1

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

ecember 3, 2020

http://ww

w.jbc.org/

Dow

nloaded from

2

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.

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

3

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

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

4

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

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

5

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.

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

6

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)

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

7

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.

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

8

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

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

9

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

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

10

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

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

11

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

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

12

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

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

13

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

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

14

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

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

15

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

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

16

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

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

17

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

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

18

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.

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

19

REFERENCES

1. Delplace, P., Bhatia, A., Cagnard, M., Camus, D., and Colombet, G. (1988)Biol. Cell. 64, 215-221

2. Lyon, J. A., and Haynes, J. D. (1986) J Immunol 136, 2245-22513. Hadley, T., Aikawa, M., and Miller, L. H. (1983) Exp. Parasitol. 55, 306-3114. Banyal, H. S., Misra, G. C., Gupta, C. M., and Dutta, G. P. (1981) J.

Parasitol. 67, 623-6265. Salmon, B. L., Oksman, A., and Goldberg, D. E. (2001) Proc Natl Acad Sci

U S A 98, 271-2766. Greenbaum, D. C., Baruch, A., Grainger, M., Bozdech, Z.,

Medzihradszky, K. F., Engel, J., DeRisi, J., Holder, A. A., and Bogyo, M.(2002) Science 298, 2002-2006

7. Le Bonniec, S., Deregnaucourt, C., Redeker, V., Banerjee, R., Grellier, P.,Goldberg, D. E., and Schrevel, J. (1999) J. Biol. Chem. 274, 14218-14223.

8. Dua, M., Raphael, P., Sijwali, P. S., Rosenthal, P. J., and Hanspal, M. (2001)Mol. Biochem. Parasitol. 116, 95-99.

9. Raphael, P., Takakuwa, Y., Manno, S., Liu, S. C., Chishti, A. H., andHanspal, M. (2000) Mol. Biochem. Parasitol. 110, 259-272.

10. Shenai, B. R., Sijwali, P. S., Singh, A., and Rosenthal, P. J. (2000) J. Biol.Chem. 275, 29000-29010.

11. Hanspal, M., Dua, M., Takakuwa, Y., Chishti, A. H., and Mizuno, A.(2002) Blood 100, 1048-1054

12. Miller, S. K., Good, R. T., Drew, D. R., Delorenzi, M., Sanders, P. R.,Hodder, A. N., Speed, T. P., Cowman, A. F., de Koning-Ward, T. F., andCrabb, B. S. (2002) J Biol Chem 277, 47524-47532

13. Delplace, P., Fortier, B., Tronchin, G., Dubremetz, J. F., and Vernes, A.(1987) Mol. Biochem. Parasitol. 23, 193-201

14. Knapp, B., Hundt, E., Nau, U., and K$upper, H. A. (1989) Mol BiochemParasitol 32, 73-83

15. Li, J., H., M., Mitamuraa, T., and Horii, T. (2002) Mol. Biochem. Parasitol.120, 177-186

16. Banerjee, R., Liu, J., Beatty, W., Pelosof, L., Klemba, M., and Goldberg, D.E. (2002) Proc Natl Acad Sci U S A 99, 990-995

17. Langreth, S. G., Jensen, J. B., Reese, R. T., and Trager, W. (1978) J.Protozool. 25, 443-452

18. Aikawa, M. (1971) Exp. Parasitol. 30, 284-320.19. Waller, R. F., Reed, M. B., Cowman, A. F., and McFadden, G. I. (2000)

EMBO J. 19, 1794-180220. Reed, M. B., Saliba, K. J., Caruana, S. R., Kirk, K., and Cowman, A. F.

(2000) Nature 403, 906-90921. Wickham, M. E., Rug, M., Ralph, S. A., Klonis, N., McFadden, G. I., Tilley,

L., and Cowman, A. F. (2001) EMBO J. 20, 1-1422. Fidock, D. A., and Wellems, T. E. (1997) Proc. Natl. Acad. Sci. USA 94,

10931-1093623. Ansorge, I., Benting, J., Bhakdi, S., and Lingelbach, K. (1996) Biochem. J.

315, 307-314.24. Rug, M., Wickham, M. E., Foley, M., Cowman, A. F., and Tilley, L. (2003)

Submitted25. Coppel, R. L., Cowman, A. F., Lingelbach, K. R., Brown, G. V., Saint, R.

B., Kemp, D. J., and Anders, R. F. (1983) Nature 306, 751-756

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

20

26. Cowman, A. F., Saint, R. B., Coppel, R. L., Brown, G. V., Favaloro, J.,Crewther, P. E., Culvenor, J. G., Bianco, A. E., Stahl, H. D., Mitchell, G. F.,Kemp, D. J., and Anders, R. F. (1985) in Modern Approaches to Vaccines ofMolecular and Chemical Basis of Resistance to Viral, Bacterial and ParasiticDiseases - Vaccines 85 (Lerner, R., Chanock, R., and Brown, F., eds), pp.13-18, Cold Spring Harbor Laboratory, Cold Spring Harbor

27. Stenzel, D. J., and Kara, U. A. (1989) Eur. J. Cell. Biol. 49, 311-31828. Lauer, S., VanWye, J., Harrison, T., McManus, H., Samuel, B. U., Hiller, N.

L., Mohandas, N., and Haldar, K. (2000) EMBO J. 19, 3556-3564.29. Ward, G. E., Miller, L. H., and Dvorak, J. A. (1993) J. Cell Sci. 106, 237-24830. Delplace, P., Bhatia, A., Cagnard, M., Camus, D., Colombet, G.,

Debrabant, A., Dubremetz, J. F., Dubreuil, N., Prensier, G., Fortier, B.,and et al. (1988) Biol. Cell 64, 215-221

31. Deguercy, A., Hommel, M., and Schrevel, J. (1990) Mol. Biochem. Parasitol.38, 233-244.

32. Dhawan, S., Dua, M., Chishti, A. H., and Hanspal, M. (2003) J Biol Chem33. Nwagwu, M., Haynes, J. D., Orlandi, P. A., and Chulay, J. D. (1992) Exp.

Parasitol. 75, 399-41434. Chulay, J. D., Lyon, J. A., Haynes, J. D., Meierovics, A. I., Atkinson, C. T.,

and Aikawa, M. (1987) J. Immunol. 139, 2768-277435. Francis, S. E., Gluzman, I. Y., Oksman, A., Knickerbocker, A., Mueller, R.,

Bryant, M. L., Sherman, D. R., Russell, D. G., and Goldberg, D. E. (1994)EMBO J. 13, 306-317.

36. Moon, R. P., Tyas, L., Certa, U., Rupp, K., Bur, D., Jacquet, C., Matile, H.,Loetscher, H., Grueninger-Leitch, F., Kay, J., Dunn, B. M., Berry, C., andRidley, R. G. (1997) Eur. J. Biochem 244, 552-560.

37. Silva, A. M., Lee, A. Y., Gulnik, S. V., Maier, P., Collins, J., Bhat, T. N.,Collins, P. J., Cachau, R. E., Luker, K. E., Gluzman, I. Y., Francis, S. E.,Oksman, A., Goldberg, D. E., and Erickson, J. W. (1996) Proc. Natl. Acad.Sci. U S A 93, 10034-10039.

38. Ring, C. S., Sun, E., McKerrow, J. H., Lee, G. K., Rosenthal, P. J., Kuntz, I.D., and Cohen, F. E. (1993) Proc Natl Acad Sci U S A 90, 3583-3587.

39. Rosenthal, P. J., Wollish, W. S., Palmer, J. T., and Rasnick, D. (1991) J. Clin.Invest. 88, 1467-1472

40. Rosenthal, P. J., Olson, J. E., Lee, G. K., Palmer, J. T., Klaus, J. L., andRasnick, D. (1996) Antimicrob. Agents Chemother. 40, 1600-1603.

41. Rosenthal, P. J., Lee, G. K., and Smith, R. E. (1993) J. Clin. Invest. 91, 1052-1056

42. Dominguez, J. N., Lopez, S., Charris, J., Iarruso, L., Lobo, G., Semenov,A., Olson, J. E., and Rosenthal, P. J. (1997) J Med Chem 40, 2726-2732.

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

21

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

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

22

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)

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

23

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).

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

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

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

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:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on Decem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from