was
stored at 708C. For the purification of sporozoites, infected
mosquitoes were dissected 24–28 d after the infective blood meal.
The salivary glands and midgut were separately collected in medium
199 on ice and then gently ground to release the sporozoites.
Ookinetes and erythrocytic-stage parasites were prepared as de-
scribed previously (Yuda et al. 1999; Kariu et al. 2002).
Genomic Southern blot hybridization. Genomic DNA of P. berghei
parasites (2 lg) was digested with ClaI, EcoRI, EcoT22I, HindIII, or
XbaI, separated on 1.2% agarose gel and then transferred to a nylon
membrane. DNA fragments were amplified by PCR using genomic
DNA as template with the following primers: 59-TGGGCAATTTTG-
CCTTTAAAAACG-39 and 59-TTTCATTGTGTTAAACGATAAGTG-
39. They were labeled with [
32
P]dCTP and used as probes.
Antibody preparation and Western blot analysis Recombinant
SPECT without signal sequence was expressed as a glutathione S-
transferase (GST)–fusion protein using the pGEX 6p-1 system
(Amersham Bioscience, Uppsala, Sweden). The recombinant protein
was purified with a GST column and used for immunization of
rabbits. Specific antibodies were affinity purified using a N-
hydroxysuccinimide-activated column (Amersham Bioscience)
Figure 7. Schematic Representation of Sporozoite Migration to and
Infection of Hepatocytes
(Left) Sporozoites migrate to the space of Disse through the Kupffer
cells. [1] The sporozoite (Sp) in the circulatory system is sequestered
to the sinusoidal endothelial cell (EC) by specific recognition of the
cell surface or glycosaminoglycans extending from the hepatocytes
(He) through fenestration. [2] The sporozoite begins to glide on the
epithelial cell surface. [3] Encountering a Kupffer cell (KC), the
sporozoite ruptures the plasma membrane, passes through the cell,
and enters into the space of Disse. Thus, the sporozoite gains access
to hepatocytes. This step requires SPECT. [4] The sporozoite infects a
hepatocyte with formation of a vacuole and develops into EEF in the
hepatocyte.
(Right) An alternative route to the hepatocyte. A small number of
sporozoites, which find gaps in the sinusoidal layer while gliding,
migrate to hepatocytes directly through the openings without need
for cell passage and infect the hepatocytes. Likewise, in Kupffer cell-
depleted rats, both wild-type and spect() sporozoites can enter
hepatocytes through numerous gaps present between the sinusoidal
endothelial cells.
DOI: 10.1371/journal.pbio.0020004.g007
PLoS Biology | http://biology.plosjournals.org January 2004 | Volume 2 | Issue 1 | Page 0082
Role of SPECT in Malarial Transmission

coupled with recombinant SPECT protein. For CS antiserum
production, the peptide DPPPPNANDPAPPNAN, corresponding to
the repeat region, was conjugated to keyhole limpet hemocyanin as a
carrier and used for the immunization of rabbits. Western blot
analysis was performed as described previously (Kariu et al. 2002).
Immunofluorescence microscopy and immunoelectron microscopy.
Immunofluorescence microscopy was performed as described pre-
viously (Kariu et al. 2002). Purified parasites were fixed in acetone for
2 min. The slides were incubated with anti-SPECT rabbit antibodies
and then with FITC-conjugated secondary antibody (Zymed. South
San Francisco, California, United States). For nuclear staining, 49,6-
diamidino-2-phenylindole (DAPI) (0.02 lg/ml final concentration) was
added to the secondary antibody solution. Immunoelectron micro-
scopy was performed as described previously (Yuda et al. 2001). In
brief, purified parasites were fixed in 1% paraformaldehyde–0.1%
glutaraldehyde for 15 min on ice. After embedding in LR Gold resin
(London Resin Company Ltd., London, United Kingdom), ultrathin
sections were incubated with anti-SPECT antibodies and then with
secondary antibody conjugated to gold particles (15 nm diameter)
(AuroProbe, Amersham Pharmacia Biotech, Uppsala, Sweden). The
samples were examined with a Hitachi H-800 transmission electron
microscope (Hitachi, Tokyo, Japan) at an acceleration voltage of 100
kV.
Targeted disruption of the spect gene. For construction of the
targeting vector, two fragments of the spect gene were amplified by
PCR using genomic DNA as template with the primer pairs 59 -
CGCGAGCTCGCAATATGGTATTAAATTTTGGGCTAGCCA-39 and
59-CGC GGATC CGGTA TTTT CATT GTGTT AAACGA TATG TGA- 39
and 59-CCGCTCGAGGTCCTATTTATCATTTTAAAATGTGTTT-
TATC-39 and 59-CGGGGTACCAATCGTCATAAATAGGAGTTAT-
GAAGT-39. These fragments were cloned into either side of the
selectable marker gene in pBluescript (Strategene, La Jolla, Califor-
nia, United States). The gene targeting experiment was performed as
described previously (Yuda et al. 1999).
Evaluation of sporozoite infectivity to rats. Sporozoites collected
from mosquito salivary glands were suspended in medium 199 and
then injected intravenously into 3-wk-old female Wistar rats (Japan
SLC, Inc., Hamamatsu, Japan) (n = 5). Before each inoculation,
sporozoites were checked for their ability to glide in vitro to confirm
that they contained over 60% motile sporozoites. Parasitemia was
checked every 12 h by a Giemsa-stained blood smear.
Measurement of the number of EEFs in the infected liver.
Sporozoites (5.0 3 10
5
) were intravenously inoculated into a 3-wk-
old female Wistar rat. After 24 h, the liver was perfused with PBS
followed by 4% paraformaldehyde. The liver was further fixed in 4%
paraformaldehyde for 6 h and frozen in liquid nitrogen. Cryostat
sections (20 lm) were prepared from the left lobe and fixed in
acetone for 2 min on a glass slide. The EEFs were detected by
immunofluorescence staining using rabbit anti-CS antiserum and
FITC-conjugated secondary antibody. At least 12 sections were
examined under an Olympus (Tokyo, Japan) BX60 fluorescence
microscope (2003) and the number of EEFs per square millimeter was
calculated.
EEF development assay in vitro. The EEF formation assay was
performed as described previously (Hollingdale et al. 1981) with
minor modifications. HepG2 cells (5.0 3 10
5
) were plated in 8-well
chamber slides. Sporozoites (5.0 3 10
3
or 5.0 3 10
4
) were suspended in
100 ll of complete medium and added to this culture. After 2 h, the
mediawerereplacedwith400ll of fresh complete medium
supplemented with 3 lg/ml glucose. The slides were incubated for 2
d with medium changed twice a day and were fixed in acetone for 2
min. The EEFs were detected by immunofluorescence staining as
described above. The number of EEFs in one-fifth of the area of each
well was counted under an Olympus BX60 fluorescence microscope
(2003).
Cell-traversing activity a ssay. The traversing activity of the
sporozoite was examined using a standard cell-wounding and
membrane repair assay (Mota et al. 2001). HepG2 cells (2.5 3 10
5
)
or HeLa cells (5.0 3 10
4
) were inoculated into 8-well chamber slides
(Nunc Inc., Napierville, Illinois, United States). Sporozoites were
added 2 d later to cells for 1 h in the presence of 1 mg/ml FITC-
labeled dextran (10,000 MW, lysine-fixable; Molecular Probes, Inc.,
Eugene, Oregon, United States). The cells were incubated for an
additional 3 h in complete culture medium and fixed with 4%
paraformaldehyde in PBS. The number of FITC-positive cells was
counted under a fluorescence microscope.
Depletion of rat Kupffer cells. For depletion of Kupffer cells, 3-wk-
old female Wistar rats were injected intravenously with 120 llof
liposome-encapsulated Cl
2
MDP or an equal volume of PBS as control.
After 48 h, sporozoites were injected into a tail vein and the
parasitemia was checked by Giemsa-stained blood smears. Cl
2
MDP
liposomes were prepared as described elsewhere (van Rooijen and
Sanders 1994). Elimination of Kupffer cells was confirme d by
immunoperoxidase staining after liver perfusion with PBS followed
by fixation with 4% paraformaldehyde in PBS. Cl
2
MDP was a gift
from Roche Diagnostics (Mannheim, Germany).
Acknowledgments
This study was supported by a grant-in-aid for Scientific Research on
Priority Areas to YC (15019042) and to MY (15019043) and for
Scientific Research (A) to YC (14207011) of the Ministry of Education,
Science, Culture, and Sports of Japan. It was also supported by a grant
from the Research for the Future Program of the Japan Society for
the Promotion of Science to YC and by a grant from the Core
Research for Evolutional Science and Technology (CREST) of the
Japan Science and Technology Agency to YC.
Conflicts of interest. The authors have declared that no conflicts of
interest exist.
Author contributions. TI, YC, and MY conceived and designed the
experiments. TI, KY, and MY performed the experiments. TI and MY
analyzed the data. TI and MY wrote the paper.
&
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