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Selective inhibition of a two-step egress of malaria parasites from the hosterythrocyte
Mark E. Wickham1, Janetta G. Culvenor2 and Alan F. Cowman1
1The Walter and Eliza Hall Institute of Medical Research
Melbourne 3050, Australia
2Department of Pathology, The University of Melbourne
Melbourne 3050, Australia
*Correspondence to:
Alan F. CowmanThe Walter and Eliza Hall Institute of Medical Research1G Royal ParadeMelbourne 3050AustraliaTelephone: 61-3-93452555Facsimile: 61-3-93470852Email: cowman@wehi.edu.au
Running title: Inhibition of P.falciparum host cell exit
Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on July 11, 2003 as Manuscript M305252200 by guest on D
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SUMMARY
Escape from the host erythrocyte by the invasive stage of the malaria parasite
Plasmodium falciparum is a fundamental step in the pathogenesis of malaria of
which little is known. Upon merozoite invasion of the host cell, the parasite
becomes enclosed within a parasitophorous vacuole, the compartment in which
the parasite undergoes growth followed by asexual division to produce 16-32
daughter merozoites. These daughter cells are released upon parasitophorous
vacuole and erythrocyte membrane rupture. To examine the process of
merozoite release we have used P.falciparum lines expressing GFP-chimeric
proteins targeted to the compartments from which merozoites must exit; the
parasitophorous vacuole and the host erythrocyte cytosol. This allowed
visualization of merozoite release in live parasites. Here we provide the first
evidence in live untreated cells that merozoite release involves a primary
rupture of the parasitophorous vacuole membrane followed by a secondary
rupture of the erythrocyte plasma membrane. We have confirmed that
parasitophorous vacuole membrane rupture occurs prior to erythrocyte
plasma membrane rupture in untransfected wild-type parasites using immuno-
electron microscopy. We have also demonstrated selective inhibition of each
step in this two-step process of exit using different protease inhibitors,
implicating the involvement of distinct proteases in each of these steps. This will
facilitate the identification of the parasite and host molecules involved in
merozoite release.
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INTRODUCTION
During the erythrocytic phase of the P.falciparum lifecycle, the merozoites
released initially from infected hepatocytes attach to and invade human
erythrocytes in the bloodstream. Upon invasion of the host cell, the parasites
become enclosed in a parasitophorous vacuole in which, as ring and
trophozoite stages, they undergo growth followed by asexual division
(schizogony) to produce 16-32 daughter merozoites. It is from both this vacuole
and the host erythrocyte that the newly formed merozoites in the schizont
must escape.
While little is known about the molecules that mediate this process, proteases
have been implicated in both parasite exit from the erythrocyte and the
subsequent invasion into erythrocytes through the use of protease inhibitors
that halt these processes (1-3). Following the demonstration that P. knowlesi
schizonts incubated in the presence of the protease inhibitors chymostatin and
leupeptin show decreased reinvasion due to inhibition of schizont maturation
(3,4), Lyon and Haynes demonstrated that P.falciparum schizonts cultured in the
presence of the inhibitors chymostatin, leupeptin, antipain and pepstatin also
fail to rupture properly (2). Recently it has been demonstrated that the protease
inhibitors E-64 and E-64d inhibit schizont maturation (5,6). The role of the
proteases involved in schizont rupture is presumably to degrade both the
parasitophorous vacuole and the erythrocyte membrane skeleton, thereby
facilitating release. However, the proteases involved have not yet been fully
characterized. A number of inhibitor-sensitive parasite proteases have been
implicated in schizont rupture, including the aspartic protease plasmepsin II
which has been demonstrated to digest spectrin, actin and protein 4.1 at neutral
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pH (7), falcipain-2; a cysteine protease that cleaves erythrocyte ankyrin and
protein 4.1 (8-11), and the cysteine protease-like SERA (serine repeat antigen)
family, the members of which possess a centrally located papain-like protease
domain (1,12-14). Interestingly, inhibition of the proteolytic processing of SERA
is possible using protease inhibitors that block schizont rupture (15). The
relevance of these protease activities to the process of merozoite release
remains to be determined, and the availability of the genome sequence of
P.falciparum has facilitated the identification of further protease-like molecules
that may play a role (12,16).
Ultrastructural evidence suggests that during schizogony the parasite plasma
membrane invaginates to surround the merozoites forming within the confines
of the parasitophorous vacuole (17), and that late in schizogony, the
parasitophorous vacuole membrane may be absent with the fully formed
merozoites free within the host erythrocyte (17,18). This suggests that escape
from the parasitophorous vacuole occurs prior to exit from the erythrocyte, but
this possibility remains uninvestigated. An alternate model for schizont rupture
has been proposed which involves escape from the host erythrocyte of the
merozoites enclosed within the parasitophorous vacuole, followed by
extraerythrocytic rupture of the vacuole and release of invasive merozoites
(Figure 1 A) (5).
Here we examine the mechanism of parasite exit from the host erythrocyte in
both wild-type and transgenic P.falciparum lines. Presented here is the first
evidence in live untreated cells that rupture of the parasitophorous vacuole
membrane occurs while the parasite is within the erythrocyte (Figure 1 B). The
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mixing of vacuolar and erythrocytic contents subsequent to this
intraerythrocytic vacuolar rupture is also detected in wild-type parasites. Each
step in the process of escape is selectively inhibitable by different protease
inhibitors, indicating that different proteases mediate each event.
EXPERIMENTAL PROCEDURES
Parasite lines.
To generate transgenic Plasmodium falciparum-infected erythrocytes expressing
KAHRP-GFP chimeric proteins we made transfection constructs in the plasmid
vector pHH2 (19,20) as described (21). A region of the KAHRP gene encoding
the first 60 amino acids, which includes a putative hydrophobic signal sequence
of eleven amino acids, was joined upstream of the GFP coding sequence in the
transfection vector pHH2 (20). Parasites stably transfected with this construct
traffic the GFP fusion into the parasitophorous vacuole throughout the asexual
lifecycle (21). These parasites are designated 3D7-His. A region of the KAHRP
gene encoding the first 123 amino acids of KAHRP was joined upstream of the
GFP coding sequence. This region of KAHRP contains both the putative
hydrophobic signal sequence required for transit to the parasitophorous
vacuole, and the histidine rich region that contains the signal required for
translocation into the erythrocyte (21). Transgenic parasites expressing this
construct traffic the GFP fusion into the erythrocyte cytosol throughout the
asexual lifecycle. These parasites are designated 3D7+His. The 3D7 cloned
P.falciparum parasites were transfected by electroporation and drug selected
using 0.25 nM WR99210 as previously described (22).
Fractionation of infected erythrocytes and western blotting.
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For permeablisation of infected red cells with streptolysin-O (SLO), haemolytic
activity of SLO was determined and 2x107 parasites were incubated in RPMI
containing 3-4 haemolytic units of SLO as described (23,24). In brief,
P.falciparum-infected erythrocytes were incubated in 3-4 haemolytic units of SLO
in RPMI for 6 min at room temperature, the supernatant resuspended in
Laemmli sample buffer, the pellet washed in RPMI and subsequently
resuspended in Laemmli sample buffer. For saponin lysis, 2x107 parasites were
incubated in 1.5 volumes of 0.15% saponin for 10 min on ice, centrifuged and
the supernatant resuspended in Laemmli sample buffer, the pellet washed in
PBS and subsequently resuspended in Laemmli sample buffer. Proteins were
separated by SDS-SAGE and transferred to PVDF and visualised by ECL using
mouse anti-GFP antiserum (1:1000).
Protease Inhibitor Treatment.
Parasitised cells were synchronised by two consecutive sorbitol treatments 4 hr
apart, cultured until middle-stage schizonts, then treated with 10 mM of L-
transepoxy-succinyl-leucylamido-(4-guanidino)butane (E-64) (Sigma) as
described (5) or a combination of 10 mg/ml each of leupeptin and antipain, or
leupeptin and chymostatin (Sigma) as described (2).
Indirect Immunofluorescence Assay.
Indirect immunofluorescence assays were performed on control and protease
treated P.falciparum-infected erythrocytes smeared on glass slides and fixed with
methanol. Slides were incubated sequentially with rabbit anti-KAHRP (1:200).
The slides were then incubated with anti-rabbit antibodies conjugated to FITC
(1:1000) in the presence of the nuclear stain 4’,6-diamino-2-phenylindole (DAPI)
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at final concentration of 2 mg/ml. Samples were viewed with a Carl Zeiss
Axioskop with a PCO SensiCam and Axiovision 3 software.
Fluorescence Microscopy.
Ring stage parasites were synchronised using two consecutive sorbitol
treatments 4 hours apart, cultured until early stage schizonts, and samples
taken hourly during the process of schizongony and merozoite release.
Protease inhibitor treated and control parasites were cultured in DAPI at final
concentration of 2 mg/ml for 30 min at 37ºC immediately prior to imaging.
Fluorescence from GFP and DAPI was observed and captured in live cells at
20ºC within 20 min of mounting the sample in culture medium under a
coverslip on a glass slide using a Carl Zeiss Axioskop with a PCO SensiCam and
Axiovision 3 software.
Immunoelectron Microscopy.
Control and protease inhibitor treated cultures were fixed in 0.25%
glutaraldehyde in 0.1 M phosphate buffer pH 7.4 for 10 min at room
temperature, followed by addition of 0.05 M NH4Cl and washed in 0.1 M
phosphate buffer. Samples were dehydrated in 70% ethanol and embedded in
L. R. White resin and polymerised for 4 hr at 50ºC or 5 days at 37ºC. Thin
sections were incubated in rabbit anti-S-Antigen antibodies in 0.05 M
phosphate, pH 7.4/0.1% Tween 20/1% BSA, washed then protein A-5 or 10 nm
gold in 0.05 M phosphate, pH 7.4/0.1% Tween 20/1% BSA, then stained in
uranyl acetate.
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RESULTS
Intraerythrocytic parasitophorous vacuole rupture in GFP-expressing
parasites
To investigate the sequence of events involved in schizont rupture transgenic
P.falciparum lines have been generated which traffic GFP fusions to the
compartments the parasite must traverse upon exit; the parasitophorous
vacuole and the host erythrocyte cytosol. To traffic GFP to the parasitophorous
vacuole, a portion of the KAHRP gene that encodes the first 60 amino acids of
the protein was joined upstream of the GFP coding sequence in the transfection
vector pHH2 (19,20). This region of KAHRP includes a putative hydrophobic
signal sequence of eleven amino acids flanked by lysine residues. Transgenic
parasites stably transfected with this construct traffic the fusion via the
canonical secretory pathway into the parasitophorous vacuole throughout the
asexual lifecycle. These parasites are designated 3D7-His. To traffic GFP into the
host erythrocyte, a region of the gene encoding the first 123 amino acids of
KAHRP was joined upstream of the GFP coding sequence. This region of
KAHRP contains both the putative hydrophobic signal sequence required for
transit to the parasitophorous vacuole, and the histidine rich region that
contains the signal required for translocation into the erythrocyte. Transgenic
parasites expressing this construct traffic the GFP fusion into the erythrocyte
cytosol throughout the asexual lifecycle via an extension of the parasite’s
canonical secretory pathway in the host cell. These parasites are designated
3D7+His.
The compartmentalization of GFP in these transgenic P.falciparum lines during
asexual division (schizogony) and escape from the host erythrocyte was
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examined. The transgenic parasites were made synchronous with regard to cell
cycle phase by sorbitol lysis and followed by fluorescence microscopy. Early in
schizogony the transgenic lines were found to exhibit GFP fluorescence in the
compartments to which the GFP fusions are trafficked; the parasitophorous
vacuole in 3D7-His parasites and the host erythrocyte cytosol in 3D7+His
parasites (Figure 2 A and B). In late trophozoites and schizonts, some GFP can
be seen in association with the food vacuole and hemozoin; GFP appears to re-
enter the parasites with erythrocyte cytoplasm ingestion (19,21). However, in
both transgenic lines late in schizogony GFP consistently localised to
compartments to which it is not targeted (Figure 2 C and D). In 3D7-His
parasites, the GFP fusion that lacks the signal required for translocation into the
host erythrocyte, was found present in the erythrocyte cytosol (Figure 2 C,
white arrow). A possible explanation for this localization is that the
parasitophorous vacuole membrane has lysed, and the GFP fusion has flooded
into the erythrocyte by free diffusion. This intraerythrocytic vacuolar rupture
and subsequent diffusion of GFP from the vacuole into the host erythrocyte has
also been observed during confocal sectioning of late schizonts of an unrelated
transgenic line that also targets GFP to the parasitophorous vacuole (Melanie
Rug, personal communication).
In 3D7+His parasites, the GFP fusion was observed both in the erythrocyte
cytosol, the compartment to which it is targeted, and immediately surrounding
the fully formed merozoites (Figure 2 C, black arrow). However, in early
schizonts the GFP fusion is excluded from the vacuole surrounding the
merozoites. This again supports an intraerythrocytic rupture of the vacuolar
membrane, allowing diffusion of GFP from the erythrocyte to surround the
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merozoites.
Fractionation of GFP-expressing parasites
To examine this intraerythrocytic rupture of the vacuolar membrane at a
population level, cell fractionation using saponin and streptolysin O was
utilized. Saponin fractionation allows analysis of the export of proteins from the
parasite as it lyses both the erythrocyte plasma and parasitophorous vacuole
membranes, leaving the parasite plasma membrane intact. Upon fractionation
with saponin, proteins retained within the parasite will be present in the pellet
and those exported from the parasite will be detected in the supernatant.
Streptolysin O fractionation allows analysis of the export of proteins from the
parasite since it permeabilises only the erythrocyte plasma membrane, leaving
the parasitophorous vacuole membrane intact. Proteins trafficked into the
vacuole will be present in the pellet and those trafficked into the host
erythrocyte present in the supernatant. In 3D7-His trophozoites the GFP fusion
is trafficked from the parasite, indicated by the predominance of the GFP fusion
in the saponin supernatant and into the parasitophorous vacuole but not
beyond, indicated by the presence of the fusion in the streptolysin O pellet but
not supernatant (Figure 2 E). Fractionation of late 3D7-His schizonts (the
lifecycle stage in M phase) shows that the GFP fusion is again trafficked from
the parasite predominantly into the parasitophorous vacuole, but is also
present in the erythrocyte cytosol, indicated by GFP fusion present in the
streptolysin O supernatant. This localisation at a population level (2x107
parasites) agrees with that observed by microscopy on individual parasites and
is consistent with intraerythrocytic rupture of the parasitophorous vacuole
membrane during schizogony and diffusion of the GFP fusion into the
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erythrocyte cytosol.
Intraerythrocytic parasitophorous vacuole rupture in wild-type parasites.
To confirm that parasitophorous vacuole rupture occurs within the erythrocyte
in wild-type parasites, immuno-electron microscopy using antibodies to S-
Antigen, a P.falciparum protein that localizes to the parasitophorous vacuole
(25,26) was performed. In untransfected trophozoites S-Antigen labelling was
observed in the parasitophorous vacuole (Figure 3 A). However, late in
schizogony the vacuolar marker is observed throughout the erythrocyte
cytosol (Figure 3 B), consistent with intraerythrocytic lysis of the
parasitophorous vacuole membrane and subsequent diffusion of the vacuolar
contents, including S-antigen, into the erythrocyte. This is consistent with the
apparent flooding of cytosolic material from the erythrocyte into the vacuole
observed previously, presumably following vacuolar lysis (27).
Intraerythrocytic lysis of the vacuole membrane in untransfected P.falciparum
lines rules out the possibility that intraerythrocytic lysis of the vacuole in
transgenic lines is an artefact of GFP expression.
Selective inhibition of parasitophorous vacuolar and erythrocyte plasma
membrane rupture
To establish that the altered localisation of GFP in late stage parasites is
attributable to intraerythrocytic lysis of the vacuolar membrane, and to exclude
the possibility that the GFP fusions are trafficked to the different compartments
late in schizogony, selective inhibition of each step in the mechanism of escape
from the host cell was attempted. It has been shown that the protease inhibitors
E-64 and E-64d inhibit lysis of the parasitophorous vacuole membrane but not
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the lysis of the erythrocyte plasma membrane (5,6). Addition of E-64 to both
3D7-His and 3D7+His transgenic lines caused an accumulation of the
parasitophorous vacuole membrane-enclosed merozoite structures observed
previously (Figure 4 A and schematically, B). The compartment surrounding
the merozoites can be identified as the parasitophorous vacuole since these
structures of the 3D7-His line, which traffics GFP to the vacuole, exhibit GFP
fluorescence (Figure 4 A, schematically, B). The lack of GFP fluorescence in
merozoite structures of the 3D7+His line, which exports GFP to the erythrocyte
cytosol, indicates that lysis of the erythrocyte plasma membrane has occurred,
releasing the GFP fusion into the culture supernatant, and that the limiting
membrane observed is of vacuolar origin. The lack of inhibition of erythrocyte
plasma membrane lysis in the presence of E-64 implicates distinct proteases in
the lysis of the two membranes.
Salmon et al. (2001) made the important observation that morphologically
similar clusters to those seen upon E-64 treatment are observed at low
frequency in untreated cultures, and proposed a two-step model for host cell
exit. The current model for schizont rupture involves a primary lysis of the
erythrocyte plasma membrane and release of the merozoites still enclosed
within the parasitophorous vacuole membrane, followed by extraerythrocytic
proteolysis of the vacuole membrane and merozoite dispersal (5). It has also
been shown that schizonts cultured in the presence of the protease inhibitors
leupeptin and antipain, like E-64, fail to rupture and remain surrounded by a
limiting membrane. Using polyclonal serum raised against human
erythrocytes, this membrane was identified as the erythrocyte plasma
membrane (2). However, since the parasitophorous vacuole may contain
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components of the erythrocyte plasma membrane either acquired upon
invasion or later internalised (28,29), it is possible that this membrane is of
vacuolar origin (5).
Determination of Membrane Origin
To determine both the origin of the limiting membrane of incompletely
ruptured schizonts formed upon leupeptin and antipain treatment, the
transgenic lines were treated with these inhibitors. Addition of leupeptin and
antipain to the 3D7-His transgenic line caused an accumulation of the limiting
membrane-enclosed merozoite structures observed previously (Figure 4 C and
schematically, D). This limiting membrane is the erythrocyte plasma membrane
since addition of leupeptin and antipain to both transgenic lines caused an
accumulation of limiting membrane-enclosed merozoite structures that exhibit
GFP fluorescence (Figure 4 C and schematically, D). A vacuolar origin of the
limiting membrane would result in membrane-enclosed merozoite structures
that did not exhibit GFP fluorescence in the 3D7+His line which traffics GFP
beyond this membrane. As with (untreated) late schizonts of the 3D7+His
transgenic line (Figure 2 C and D), GFP fluorescence was observed immediately
surrounding the fully formed merozoites in the 3D7+His erythrocyte
membrane-enclosed merozoite structures. Again, this supports an
intraerythrocytic rupture of the vacuolar membrane, allowing diffusion of GFP
from the erythrocyte to surround the merozoites. Therefore leupeptin and
antipain inhibit the lysis of the erythrocyte plasma membrane without
inhibiting lysis of the parasitophorous vacuole. The lack of inhibition of
parasitophorous vacuole membrane lysis in the presence of leupeptin and
antipain indicates that the process of escape is a two-step event involving
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distinct proteases, both steps of which can be selectively inhibited.
To confirm the origin of the limiting membrane in protease inhibitor treated
cells, immunoelectron microscopy and indirect immunofluorescence assays
using antibodies to parasite proteins exported from the parasite were
performed. Immunoelectron microscopy was performed using antibodies to
the vacuolar protein S-Antigen on thin sections of leupeptin and chymostatin
treated wild-type parasites. In early schizonts we observe S-antigen labelling in
the parasitophorous vacuole (Figure 3 C). However, late in schizogony the
vacuolar marker is observed throughout the host erythrocyte cytosol (Figure 3
D) consistent with selective inhibition of erythrocyte plasma membrane, but
not vacuolar, lysis. Using indirect immunofluorescence, PfEMP-3, a parasite
protein that localizes under the host erythrocyte plasma membrane is present
in merozoite clusters produced by leupeptin and antipain, but not E-64,
treatment (Figure 4 E). This indicates that the origin of the limiting membrane
in leupeptin and antipain-treated cells is the erythrocyte plasma membrane.
DISCUSSION
These data provide the first direct evidence that P.falciparum merozoites escape
from the host erythrocyte is a two-step process involving a primary exit from
the vacuole it acquires upon entry, followed by a secondary exit from the
erythrocyte itself (Figure 1 B).
It has been shown ultrastructurally that early in schizogony the parasite plasma
membrane invaginates to surround the merozoites forming within the confines
of the parasitophorous vacuole (17). Late in schizogony the parasitophorous
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vacuole membrane may be absent with the fully formed merozoites free within
the host erythrocyte (17,18). This is consistent with an intraerythrocytic rupture
of the parasitophorous vacuole membrane. Indeed late stage schizonts have
been observed with the parasitophorous vacuole either intact or partially intact
with material of the same density either side of the parasitophorous vacuole
membrane (Ross Waller, personal communication). While it is difficult to argue
on the basis of density of the material either side of the vacuolar membrane
that the parasitophorous vacuole has become permeable to the host
erythrocyte cytosol in these cells, this in combination with the localisation of the
vacuolar marker S-Antigen to the host erythrocyte cytosol (Figure 3), provides
ultrastructural evidence for intraerythrocytic rupture of the vacuole membrane.
This sequence of events clearly occurs at the population level, as detected by the
release of parasitophorous vacuolar contents into the host erythrocyte cytosol.
Both steps in this two-step process can be selectively inhibited; the primary
vacuolar lysis by E-64 and the secondary erythrocyte membrane rupture by
leupeptin and antipain, indicating that independent proteases mediate each
step. Since E-64 is an irreversible inhibitor of cysteine proteases that does not
inhibit serine proteases, it is likely that the protease(s) involved in
parasitophorous vacuole lysis is a cysteine protease. The components of the
parasitophorous vacuole that such a protease(s) would proteolytically process
remain to be identified. The protease inhibitors shown to inhibit erythrocyte
plasma membrane rupture are leupeptin, antipain, chymostatin and pepstatin
(2,3,30). Leupeptin and antipain, used in this study at 10 mg/ml, each inhibit
both trypsin-like serine proteases and cysteine proteases. Pepstatin is a non-
selective inhibitor of aspartic proteases, and chymostatin inhibits both serine
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proteases and cysteine proteases – both of these inhibit schizont maturation at
10 mg/ml (2). It is therefore possible that the protease(s) mediating erythrocyte
plasma membrane rupture is a cysteine or serine protease. However, it is likely
that the rupture of each membrane involves a cascade of events that may be
inhibited at different points using inhibitors with different specificities. As such
it remains possible that erythrocyte plasma membrane rupture involves both
an aspartic protease and a cysteine or serine protease.
It is important to note that E-64, which inhibits parasitophorous vacuolar lysis,
appears to do so only when added to middle stage schizonts (15) - no inhibition
is observed with late stage schizonts (5). One explanation for this lack of
inhibition of vacuolar rupture in late stage schizonts is that the parasitophorous
vacuole membrane degradation has already commenced in these parasites.
That leupeptin and antipain do not inhibit parasitophorous vacuole rupture
may suggest that these inhibitors do not access the parasitophorous vacuole of
infected erythrocytes. However, it has been demonstrated that leupeptin added
to parasite cultures inhibits the processing of SERA, which localises to the
parasitophorous vacuole, from a 56 kDa fragment to a 50 kDa fragment (15),
indicating that leupeptin is indeed able to access the parasitophorous vacuole of
P.falciparum infected erythrocytes. Additionally, the more membrane
permeable analogue of E-64, E-64d, that lacks charged groups, has been
demonstrated to block schizont development (6) again suggesting that the site
of action of these inhibitors does not result from permeability differences.
The inhibitor-sensitive proteases identified so far which may be involved in
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erythrocyte plasma membrane rupture are the aspartic protease plasmepsin II,
which cleaves erythrocyte spectrin, actin and protein 4.1 (31), the cysteine
protease falcipain-2, which cleaves erythrocyte ankyrin and protein 4.1 (8,32),
the putative serine protease ABRA (33) or the SERA/SERPH family of serine
protease-like molecules. These molecules have been shown to localize to the
parasitophorous vacuole and some, such as ABRA and the SERA/SERPH
family, have been shown to be weakly associated with the merozoite surface
(34). It has been recently demonstrated that SERA is associated with the
parasitophorous vacuole membrane (15). Additionally, processing of
baculovirus-expressed SERA is can be inhibited by a number of protease
inhibitors, including those shown to inhibit rupture of the vacuole and
erythrocyte plasma membrane (such as E-64 and leupeptin), and that the
proteases responsible for this processing also appear membrane associated (15).
It is possible that degradation of the parasitophorous vacuole membrane is
required for these proteases to gain access to their substrates at the erythrocyte
plasma membrane, but the degradation of the erythrocyte plasma membrane
in the presence of E-64 suggests that the proteases responsible may be actively
trafficked across the vacuolar membrane.
The identification of the molecules mediating the process of schizont rupture is
complicated by correlating inhibitor studies based on cell-free assays with
experiments on P.falciparum parasite culture. For instance, leupeptin inhibits
schizont rupture at concentrations of up to 68 mg/ml (15) through the inhibition
of erythrocyte plasma membrane rupture (2). At higher concentrations (678
mg/ml), leupeptin inhibits both the proteolytic processing of SERA in cell-free
assays (15), and the cleavage of ankyrin by falcipain-2 (9). Likewise E-64, which
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clearly inhibits parasitophorous vacuolar rupture at 10 mM (5), also inhibits
SERA and ankyrin cleavage at higher concentrations (100 mM and 10-100 mM,
respectively) (9,15). Consequently, the roles of these candidate protease
activites in the process of escape remain to be elucidated.
There has already been some success in the design of inhibitors selective for the
aspartic proteases plasmepsin I and II (35-37) and the papain-family cysteine
proteases known as the falcipains (6,11,38-42). Most recently, a screening of
chemical libraries has identified inhibitors of falcipain-1 that have facilitated the
identification of the role of falcipain-1 in red blood cell invasion (6). Parasites
expressing GFP will prove an invaluable tool both in the search for the parasite
molecules mediating the process of escape, and for the screening of inhibitors
or combinations of inhibitors targeting this process.
ACKNOWLEDGEMENTS
We thank H-G. W. Meyer, S. Bhakdi and A. Hibbs for the generous gift of SLO.
We thank M. Duraisingh, B. Crabb, M. Rug, and A. Maier for helpful
discussions. We thank the Red Cross Blood Service (Melbourne, Australia) for
supply of red cells and serum. A.C. is supported by a Howard Hughes
International Research Fellowship from the Howard Hughes Medical Institute.
This work was supported by a grant from the National Institutes of Health USA
(RO1 AI44008) and the National Health and Medical Research Council of
Australia. M.W. is supported by an Australian Postgraduate Research Award.
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Figure 1: Models of merozoite escape from the host erythrocyte.
(A) Primary rupture of the erythrocyte plasma membrane late in schizogony
results in parasitophorous vacuole membrane enclosed merozoite structures
(PEMS). Following a secondary extraerythrocytic rupture of the
parasitophorous vacuole membrane, invasive merozoites are released. (B)
Primary rupture of the parasitophorous vacuole membrane late in schizogony
results in erythrocyte plasma membrane enclosed merozoite structures, and
mixing of vacuolar and erythrocyte cytosolic contents. Following a secondary
rupture of the erythrocyte plasma membrane, invasive merozoites are
released.
Figure 2: P.falciparum ruptures the parasitophorous vacuole within the host
erythrocyte during asexual division.
(A) Early 3D7-His schizonts traffic GFP (green) to the parasitophorous vacuole
where it surrounds the fully formed daughter merozoites. Early 3D7+His
schizonts traffic GFP into the host erythrocyte, and it is not observed in the
vacuole surrounding the daughter merozoites. Parasite nuclei are stained with
DAPI (blue). (C) Maturation of the schizont involves lysis of the
parasitophorous vacuole; following vacuolar lysis, late 3D7-His schizonts
display GFP fluorescence immediately surrounding the merozoites in addition
to the erythrocyte cytosol (white arrow). Late 3D7+His schizonts traffic GFP
beyond the vacuole into the host erythrocyte, and GFP fluorescence is
observed immediately surrounding the daughter merozoites (black arrow).
This localisation of GFP during early and late schizogony is represented
schematically in B and D, respectively. (E) Examination of intraerythrocytic
rupture of the parasitophorous vacuole at a population level. Parasites that
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traffic GFP to the vacuole were fractionated before M phase (T, Trophozoites)
and during schizogony (S). Upon saponin fractionation (Sap), GFP is detected in
the pellet (P) and predominantly in the supernatant (Sn), indicating that GFP is
exported from the parasite in both stages. Upon Streptolysin O fractionation
(SLO) GFP is detected in the pellet in both stages, indicating that the destination
of export is the vacuole. The presence of GFP in the SLO supernatant in
schizonts indicates the presence of GFP in the erythrocyte cytosol during
schizogony.
Figure 3: Intraerythrocytic rupture of the parasitophorous vacuole membrane
during schizogony of untransfected P.falciparum.
Detection of S-Antigen by immunogold labelling with anti-S-Antigen antibodies
on ultra-thin sections of wild-type P.falciparum parasites. (A) In untreated early
schizonts S-Antigen localizes to the parasitophorous vacuole. (B) In untreated
late schizonts S-Antigen localizes throughout the erythrocyte cytosol. (C) In
leupeptin and chymostatin treated early schizonts S-Antigen localizes to the
parasitophorous vacuole. (D) In leupeptin and chymostatin treated late
schizonts, S-Antigen localizes throughout the erythrocyte cytosol.
Figure 4: Selective inhibition of the process of P.falciparum exit from the host
erythrocyte.
(A) Treatment of GFP expressing parasites with E-64 inhibits vacuolar, but not
erythrocyte membrane, lysis resulting in PVM-enclosed merozoite structures
(PEMS). 3D7-His, but not 3D7+His PEMS display GFP fluorescence (green).
Parasite nuclei are stained with DAPI (blue). The merge of the 3 channels is
shown on the right. These structures are represented schematically in B. (C)
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Treatment of GFP expressing parasites with leupeptin and antipain inhibits
erythrocyte, but not vacuolar membrane lysis. Both 3D7-His and 3D7+His
clusters of merozoites display GFP fluorescence. This localisation of GFP is
represented schematically in D. (E) Identification of limiting membrane in
protease inhibitor treated P.falciparum parasites. Indirect immunofluorescence
assay with anti-KAHRP antibodies to determine the origin of the limiting
membrane in E-64, and leupeptin and antipain treated parasites. Fixed blood
smears were reacted with anti-KAHRP antibodies (green). Parasite nuclei are
stained with DAPI (blue).
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B
A
Primary rupture of parasitophorous vacuole membrane
Merozoite ReleaseLate Schizont
Primary rupture of erythrocyte
Secondary rupture oferythrocyte plasma
Secondary rupture of parasitophorous vacuole membrane
Wickham et al. Figure 1
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Mark E. Wickham, Janetta G. Culvenor and Alan F. Cowmanerythrocyte
Selective inhibition of a two-step egress of Malaria parasites from the host
published online July 11, 2003J. Biol. Chem.
10.1074/jbc.M305252200Access the most updated version of this article at doi:
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