Invited review

Parasite ligand–host receptor interactions during invasion

of erythrocytes by Plasmodium merozoites

Deepak Gaur, D.C. Ghislaine Mayer, Louis H. Miller*

Laboratory of Malaria and Vector Research (LMVR), National Institutes of Allergy and Infectious Diseases (NIAID),

National Institutes of Health (NIH), 12735 Twinbrook Parkway, Building Twinbrook III/Room 3E-32D, Bethesda, MD 20892-8132, USA

Received 2 September 2004; received in revised form 11 October 2004; accepted 11 October 2004

Abstract

Malaria parasites must recognise and invade different cells during their life cycle. The efficiency with which Plasmodium falciparum

invades erythrocytes of all ages is an important virulence factor, since the ability of the parasite to reach high levels of parasitemia is often

associated with severe pathology and morbidity. The merozoite invasion of erythrocytes is a highly complex, multi-step process that is

dependent on a cascade of specific molecular interactions. Although many proteins are known to play an important role in invasion, their

functional characteristics remain unclear. Therefore, a complete understanding of the molecular interactions that are the basis of the invasion

process is absolutely crucial, not only in improving our knowledge about the basic biology of the malarial parasite, but also for the

development of intervention strategies to counter the disease. Here we review the current state of knowledge about the receptor–ligand

interactions that mediate merozoite invasion of erythrocytes.

q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Erythrocyte; Plasmodium; Cell invasion; Merozoite surface proteins; Duffy binding protein

1. Introduction

Malaria is a major human disease that accounts for

almost 2 million deaths annually. The malaria parasites have

a complex life cycle involving both a vertebrate and an

invertebrate host. Survival and transmission depends on the

ability of the invasive stages of the parasite to recognise and

invade the appropriate host cell types. The asexual

erythrocytic phase of the Plasmodium life cycle is

responsible for producing the clinical features and patho-

logy associated with malaria. To begin the erythrocytic

phase, the exoerythrocytic schizonts in the liver release

merozoites that invade erythrocytes and develop there

through the ring, trophozoite and schizont stages. Numerous

molecules that are implicated in the invasion process

have been identified in apical organelles (rhoptry, micro-

nemes) and on both the merozoite and erythrocyte surfaces

(Table 1). The functions of only a few of these molecules

0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by

doi:10.1016/j.ijpara.2004.10.010

* Corresponding author. Tel.: C1 301 435 2177; fax: C1 301 402 2201.

E-mail address: lmiller@niaid.nih.gov (L.H. Miller).

are well characterised and the molecular mechanisms

involved at each step of invasion are not well understood.

2. The steps in the invasion process

The invasion of erythrocytes by Plasmodium merozoites

is a complex, multistep process and the sequence of invasive

steps is probably similar for all Plasmodium species. The

description of invasion is based on videomicroscopy

(Dvorak et al., 1975) and ultrastructural analysis (Aikawa

et al., 1978, 1981; Miller et al., 1979; Aikawa and Miller,

1983; Bannister and Dluzewski, 1990; Bannister et al.,

2000). In the first step, the merozoite attaches reversibly to

the erythrocyte surface followed by apical reorientation,

formation of an irreversible junction, a parasitophorous

vacuole, entry into the vacuole by movement of the junction

and resealing of the vacuolar and erythrocytic membranes.

Our review focuses only on the molecular interactions that

mediate the early steps of invasion from initial attachment

to junction formation. The steps in the formation of

International Journal for Parasitology 34 (2004) 1413–1429

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Table 1

Parasite ligands and host receptors involved during invasion of erythrocytes

by Plasmodium merozoites

Parasite ligand Erythrocyte receptor

Plasmodium falciparum

EBA-175 Glycophorin A

BAEBL (Dd2/NM) Glycophorin C

BAEBL (three other variants) Receptor unknown

(not glycophorin C)

JESEBL Receptor unknown

EBL-1 Receptor unknown

PfMSP-1 Band 3

PfAMA-1 Receptor unknown

PfRH1 Receptor unknown

PfRH2b Receptor unknown

Ligand unknown Glycophorin B

Plasmodium vivax

Duffy binding protein (PvDBP) Duffy blood group antigen

(DBGA)

PvRBP1&2 Receptor unknown (Reticulocytes)

Plasmodium knowlesi

DBP a-protein Duffy blood group antigen

(human and rhesus)

b-protein, g-protein Receptor unknown, rhesus

(chymotrypsin resistant, rhesus)

Plasmodium yoelli

Py235 (RBL like, 14 copies) Receptor unknown (sialic acid-

independent, trypsin sensitive)

135 kDa protein (DBL like) Duffy blood group antigen

(DBGA)

D. Gaur et al. / International Journal for Parasitology 34 (2004) 1413–14291414

a parasitophorous vacuole, modification of the cytoskeleton

of the host cell, signaling and entry by apicomplexan

parasites are described elsewhere (Sibley et al., 1998;

Morrissette and Sibley, 2002; Opitz and Soldati, 2002;

Harrison et al., 2003; Sibley, 2004). However, these steps

are not well described for Plasmodium. The specific

molecular interactions between parasite receptors on the

merozoite and host receptors on the erythrocyte membrane

during the early steps of erythrocyte invasion are discussed

in the following sections.

2.1. Step 1: initial attachment of the merozoite

to the erythrocyte surface

In the first step of invasion, the merozoite attaches to the

erythrocyte surface. The initial attachment appears to be

host cell specific as Plasmodium knowlesi merozoites attach

only to erythrocytes from susceptible hosts (rhesus, human)

and do not attach to erythrocytes from non-susceptible hosts

like guinea pig, chicken (Miller et al., 1979). This suggests

that initial attachment of the merozoite involves specific

molecular interactions. This initial contact may occur on

any part of the merozoite surface. Videomicroscopy has

shown that the merozoite can even attach by the surface

opposite the apical pole (Dvorak et al., 1975). This process

of initial attachment is reversible as observed with the

attachment and detachment of P. knowlesi merozoites

from Duffy blood group negative human erythrocytes

(L.H. Miller, unpublished results). It is logical to believe

that molecules present on the merozoite surface mediate this

initial attachment, although proof for such parasite receptor

interactions during initial attachment is lacking.

A number of merozoite surface proteins (MSPs) have

been identified, but their role in invasion still remains to be

defined. Merozoite surface proteins (MSP-1, 2, 4, 5, 8 and 10)

link to the membrane via a glycosylphosphatidylinositol

(GPI) membrane anchor (Gerold et al., 1996; Marshall et al.,

1997, 1998; Black et al., 2001, 2003). Other MSPs such as

MSP-3, MSP-6, MSP-7 and MSP-9 are soluble and are, in

part, associated with the merozoite surface (Stahl et al., 1986;

Weber et al., 1988; McColl and Anders, 1997; Pachebat et al.,

2001; Trucco et al., 2001). The GPI-anchored MSPs in

Plasmodium falciparum, except for MSP-2, have one or two

epidermal growth factor (EGF)-like domains at the carboxy-

terminus. The possibility exists, based on analogy with other

EGF-like domains, that they may have a role in mediating

key protein–protein interactions. The EGF-like domains are

targets of protective immune responses of the host. However,

the function of the EGF-like domains on merozoites remains

unknown.

MSP-1 was the first Plasmodium MSP to be discovered

and has been the most well characterised member of the

MSP family. It appears that MSP-1 is essential for invasion

and survival of the malarial parasite as it has not been

possible to knock out the gene. As MSP-1 is evenly

distributed on the merozoite surface, it may mediate the

initial interaction with the erythrocyte during the invasion

process. It was previously suggested that full length MSP-1

binds erythrocytes in a sialic acid-dependent manner

(Perkins and Rocco, 1988). Recently, it has been reported

that MSP-142 and MSP-9 form a co-ligand complex that

binds to band 3, a membrane transport glycoprotein present

on the erythrocyte surface (Goel et al., 2003; Li et al., 2004).

Plasmodium falciparum MSP-9 is a highly conserved

antigen that includes an unusual C-terminal acidic–basic

tandem repeat region, an N-terminal hexapeptide tandem

repeat region, and four cysteine residues in the N-terminus

(Stahl et al., 1986; Weber et al., 1988; Kushwaha et al.,

2000). MSP-9 is also known as acidic–basic repeat antigen

(ABRA) and it was shown that the N-terminal cysteine-rich

region of MSP-9 interacts with band 3 (Kushwaha et al.,

2002). While these reports are interesting, more work is

required to conclusively prove the functional role of

merozoite surface proteins during invasion.

As shown clearly in studies of P. falciparum and

P. knowlesi, MSP-1 undergoes extensive proteolytic proces-

sing during late schizogony and after merozoite release

(Holder and Freeman, 1982, 1984; Freeman and Holder,

1983; David et al., 1984). The molecule is cleaved into four

proteolytic fragments that remain bound as a non-covalent

complex on the merozoite surface (McBride and Heidrich,

1987). The fragment masses as described in P. falciparum

are 83 kDa (N-terminus), 30 and 38 kDa (central regions),

and 42 kDa (C-terminus) (Holder et al., 1987). The soluble

D. Gaur et al. / International Journal for Parasitology 34 (2004) 1413–1429 1415

merozoite proteins MSP-6 and MSP-7 bind to MSP-1

(Pachebat et al., 2001; Trucco et al., 2001). In Plasmodium

yoelii, MSP-7 and two related proteins (PyMSRP-1,

PyMSRP-2) have been found to interact with the amino-

terminal region of the 83 kDa fragment of PyMSP-1 (Mello

et al., 2002). These proteins belong to a multigene family of

MSP-7 related proteins that also include six homologues

found in P. falciparum. PfMSRP-1 and PfMSRP-2 have been

demonstrated to interact directly with MSP-1 by an in vitro

binding assay (Mello et al., 2002). While MSP-1 presumably

is involved in the process of erythrocyte invasion, the role of

these interactions in the biology of the parasite remains a

mystery.

The 42 kDa C-terminal fragment of MSP-1 (MSP-142) is

attached to the plasma membrane of the merozoite by a

GPI-anchor (Gerold et al., 1996). In the final proteolytic

step, the 42 kDa fragment is cleaved into a 33 kDa soluble

fragment that is shed and a 19 kDa membrane bound

fragment that remains attached to the merozoite surface

after invasion (Blackman et al., 1991). This 19 kDa

fragment is observed around the merozoite that has

entered the parasitophorous vacuole (Blackman et al.,

1990, 1996). Similar proteolytic processing steps are also

believed to occur for the MSP-1 molecules of Plasmodium

vivax (Longacre et al., 1994) and Plasmodium cynomolgi

(Longacre, 1995). The 19 kDa carboxyl fragment of MSP-1

contains two EGF-like domains (Blackman et al., 1991).

Antibodies directed against MSP-119 block erythrocyte

invasion in vitro and immunisation with the recombinant

MSP-119 in mice and monkeys elicits protection against

parasite challenge (Daly and Long, 1993; Ling et al., 1994;

Kumar et al., 1995). Similar results have been reported with

antibodies raised against the C-terminal 42 kDa of MSP-1

that contains MSP-119 (Singh et al., 2003). Immunisation

with the Escherichia coli produced MSP-142 elicits high

titer antibodies in Aotus monkeys and leads to significant

protection against a lethal P. falciparum in vivo challenge

(Singh et al., 2003). Invasion inhibitory antibodies against

MSP-119 block the proteolytic processing of MSP-142

(Blackman et al., 1994). These results show that MSP-1

has a crucial role in erythrocyte invasion. However, its exact

function including host cell molecular interaction remains

unknown.

2.2. Step 2: apical reorientation of the merozoite

Apical reorientation is a necessary step before mero-

zoites can enter the erythrocyte. During the process of initial

attachment, the apical pole of the merozoite is usually not in

direct apposition to the erythrocyte membrane. Thus, the

merozoite must undergo apical reorientation. The molecular

interactions that mediate this step in invasion are not well

understood. However, a recent study has presented data that

Apical Membrane Antigen 1 (AMA-1) may be directly

responsible for apical reorientation (Mitchell et al., 2004). It

was reported that in the presence of an invasion inhibitory

rat monoclonal antibody (MAb R31C2), P. knowlesi

merozoites attached normally to the erythrocyte surface,

but did not undergo apical reorientation (Mitchell et al.,

2004). This study would have been more convincing if the

parasites had been treated with cytochalasin because

P. knowlesi does not remain bound to Duffy-negative

erythrocytes except in the presence of cytochalasin. In the

presence of cytochalasin, P. knowlesi merozoites reorient

apically to Duffy-negative erythrocytes and remain bound,

despite the absence of a junction. Presumably a later step in

invasion such as the moving junction causes release of

merozoites from the erythrocyte in the absence of a

junction.

AMA-1 is a Type I integral membrane protein (Hodder

et al., 1996). The C-terminal cytoplasmic domain is highly

conserved in Plasmodium species suggesting that it is

important for a cytoplasmic interaction (e.g. signaling or

moving junction). The ectodomain of AMA-1 contains 16

cysteine residues, which are conserved within all Plasmo-

dium species (Hodder et al., 1996). The bonding pattern of

these cysteine residues suggests that there are three

disulfide-constrained regions forming the extracellular

part of the AMA-1 protein (Hodder et al., 1996). The

3-dimensional structure of AMA-1 has not yet been solved.

AMA-1 is expressed in the late schizont stage of the asexual

life cycle of the parasite (Deans et al., 1984). The

P. falciparum AMA-1 (PfAMA-1) molecule is synthesised

as an 83 kDa precursor, from which an N-terminal

prodomain is cleaved (Narum and Thomas, 1994; Howell

et al., 2001). In other Plasmodium species AMA-1 is

synthesised as a 66 kDa protein. Although, it was initially

reported that AMA-1 is localised in the neck of rhoptries in

mature merozoites (Narum and Thomas, 1994), it has been

recently shown that the mature 66 kDa form of PfAMA-1 is

present in the micronemes (Healer et al., 2002; Bannister

et al., 2003). Like MSP-1, AMA-1 is also proteolytically

processed into smaller fragments (Howell et al., 2001).

AMA-1 is processed to a 44–48 kDa molecule during the

translocation of the molecule to the merozoite surface

(Howell et al., 2001, 2003; Dutta et al., 2003).

The role of AMA-1 in invasion of erythrocytes is

supported by several studies. Antibodies against AMA-1

inhibit in vitro erythrocyte invasion by P. knowlesi and

P. falciparum (Deans et al., 1982; Triglia et al., 2000).

PfAMA-1 derived peptides inhibit merozoite invasion of

erythrocytes by P. falciparum (Urquiza et al., 2000). Like

MSP-1, it has not been possible to knockout the PfAMA-1

gene (Triglia et al., 2000). However, the function of

PfAMA-1 could be complemented with an AMA-1

transgene (PcAMA-1) from Plasmodium chabaudi, a rodent

parasite (Triglia et al., 2000). Interestingly, the functional

expression of PcAMA-1 in P. falciparum increased the

parasite’s ability to invade mouse erythrocytes by 60%

compared with the wild type parasite (Triglia et al., 2000).

The Toxplasma gondii homologue of Plasmodium AMA-1

has also been shown to be involved in host cell invasion

D. Gaur et al. / International Journal for Parasitology 34 (2004) 1413–14291416

(Hehl et al., 2000). These results indicate an important role

for AMA-1 in the invasion of erythrocytes across divergent

Plasmodium species and suggest a host specific role in

binding to erythrocytes.

Different domains of the P. yoelii AMA-1 protein have

been expressed on the surface of transfected COS-7 cells

and have been shown to bind mouse erythrocytes (Fraser

et al., 2001). The domains of P. yoelii AMA-1 bind only

mouse and rat erythrocytes; but not to human erythrocytes.

Out of the three domains of AMA-1, the domains 1/2

expressed as a contiguous product had the highest

erythrocyte binding activity. When the two domains were

expressed separately, domains 1 and 2 mediated only

34–47% binding activity compared to that of contiguous

domains 1/2. Similarly, domains 2/3 only had a 33%

binding relative to domains 1/2. The full-length ectodomain

consisting of all three domains (1/2/3) showed no

erythrocyte binding (Fraser et al., 2001). In another study,

it was observed that P. falciparum AMA-1 binds poorly to

human erythrocytes but surprisingly binds to rodent

erythrocytes (J.H. Adams, personal communication). It

may be possible that the receptor for PfAMA-1 is not highly

exposed on human erythrocytes, but is exposed on the

surface of mouse erythrocytes. These studies showed that

multiple domains of AMA-1 could bind erythrocytes. The

physiological significance of these studies remains to be

determined.

2.3. Step 3: events around junction formation

Once the apical prominence of the merozoite is apposed

directly towards the erythrocyte membrane, a characteristic

junction is formed between the invading merozoite and the

erythrocyte membrane. The junction is characterised by an

increased electron-dense thickening under the erythrocyte

membrane at the site of contact between the merozoite

and erythrocyte (Aikawa et al., 1978; Miller et al., 1979).

A slight indentation of the erythrocyte membrane is

observed in which the apical pole of the merozoite becomes

positioned. While the initial contacts between the merozoite

and erythrocyte are reversible, the development of the

junction is an irreversible step that commits the parasite to

invasion. Once the merozoite–erythrocyte junction is

initiated, the next phase begins with the movement of the

junction around the penetrating merozoite. This involves a

number of molecular events that allow the merozoite to gain

physical entry into the erythrocyte within the parasitophor-

ous vacuole. The parasite ligand and erythrocyte receptor

molecules that function after apical reorientation, and lead

to junction formation, are better characterised. Initial studies

on P. knowlesi/P. vivax had identified the Duffy blood group

antigen as a receptor for erythrocyte invasion and showed its

functional role in junction formation. These findings laid the

basis for identification of erythrocyte binding parasite

proteins that mediate erythrocyte invasion.

3. Duffy blood group antigen as receptor in erythrocyte

invasion

The human malaria parasite, P. vivax and the related

simian parasite P. knowlesi invade human erythrocytes

using the Duffy blood group antigen as the receptor (Miller

et al., 1975, 1976; Barnwell et al., 1989). The Duffy blood

group antigen is a chemokine receptor (Chaudhuri et al.,

1993; Horuk et al., 1993) that binds a family of chemokines

including IL-8 and MGSA, melanoma growth stimulatory

activity (Horuk et al., 1993; Hesselgesser et al., 1995). The

evidence that junction formation precedes invasion is

derived from the data on the interaction between Duffy

blood group-negative human erythrocytes and P. knowlesi

merozoites (Miller et al., 1975, 1979). Duffy-negative

human erythrocytes that lack the Duffy blood group antigen

are refractory to invasion by P. knowlesi (Miller et al., 1975;

Mason et al., 1977). When P. knowlesi merozoites interact

with Duffy-negative human erythrocytes, initial attachment

and apical reorientation occur normally; however, the

junction is not formed (Miller et al., 1979). The use of

cytochalasin B allowed the isolation of the attachment phase

from the erythrocyte entry phase of merozoite invasion, as

cytochalasin B blocks movement of the junction, a later step

in invasion (Miller et al., 1979). Cytochalasin B-treated

P. knowlesi merozoites attach and form a junction with the

erythrocyte, but the invasion process does not advance

further and there is no entry of the merozoite into the

erythrocyte. Cytochalasin B-treated P. knowlesi merozoites

attached equally well to both Duffy-positive and Duffy-

negative erythrocytes (Miller et al., 1979). However,

ultrastructural studies have shown that no junction was

observed with the Duffy-negative erythrocytes. Instead the

merozoite remained bound by filaments extending from the

edge of the apical end of the merozoite to the erythrocyte

(Miller et al., 1979). While this data clearly shows that the

Duffy blood group antigen has a functional role in junction

formation in P. knowlesi, it still does not prove that the

Duffy blood group antigen directly forms the junction. It is

possible that interactions with the Duffy receptor help in

positioning other molecules that form the junction.

Similarly, the evidence that P. vivax also uses the

Duffy blood group antigen as a receptor during erythro-

cyte invasion comes from studies which showed that

Duffy blood group-negative individuals were resistant to

P. vivax infection (Miller et al., 1976, 1978; Spencer

et al., 1978). The Duffy-negative phenotype is rare among

the White and Asian populations, whereas it is has a high

prevalence among the black population especially those

originating from West Africa (Sanger et al., 1955). The

incidence of P. vivax infection in the West African

population is very low (Boyd and Stratman-Thomas,

1933; Young et al., 1955). In invasion assays using

P. vivax parasites obtained from squirrel monkeys, it was

demonstrated in vitro that P. vivax does not invade

Duffy-negative erythrocytes (Barnwell et al., 1989).

D. Gaur et al. / International Journal for Parasitology 34 (2004) 1413–1429 1417

Hence, invasion of human erythrocytes by P. vivax/

P. knowlesi is completely dependent on the presence of

the Duffy blood group antigen on the surface of the

erythrocyte and its interaction with the parasite Duffy-

binding protein. Unlike P. knowlesi, there is no data that

demonstrates the role of the Duffy blood group antigen in

junction formation in P. vivax. Based on analogy with

P. knowlesi, it is assumed that in P. vivax the Duffy blood

group antigen is playing a role in junction formation.

However, erythrocyte receptors for P. knowlesi may

not be completely restricted to the Duffy blood group

antigen as the Duffy-negative human erythrocytes were

invaded after treatment with trypsin or neuraminidase

(Mason et al., 1977). Similarly, the cytochalasin B-treated

merozoites of P. knowlesi could form a junction with

trypsin treated Duffy-negative erythrocytes (Miller et al.,

1979). The enzymatic treatment of Duffy-negative human

erythrocytes probably decreases the steric hindrance or

charge interference in the interaction between merozoites

and the erythrocyte and thus, allows invasion through

another receptor. Another interesting observation is that

while P. vivax invades Saimiri monkey (squirrel monkey)

erythrocytes, the P. vivax Duffy-binding protein does not

bind Saimiri monkey erythrocytes (Wertheimer and

Barnwell, 1989). This suggests that the parasite is using

an invasion pathway independent of the Duffy blood

group antigen for junction formation and invasion of the

Saimiri erythrocytes.

Unlike P. vivax that invades only Duffy blood group-

positive reticulocytes, P. falciparum exhibits redundancy

in erythrocyte invasion and invades all human erythro-

cytes. This restriction in erythrocyte invasion by P. vivax

led to the discovery of two families of erythrocyte binding

parasite proteins that mediate the invasion process. The

first family is known as the DBL Superfamily and consists

of the P. vivax Duffy-binding protein (PvDBP) that binds

to the Duffy blood group antigen and its homologous

Duffy-binding-like (DBL) proteins of P. falciparum,

P. knowlesi and P. yoelii. The DBL Superfamily is

named after the region of PvDBP that binds the Duffy

blood group antigen on the erythrocyte surface. It consists

of the erythrocyte-binding-like (EBL) proteins and the Var

family of variant surface antigens. The variant surface

antigens also contain multiple cysteine rich DBL domains.

The second family consists of the P. vivax reticulocyte

binding protein that specifically targets the reticulocytes

for invasion and its homologous reticulocyte-binding-like

(RBL) proteins of P. falciparum, P. knowlesi and

P. yoelii. The classification of Plasmodium proteins as

DBL and RBL refers to a family of homologous parasite

proteins and does not relate to a similar erythrocyte

binding specificity of these proteins. The different DBL

and RBL proteins may recognise erythrocyte receptors

other than the Duffy blood group antigen or the

reticulocyte receptor molecule.

4. DBL family

The P. vivax/P. knowlesi Duffy-Binding Protein (DBP)

that binds to the Duffy blood group antigen on the

erythrocyte surface is characterised by the presence of a

cysteine-rich region near the N-terminus, a low complexity

intermediate region, followed by another cysteine-rich

region, a transmembrane and a short cytoplasmic tail.

Although P. falciparum invades Duffy-negative erythro-

cytes and is not dependent on the Duffy blood group antigen

for erythrocyte invasion, a family of erythrocyte binding

proteins have been identified in P. falciparum, consisting of

EBA-175, BAEBL/EBA-140, JESEBL/EBA-181 and

EBL-1, that share a similar cysteine rich binding domain

as P. vivax/P. knowlesi DBP and belong to the Duffy-

binding-like (DBL) Superfamily (Adams et al., 2001). Like

the P. vivax/P. knowlesi DBP, these proteins have two

cysteine-rich domains, an N-terminal DBL domain and

C-terminal domain adjacent to a transmembrane domain. In

contrast to the N-terminal cysteine-rich region of the

P. vivax and P. knowlesi DBP, the N-terminal cysteine-

rich region of the P. falciparum proteins is duplicated to

form an F1 and F2 region (Adams et al., 1992).

4.1. Plasmodium vivax and Plasmodium knowlesi DBLs

Two Duffy-binding proteins of molecular masses 140

and 135 kDa have been identified in P. vivax and

P. knowlesi, respectively (Haynes et al., 1988; Wertheimer

and Barnwell, 1989; Adams et al., 1990). While, the

P. vivax Duffy-binding protein (DBP) specifically binds the

human Duffy blood group antigen, the P. knowlesi DBP

binds both the human and rhesus Duffy blood group antigen

(Chitnis and Miller, 1994). The erythrocyte-binding domain

has been shown to lie within a conserved N-terminal

cysteine rich region, termed as region II (Chitnis and Miller,

1994; Chitnis et al., 1996). Region II of P. vivax DBP

contains 330 amino acids and the critical binding residues

have been recently mapped to a 170-residue stretch between

amino acids 291 and 460 (Ranjan and Chitnis, 1999). It has

been reported that while the positions of the cysteine

residues are conserved, other amino acids are highly

polymorphic (Xainli et al., 2000). Almost all polymorph-

isms (93%) were found in the 170 amino acid critical

binding region (Xainli et al., 2000). These polymorphisms

did not alter the erythrocyte-binding function of PvDBP.

Since this region of the parasite molecule is a critical point

of contact between the parasite and the host, and would

elicit an antibody response, it was suggested that the high

polymorphism in this region of the molecule arose to enable

the parasite to evade the host immune response (Xainli

et al., 2000).

The 135 kDa P. knowlesi DBP is encoded by a a-gene in

the genome of P. knowlesi and is also denoted as the aprotein (Chitnis and Miller, 1994). Plasmodium knowlesi

also has two other highly homologous genes—b and g that

D. Gaur et al. / International Journal for Parasitology 34 (2004) 1413–14291418

encode the b and g proteins, respectively. Region II of the band g proteins have different binding specificities compared

to the a protein. Region II of the b and g proteins bound

rhesus erythrocytes but did not bind either Duffy-positive or

-negative human erythrocytes (Chitnis and Miller, 1994). In

addition, region II of the b and g proteins bound

chymotrypsin treated rhesus erythrocytes, which have lost

the Duffy blood group antigen (Chitnis and Miller, 1994).

Plasmodium knowlesi is known to invade untreated and

chymotrypsin treated rhesus erythrocytes at the same rate,

indicating that P. knowlesi has alternate pathways for

invasion of rhesus erythrocytes that do not depend on the

Duffy blood group antigen. The P. knowlesi b and g proteins

appear to mediate this Duffy antigen independent pathway

of invasion of rhesus erythrocytes (Chitnis and Miller,

1994).

4.2. Plasmodium falciparum DBLs

The homologous proteins of PvDBP in P. falciparum have

not been directly shown to play a role in junction formation.

However, by analogy with the P. vivax/P. knowlesi DBP, it is

speculated that these proteins are involved in junction

formation as the characteristic erythrocyte binding DBL

domains are conserved in these proteins.

4.2.1. EBA-175

A 175 kDa erythrocyte binding antigen, EBA-175, was

the first erythrocyte binding protein to be identified in

P. falciparum (Camus and Hadley, 1985). It is localised in

the micronemes. In an erythrocyte binding assay using

P. falciparum culture supernatants, EBA-175 was observed

to bind normal erythrocytes but not neuraminidase treated

erythrocytes suggesting that binding of EBA-175 was sialic

acid-dependent (Camus and Hadley, 1985). EBA175 was

shown to specifically bind glycophorin A, a sialoglycopro-

tein on the erythrocyte surface (Sim et al., 1994). Although,

glycophorin B contains the identical 11 O-linked oligosac-

charides found on glycophorin A, EBA-175 does not bind to

glycophorin B (Sim et al., 1994). En(a-) erythrocytes that

lack glycophorin A are not bound by EBA-175 despite the

presence of glycophorin B on these erythrocytes (Sim et al.,

1994). It was shown that the binding specificity of EBA-175

is not solely determined by the presence of sialic acid

residues but the amino acid sequence of glycophorin A also

contributes to this specificity (Sim et al., 1994).

Antibodies against EBA-175 blocked erythrocyte inva-

sion in vitro by about 90% (Pandey et al., 2002). These data

further substantiate that EBA-175 plays a role in erythrocyte

invasion by P. falciparum. Targeted disruption of EBA-175

in the parasite clone W2mef, whose invasion is sialic acid-

dependent, was found to be associated with a switch to a

sialic acid-independent pathway of invasion (Reed et al.,

2000; Duraisingh et al., 2003a). This switch was observed

specifically in the P. falciparum clone W2mef (Dd2) only.

EBA-175 was also disrupted in a cloned line, Dd2/Nm, Dd2

selected on neuraminidase treated erythrocytes (Kaneko

et al., 2000). The transfected parasites showed the same

ability to invade neuraminidase treated erythrocytes as the

wild type Dd2/Nm parasites (Kaneko et al., 2000). These

results suggest that P. falciparum can invade erythrocytes

without EBA-175 except in one parasite clone where

disruption of EBA-175 produced a switch in the parasite’s

invasion phenotype.

The cytoplasmic domain of type I transmembrane

proteins in the Apicomplexan parasite, T. gondii, contain

targeting signals that are responsible for sorting the proteins

to the correct subcellular location (Joiner and Roos, 2002).

The cytoplasmic domain of EBA-175 has been shown not to

be essential for localisation of EBA 175 to the micronemes

(Gilberger et al., 2003a). Interestingly, it was shown that the

cytoplasmic domain of TRAP, a sporozoite stage protein

that is essential for invasion of liver cells, could substitute

for the cytoplasmic domain of EBA-175. The transfected

parasites expressing the EBA-175TRAP chimeric protein

invaded erythrocytes at the same efficiency as wild type

parasites. The functional homology of the EBA-175

cytoplasmic tail with that of TRAP suggests that at different

stages of the parasite life cycle similar protein–protein

interactions link these proteins to the invasion molecular

machinery (Gilberger et al., 2003a).

4.2.2. Paralogues of EBA-175

The P. falciparum genome project has enabled the

identifications of four paralogues of EBA-175 (Adams et al.,

2001). They are named BAEBL (EBA-140), JESEBL

(EBA-181), EBL-1 and PEBL (EBA-165). BAEBL is

expressed in late schizogony and has an apparent molecular

mass of 140 kDa. JESEBL is a 181 kDa protein, whose gene

is present on chromosome 1 (Adams et al., 2001; Gilberger

et al., 2003b; Mayer et al., 2004). Like EBA-175, both

BAEBL and JESEBL have been localised to the micro-

nemes. PEBL or EBA-165 is transcribed but does not appear

to be expressed at the protein level due to frameshift

mutations in the coding region (Triglia et al., 2001). Thus,

PEBL is a pseudogene that may not play a role in merozoite

invasion of human erythrocytes. Despite the lack of

significant nucleotide identity among the five ebl genes

the amino acid sequence identity in the DBL domain is

w20–36%, with the highest homology being between

BAEBL and EBA-175. The function of the carboxyl

cysteine-rich domain is unknown despite it being more

conserved than the DBL domain. Each paralogue appears as

a single copy gene. The characteristics of these proteins are

summarised in Table 2. Recent studies have focused on the

characterisation of BAEBL and JESEBL.

4.2.3. Polymorphism in the erythrocyte-binding domain

of BAEBL/EBA-140

A combination of studies using enzyme-treated erythro-

cytes as well as mutant erythrocytes deficient in different

surface molecules have identified the erythrocyte receptor

Table 2

Characteristics of Plasmodium falciparum erythrocyte binding proteins

EBL Chromo-

some

Mass Location Function Effect of gene

disruption/deletion

References

1. EBA-175a 7 175 kDa Micronemes Erythrocyte binding: binds to

glycophorin A

Switch to sialic acid-

independent invasion

pathway; reduced

invasion of chymo-

trypsin treated

erythrocytes

Camus and Hadley (1985), Sim

et al. (1994), Kaneko et al. (2000),

Reed et al. (2000), Duraisingh

et al. (2003a) and Gilberger et al.

(2003a)

2. BAEBLa

(EBA-140)

13 140 kDa Micronemes Erythrocyte binding; polymorh-

ism affects binding specificity;

binds to glycophorin C and other

receptors depending on mutation

in BAEBL

No effect Adams et al. (2001), Mayer et al.

(2001), Thompson et al. (2001),

Maier et al. (2002), Mayer et al.

(2003) and Lobo et al. (2003)

3. JESEBLa

(EBA-181)

1 181 kDa Micronemes Erythrocyte binding; binds to

trypsinised erythrocytes; poly-

morhism affects binding speci-

ficity

No effect Adams et al. (2001), Gilberger

et al. (2003b) and Mayer et al.

(2004)

4. EBL-1 13 304 kDa Not reported Not reported Not reported Peterson et al. (1995), Peterson

and Wellens (2000) and Adams

et al. (2001)

5. PEBL 4 Not

translated

N/A Pseudogene No effect Triglia et al. (2001)

a Maximum expression of these proteins is in late schizont stage.

D. Gaur et al. / International Journal for Parasitology 34 (2004) 1413–1429 1419

of BAEBL from the P. falciparum clone Dd2/Nm as

glycophorin C/D (Mayer et al., 2001, 2002; Lobo et al.,

2003; Maier et al., 2003). The binding domain for BAEBL

on glycophorin C is located between residues 14 and 22

(Lobo et al., 2003). Another study with the P. falciparum

strain E12 has reported that binding of BAEBL to

erythrocytes is sialic acid-dependent and resistant to trypsin,

proteinase K and pronase (Thompson et al., 2001). The

protease resistant properties of the erythrocyte receptor

suggest that it is neither glycophorin A, B, C or D.

The disparity in the erythrocyte-binding phenotype of

BAEBL from different P. falciparum clones was investi-

gated. Region II was sequenced from 25 P. falciparum

clones (Mayer et al., 2002). Five BAEBL variants resulting

from single point mutations at four positions in the first DBL

domain were observed. These mutations occurred at

positions 185, 239, 261 and 285 of the F1 region. These

polymorphisms were found to lead to a variation in the

erythrocyte binding specificity of BAEBL. The residues

involved and the binding specificities of the variants are

reported elsewhere (Mayer et al., 2002). The difference in

erythrocyte-binding phenotype of the two BAEBL variants

can be attributed to the amino acid changes in the region II

of BAEBL. This study has revealed that single amino acid

substitutions can change the erythrocyte molecule recog-

nised by BAEBL and suggests that a single P. falciparum

ligand can function in multiple invasion pathways. A study

using field isolates from Africa has revealed that in addition

to the point mutation in the F1 region of BAEBL, an

additional amino acid substitution was detected in the F2

domain (Baum et al., 2003b). The low-frequency poly-

morphisms in BAEBL is in sharp contrast to the large

number of polymorphisms found in region II of EBA-175,

suggesting a strong immune selective pressure on the latter

but not on the former.

Comparison of the merozoite invasion for the

P. falciparum strain E12 (that expresses BAEBL) and

another P. falciparum strain D10 (that lacks BAEBL) did

not identify a pathway with the functional properties

expected of the EBA-140 molecule (Thompson et al.,

2001). BAEBL has been knocked out in the Pf clones 3D7,

W2mef and the invasive ability of the transfected parasites

were similar to the wild type parasites (Maier et al., 2003).

4.2.4. Polymorphism in the erythrocyte-binding

domain of JESEBL (EBA-181)

The characterisation of JESEBL demonstrated its

erythrocyte-binding specificity to be dependent on a trypsin

resistant, chymotrypsin and neuraminidase sensitive mol-

ecule different from glycophorin B (Gilberger et al., 2003b;

Mayer et al., 2004). Furthermore, targeted disruption of the

eba-181 gene had no effect on the invasion phenotype of the

parasite (Gilberger et al., 2003b). Recently, the erythrocyte-

binding domain of JESEBL from 20 clones from various

parts of the world was sequenced. Eight polymorphisms, all

leading to changes in amino acids, were identified at

positions 359, 363, 414, 443, and 637 (Mayer et al., 2004).

No synonymous mutations were observed. Three base

substitutions in region II of JESEBL/EBA-181 occurred in

the F1 domain, two in the F2 domain, whereas the

polymorphisms in BAEBL are mostly restricted to the

amino-terminal segment of the F1 region (Mayer et al.,

2004). Four erythrocyte-binding patterns were observed

indicating at least four different erythrocyte receptors

D. Gaur et al. / International Journal for Parasitology 34 (2004) 1413–14291420

(Mayer et al., 2004). The positions of the amino acid

variations and the erythrocyte-binding specificities of each

variant have been described elsewhere (Mayer et al., 2004).

There are fewer mutations in region II of BAEBL and

JESEBL as compared to EBA-175 (Mayer et al., 2002,

2004). In contrast to BAEBL and JESEBL, multiple non-

synonymous mutations were identified throughout region II

of the Duffy-binding protein of P. vivax (Xainli et al., 2000)

and in EBA-175 of P. falciparum (Liang and Sim, 1997).

Unlike BAEBL and JESEBL, the mutations in EBA-175 are

scattered throughout the DBL domain and do not change its

requirement for sialic acid on erythrocytes (Liang and Sim,

1997). In contrast, each point mutation in BAEBL and

JESEBL led to the recognition of different receptors on the

erythrocyte. The variants of BAEBL and JESEBL seem to

have arisen by single point mutations that can be linked. It is

not known why all the mutations in regions II of BAEBL

and JESEBL affect their receptor specificities. We can

speculate that JESEBL and BAEBL are not critical in

erythrocyte invasion or that they are less exposed to the

immune system. It is also possible that polymorphisms in

the erythrocyte-binding domain of BAEBL and JESEBL/

EBA-181 provide the parasite population with a survival

advantage in a genetically diverse human population.

4.2.5. EBL-1

EBL-1 was identified as a second member of the DBL-

EBP family on the basis of consensus sequence homology

(Peterson et al., 1995). It is expressed during the late

schizont stage and its function has not been fully

characterised (Blair et al., 2002). In two genetic crosses,

inheritance of EBL-1 was linked to the rapid proliferation

phenotype of the progeny suggesting a role in erythrocyte

invasion efficiency (Wellems et al., 1987; Peterson and

Wellems, 2000). Thus, like other P. falciparum EBLs,

EBL-1 is also probably involved in erythrocyte receptor

recognition playing either a synergistic or an alternative role

in the invasion process.

4.3. Plasmodium yoelii DBL

The murine homologue of the human Duffy blood group

antigen was shown to be a receptor for P. yoelii invasion of

mature erythrocytes (Swardson-Olver et al., 2002). This was

demonstrated as the P. yoelii non-lethal 17X strain invaded

normal mouse erythrocytes almost eight-fold more than

erythrocytes from Duffy blood group knock out (DKO)

mice. On the other hand, P. yoelii invaded the DKO

reticulocytes almost equally to normal reticulocytes, thus

showing that P. yoelii has an alternate Duffy-independent

pathway for reticulocyte invasion (Swardson-Olver et al.,

2002). In contrast to P. vivax that is totally dependent on the

Duffy blood group antigen for erythrocyte invasion,

P. yoelii requires the Duffy blood group antigen only for

invasion of mature erythrocytes. P. yoelii seems to have a

separate Duffy-independent pathway for invasion of

reticulocytes. This is the first report that demonstrates the

same Plasmodium species uses different pathways for

invasion of mature erythrocytes and reticulocytes.

The parasite ligand for the murine Duffy blood group

antigen has not been described. A P. yoelii protein of

135 kDa has been identified in parasite culture supernatants

and was observed to bind murine erythrocytes (Ogun et al.,

2000). Similar to the binding of the P. vivax/P. knowlesi

DBP, the binding of this P. yoelii 135 kDa protein was

abolished by chymotrypsin treatment, while neuraminidase

and trypsin had no significant effect (Ogun et al., 2000). It

thus appears that the 135 kDa P. yoelii protein may be the

P. yoelii homologue of the P. vivax/P. knowlesi DBP.

However, more studies are required to prove this and also to

demonstrate that the 135 kDa P. yoelii protein is the parasite

ligand that binds the murine Duffy blood group antigen.

The Duffy blood group antigen is considered to play a

role during the junction formation step in erythrocyte

invasion. However, the invasion of Duffy-knockout reticu-

locytes suggests that P. yoelii is able to form a junction in

the absence of the Duffy blood group antigen. Thus, a

molecule other than DBP is involved in the formation of the

junction formation of P. yoelli merozoites with mouse

reticulocytes. As only one DBL gene is found in the P. yoelii

genome, a gene other than the DBL gene family is involved

in junction formation. Other erythrocyte binding proteins

are known to exist in P. yoelii. One is the Py235 protein and

another is MAEBL, a chimeric protein that shares homology

with AMA-1 in the amino-terminal region and with the

Duffy-binding protein in the carboxy-terminus (Kappe

et al., 1998). The invasion of Duffy-knockout reticulocytes

by P. yoelii was sensitive to separate chymotrypsin and

trypsin treatments of the erythrocytes. The erythrocyte

binding of one Py235 protein (Ogun et al., 2000) and

MAEBL (Swardson-Olver et al., 2002) was also found to be

sensitive to chymotrypsin and trypsin. These proteins may

be involved in junction formation during reticulocyte

invasion, as P. yoelii invasion of reticulocytes is also

sensitive to both enzymes.

5. Reticulocyte binding like (RBL) protein family

Plasmodium vivax is known to invade only reticulocytes

and not mature erythrocytes. Since reticulocytes comprise a

minority (w1%) of the total erythrocyte population,

P. vivax merozoites would interact with numerous mature

erythrocytes in circulation before encountering a reticulo-

cyte. Junction formation irreversibly commits the parasite to

invade the erythrocyte and involves the Duffy blood group

antigen expressed on all circulating erythrocytes. Barnwell

and coworkers suggested that a premature interaction

between the parasite’s Duffy-Binding Protein and the

Duffy blood group antigen on a mature erythrocyte

prior to encountering a reticulocyte could be detrimental

to P. vivax survival (Galinski et al., 1992; Barnwell

D. Gaur et al. / International Journal for Parasitology 34 (2004) 1413–1429 1421

and Galinski, 1998). Since junction formation always leads

to invasion, then why should P. vivax not invade mature

erythrocytes as the Duffy blood group antigen, the

erythrocyte receptor for junction formation, is found on

all erythrocytes? It was suggested that a host cell selection

step occurs prior to merozoite–erythrocyte junction for-

mation and prevents the parasite from invading a non-target

cell (Galinski et al., 1992; Barnwell and Galinski, 1998).

Two high molecular mass proteins in P. vivax, PvRBP-1 and

PvRBP-2 were identified that bound only to reticulocytes,

indicating that these proteins accounted for the selective

invasion of reticulocytes (Galinski et al., 1992). If the above

supposition is correct in that the reticulocyte preference

must come before junction formation, these proteins must

trigger junction formation. It is also possible that these

parasite receptors are involved in apical reorientation that

precedes junction formation and invasion. PvRBP-1 and

PvRBP-2 are expressed at the apical pole of the merozoite

(Galinski et al., 1992). Both PvRBP-1 (325 kDa) and

PvRBP-2 (330 kDa) have transmembrane domains at the

C-termini with short cytoplasmic domains. PvRBP-1 is a

dimer covalently associated by one or more disulfide bonds,

and together PvRBP-1 and PvRBP-2 form a protein

complex through non-covalent interactions. In erythrocyte

binding assays these two proteins have been shown to bind

specifically to human erythrocytes enriched for reticulo-

cytes. Their adhesion to the reticulocytes is independent of

the Duffy phenotype of the erythrocytes. However, the

receptor molecule present on the reticulocyte to which

PvRBP-1 and 2 bind is yet unknown (Galinski et al., 1992).

Homologues of the P. vivax reticulocyte binding proteins

have been found in P. falciparum and P. yoelii. Although

these Plasmodium species do not exhibit a strong preference

for invading only reticulocytes, the homologous proteins

Table 3

Characteristics of Plasmodium falciparum reticulocyte binding-like homologue (

PfRh Chromosome Mass Location Function

1. PfRh1

(PfNBP1)

4 350 kDa Apical pole

(IFA)

Erythrocyt

binds to try

ted but not

nidase trea

erythrocyte

2. PfRh2aa

(PfNBP2a;

PfRBP2-Ha)

13 370 kDa Rhoptries

(EM)

Not determ

3. PfRh2ba

(PfNBP2b;

PfRBP2-Hb)

13 370 kDa Rhoptries

(EM)

Mediates i

through a t

neuraminid

ant, chymo

sensitive p

4. PfRh3

(PfNBP3)

12 Not translated

(frameshift

mutation)

N/A Pseudogen

5. PfRh4

(PfNBP4)

4 220 kDa Micronemes

(IFA)

Not determ

a Contiguous, highly homologous genes (duplicated); N/A, not applicable; N, n

may be playing a role in apical interaction and signaling the

formation of the junction. It is speculated that the

P. falciparum RBL homologues (PfRh) may be involved

in recruitment of high affinity receptors like EBA-175 by

signaling release of the micronemal proteins. This has also

been suggested for the role of the Py235 protein family in

P. yoelii invasion of erythrocytes. Thus, in addition to the

DBL Superfamily, a second family of high molecular

weight erythrocyte binding proteins designated as the RBL

(reticulocyte binding-like) family has been identified in

P. vivax, with homologues present in P. falciparum and

P. yoelii. The characteristics of the PfRH proteins are

summarised in Table 3.

PfRh1, a P. falciparum orthologue of PvRBP-1, has been

identified (Rayner et al., 2001) on chromosome 4 and

located at the apical pole of the merozoite. PfRh1 has been

shown to be involved in erythrocyte binding and thus is

implicated to play a role in erythrocyte invasion. PfRh1

binds to erythrocytes in a trypsin resistant, sialic acid-

dependent manner and thus, cannot bind to either

Glycophorin A, C, D or Receptor X (Rayner et al., 2001).

Although glycophorin B is trypsin resistant, it is not

the receptor for PfRh1 as PfRh1 binds glycophorin B

deficient erythrocytes. Hence, PfRh1 is the parasite ligand

that binds an unknown, trypsin resistant receptor, termed

Receptor Y (Rayner et al., 2001).

Two P. falciparum orthologues of PvRBP-2, P. falci-

parum reticulocyte homologues 2a and 2b (PfRh2a and 2b)

have been identified (Rayner et al., 2000). The

P. falciparum genes are contiguous to each other on

chromosome 13 and have a highly homologous sequence

that suggests that they probably result from a duplication

event, as they share more than 8 KB of nucleotide sequence

(Rayner et al., 2000). However, the two genes have

PfRh) proteins

Effect of gene disruption/

deletion

References

e binding;

psin trea-

neurami-

ted

s

Not reported Rayner et al. (2001) and

Taylor et al. (2002)

ined No effect Rayner et al. (2000, 2001),

Taylor et al. (2002) and

Duraisingh et al. (2003b)

nvasion

rypsin/

ase resist-

trypsin

athway

Lower invasion of NCT

treated erythrocytes com-

pared to wild type; higher

invasion of TCCT and CT

treated erythrocytes

Rayner et al. (2000, 2001),

Taylor et al. (2002) and

Duraisingh et al. (2003b)

e No effect Taylor et al. (2001, 2002)

ined Not reported Kaneko et al. (2000)

euraminidase; T, trypsin; CT, chymotrypsin.

D. Gaur et al. / International Journal for Parasitology 34 (2004) 1413–14291422

divergent C-termini. Both the PfRH2a/2b proteins are large

hydrophilic proteins that are predicted to be more than

350 kDa and possess an N-terminal signal sequence and a

single transmembrane domain near their C-termini (Rayner

et al., 2000). They are located at the apical end of the

merozoite and have been further shown by immunoelectron

microscopy to be located in the neck of the rhoptries of

merozoites (Taylor et al., 2002; Duraisingh et al., 2003b). It

has been proposed that PfRH2a/2b play an important

adhesion function in erythrocyte invasion (Rayner et al.,

2000; Duraisingh et al., 2003b). PfRH2a/2b were knocked

out by targeted gene disruption in the clone 3D7

(Duraisingh et al., 2003b). The transfected parasites lacking

PfRH2b showed a significantly altered invasion phenotype

with enzymatically treated erythrocytes compared to the

wild type clone, whereas the knockout parasite lacking

PfRH2a showed no apparent difference to the invasion

phenotype of the wild type clone, 3D7 (Duraisingh et al.,

2003b). The PfRh2b knockout parasite invaded erythro-

cytes, sequentially treated with two enzymes, neuramini-

dase and trypsin, at a lower efficiency compared to wild type

parasites. The PfRh2b knockout parasites invaded chymo-

trypsin treated erythrocytes better than the wild type

parasites. A similar result was observed with erythrocytes

treated sequentially with trypsin and chymotrypsin. On this

basis, the PfRH2b ligand has been suggested to mediate

invasion through a chymotrypsin sensitive, trypsin/

neuraminidase resistant receptor, which is referred to as

Receptor Z (Duraisingh et al., 2003b). Erythrocyte binding

activity of PfRh2a/2b has not been demonstrated.

Two more RBP homologues have been identified in

P. falciparum. One, PfRh4 is present on chromosome 4 and

encodes a 220 kDa type I transmembrane protein, similar to

the known members of this multigene family (Kaneko et al.,

2002). PfRh4 is more similar to PvRBP-1 than PvRBP-2.

Immunoflourescence (IFA) studies have shown that it is

located in the micronemes (Kaneko et al., 2002). However,

proof of its location in micronemes must await ultrastruc-

tural studies. The structural similarity with PvRBP-1 and

apical localisation implicate PfRh4 as a potential erythro-

cyte binding protein. Like PfRh2a/2b, erythrocyte-binding

activity of PfRh4 has also not been demonstrated. The other,

PfRh3 is a pseudogene that is transcribed but not translated

due to frameshifts at the 5 0 end of the gene (Taylor et al.,

2001).

In the rodent malaria parasite P. yoelii, a group of high

molecular mass rhoptry proteins of 235 kDa have been

identified (Holder and Freeman, 1981; Oka et al., 1984).

The genes that encode Py235 are present as a multigene

family (Keen et al., 1990; Holder et al., 1994). They are

large with a size greater than 8 KB (Keen et al., 1994;

Sinha et al., 1996) and have up to 14 copies (Carlton

et al., 2002). As mentioned above, Py235 homologues

have been conserved over a wide range of Plasmodium

species and this indicates an important role for this protein

in parasite survival. The importance of Py235 protein has

been further substantiated by the recent finding that

distinct subsets of Py235 genes are expressed in

sporozoites and infected hepatocytes and that antibodies

to Py235 inhibited sporozoite invasion of hepatocytes

(Preiser et al., 2002). As a result Py235 appear to be

important in the recognition and invasion of mosquito

salivary glands and the liver.

Py235 is involved in a mechanism of gene expression by

which the merozoites originating from a single schizont

express distinct members of this multigene family (Preiser

et al., 1999). This type of clonal phenotypic variation

provides the parasite with a survival strategy in the

mammalian host and contributes to the observed chronicity

of malarial infections (Preiser et al., 1999). This phenom-

enon is genetically and functionally distinct from the

classical antigenic variation, which is mediated by the var

multigene family of P. falciparum.

It was reported that among the group of Py235 proteins,

at least two proteins were released in a soluble form in

culture supernatants of P. yoelii and only one of these two

proteins was observed to bind mouse erythrocytes (Ogun

and Holder, 1996). Chymotrypsin and trypsin treatment of

the mouse erythrocytes reduced its binding by about

40–50%, whereas neuraminidase treatment had no signifi-

cant effect (Ogun et al., 2000). Thus, it appears that a sialic

acid-independent, trypsin and chymotrypsin sensitive

receptor is involved in binding of this single Py235 protein

to mouse erythrocytes. However, it is not the only

determinant by which Py235 attaches to erythrocytes,

which is clearly demonstrated as the binding of the protein

is only partially affected by treatment with these enzymes.

The functional role of the other Py235 proteins remains to

be determined. The presence of Py235 proteins as a

multigene family and the variation within these proteins

could result in a different erythrocyte binding phenotype or

function for each of these proteins.

6. Redundancy in invasion of Plasmodium falciparum

Numerous studies have indicated that malarial mero-

zoites, especially from P. falciparum and P. knowlesi, have

the ability to invade erythrocytes through several invasion

pathways. Evidence for alternative invasion pathways was

provided by P. falciparum strains that invaded glycophorin

A-deficient erythrocytes (Miller et al., 1977; Pasvol et al.,

1982a) and sialic acid-deficient erythrocytes (Mitchell et al.,

1986). These studies using different enzymatically treated

target erythrocytes showed that P. falciparum is not totally

dependent on sialic acids or glycophorin A for invasion

(Mitchell et al., 1986; Hadley et al., 1987; Dolan et al.,

1994; Gaur et al., 2003). The alternative invasion pathways

are classified on the basis of the nature of erythrocyte

receptor involved in invasion, which in turn is defined by the

enzymatic treatments and erythrocytes null for specific

surface proteins.

Table 4

Invasion pathways of Plasmodium falciparum merozoites

Pathway Parasite ligands Erythrocyte recep-

tors

1. Sialic acid-dependent,

trypsin-sensitive

EBA-175 Glycophorin A

BAEBL (Dd2/Nm) Glycophorin C

JESEBL (PNG5) Receptor unknown

2. Sialic acid-dependent,

trypsin-resistant

Ligand unknown Glycophorin B

JESEBL

(Dd2/Nm)

Receptor unknown

PfRh1 Receptor unknown

(Y)a

3. Sialic acid-dependent;

trypsin, proteinase

K andpronase-resistant

BAEBL (E12) Receptor

unknowns

4. Sialic acid-independent,

trypsin-sensitive

Ligand unknown Receptor unknown

(X)a

BAEBL (PNG4) Receptor unknown

JESEBL (HB3) Receptor unknown

5. Sialic acid-independent,

trypsin-resistant

BAEBL

(M. Camp)

Receptor unknown

JESEBL (3D7) Receptor unknown

PfRh2b Receptor unknown

(Z)a

a The unknown receptors are referred in literature by the letters in

parenthesis.

D. Gaur et al. / International Journal for Parasitology 34 (2004) 1413–1429 1423

Most P. falciparum clones, with the exception of 7G8,

invade trypsin treated erythrocytes and thus, are able to

invade using the trypsin-resistant pathway (Dolan et al.,

1994; Gaur et al., 2003). The invasion of trypsinised

erythrocytes has been attributed to the presence of

glycophorin B (GPB), a trypsin-resistant surface molecule.

The parasite ligand that binds to glycophorin B has not been

identified. Not all P. falciparum clones are dependent on

glycophorin B for invasion through the trypsin resistant

pathway (Gaur et al., 2003). The Indochina I strain invaded

trypsinised glycophorin B-deficient (S-s-U-) erythrocytes at

an invasion rate similar to trypsinised normal erythrocytes

(Gaur et al., 2003). This indicated that this strain could

invade through a glycophorin B-independent, trypsin

resistant pathway (Gaur et al., 2003).

Invasion studies with MkMk erythrocytes that lack both

glycophorins A and B showed that P. falciparum parasites

can invade erythrocytes through pathways independent of

both glycophorins A and B (Hadley et al., 1987). Many

P. falciparum clones with the exception of Camp, Dd2 and

FCR3 invade neuraminidase treated erythrocytes and are

known to be sialic acid-independent (Mitchell et al., 1986;

Hadley et al., 1987; David et al., 1984). These three clones

are thus completely dependent on sialic acid residues for

invasion. On the other hand, a number of P. falciparum

strains can invade erythrocytes using a sialic acid-

independent, trypsin-sensitive receptor. The identity of

this receptor has not been elucidated and, it is thus, referred

to as Receptor X (Dolan et al., 1994). The parasite ligand

that binds to Receptor X also remains unknown.

In addition, P. falciparum parasites have the ability to

switch their invasion from sialic acid-dependent to sialic

acid-independent pathways (Dolan et al., 1990). Such a

phenomenon was observed when the P. falciparum

neuraminidase sensitive strain Dd2 was cultured for several

cycles in neuraminidase-treated erythrocytes and the

parasite lines recovered, denoted as Dd2/Nm, were capable

of invading erythrocytes lacking sialic acid residues (Dolan

et al., 1990). The genetic basis of the switch remains

unknown. A similar switch in invasion phenotype of W2mef

(Dd2) was observed when EBA-175 was disrupted (Reed

et al., 2000; Duraisingh et al., 2003a).

Alternative invasion pathways seem to be commonly

used by P. falciparum field isolates from India and Africa,

although the parasites obtained from Gambia, West Africa

seem to have a higher dependence on sialic acid residues

(Okoyeh et al., 1999; Baum et al., 2003a). The invasion

pathways of P. falciparum, so far identified depend on

glycophorins A, B, C/D, Receptors X, Y and Z as

erythrocyte receptors. These invasion pathways have been

summarised in Table 4. Of these receptors, the parasite

ligands that bind to glycophorin B and receptor X are not

known. There are a large number of parasite molecules—

JESEBL, EBL-1, AMA-1, PfRH1 and PfRH2b that play a

role in merozoite invasion and whose corresponding

erythrocyte receptor remains unknown.

Redundancy in invasion could afford the parasite

several advantages. The first advantage is for immune

evasion. Many EBPs have immunodominant epitopes that

elicit an immune response, and thus, having multiple

invasion pathways involving several molecules helps in

maintaining the invasion process even when an immune

response is built against some invasion molecules.

Secondly, redundancy may contribute to the ability of P.

falciparum to invade erythrocytes of all ages. Thirdly, it

may provide P. falciparum the ability to invade a wide

variety of human erythrocytes such that no human

erythrocyte is known to be totally refractory to invasion

by P. falciparum.

7. Erythrocyte receptor polymorphism

The glycophorins represent examples of erythrocyte

receptor polymorphism that appears to influence suscepti-

bility to malaria. As mentioned previously different

glycophorin molecules (A, B, C/D) play important roles

as erythrocyte receptors in the invasion process. However,

none of the erythrocytes containing different polymorphic

forms of the glycophorins emulate the Duffy system. All

glycophorin polymorphisms decrease invasion partially,

but do not block it completely. For example, invasion into

the En(a-) erythrocytes, which lack glycophorin A, is

significantly reduced but not abolished (Miller et al., 1977;

Pasvol et al., 1982b). En(a-) is a rare blood type. On the

other hand, higher frequencies of erythrocytes exhibiting

the S-s-U- phenotype, characterised by the absence of

D. Gaur et al. / International Journal for Parasitology 34 (2004) 1413–14291424

glycophorin B, have been found in Africa. The S-s-U-

phenotype is highly prevalent among the pygmies in

African countries (Vos et al., 1971; Lowe and Moores,

1972) and like the En(a-) cells, the S-s-U- erythrocytes are

also partially resistant to malaria invasion (Pasvol et al.,

1982a; Facer, 1983). Another variation observed in the

glycophorin system is the Dantu phenotype (Contreras

et al., 1984). Dantu is an antigen found in low frequencies

in Southern African populations. It is formed as a result of

misalignment of the tandem genes for glycophorins A and

B during meiosis. The protein product of the chimeric gene

is a hybrid glycophorin consisting of the N-terminus of

glycophorin B and the C-terminus of glycophorin A

(Contreras et al., 1984). Like the S-s-U- cells, the invasion

of P. falciparum into Dantu erythrocytes is severely

compromised (Field et al., 1994). The existence of variant

glycophorin variants that appear to be of African origin has

led to the speculation that these may have been selected as

a result of relative resistance that they confer to

P. falciparum malaria. Likewise, deficiency in glycophorin

C also reduces invasion by P. falciparum (Pasvol et al.,

1984). The same gene with use of alternative start codons

encodes both glycophorin C and glycophorin D. There are

three mutations of the glycophorin C/D gene that lack high-

incidence antigens (Colin, 1995). Erythrocytes that are null

for these proteins are termed as Leach. Gerbich and Yus

erythrocytes contain exon 3 and exon 2 deletions,

respectively. Each deletion leads to a truncated glycophorin

C and missing glycophorin D (Reid and Spring, 1994;

Colin, 1995). The Gerbich phenotype is found at high allele

frequencies in some regions of Papua New Guinea (Booth

et al., 1970; Serjeantson et al., 1994). It was reported that

Melanesians in Papua New Guinea (PNG) with a Gerbich-

negative phenotype had a lower combined parasitemia rate

of P. falciparum and P. vivax infection compared to those

with the Gerbich-positive phenotype (Serjeantson, 1989).

These results suggested that Gerbich-negativity might

confer some selective advantage in malaria endemic

areas. The direct correlation between Gerbich-negativity

and malaria resistance may be obscured by the presence of

other genetic polymorphisms in the study population that

also have a protective effect against malaria. As such

hereditary ovalocytosis due to a deletion in the gene of

band 3 (Jarolim et al., 1991) is widespread in PNG and

provides protection against severe malaria (Serjeantson

et al., 1977; Allen et al., 1999). A reduced invasion of such

ovalocytes by P. falciparum has been demonstrated in vitro

(Kidson et al., 1981). Gerbich erythrocytes are also known

to have decreased membrane stability (Reid et al., 1987)

with an increased deformability (Kuczmarski et al., 1987).

While a significant association of Gerbich-negativity with

increased ovalocytosis was found in a malaria holoendemic

region in PNG, it was not associated with differences in

malaria infection (Patel et al., 2001). A recent study has

reported that the frequency of genetic polymorphisms of

band 3 and glycophorin C differed in two ethnically

and geographically distinct malaria endemic regions

of PNG (Patel et al., 2004). The mutations were

independently distributed in the two study populations

and neither mutation was found to influence asymptomatic

P. falciparum or P. vivax infection. These results suggested

that while Gerbich-negativity has no effect on blood stage

infection, it may produce a difference at the level of severe

malaria morbidity as has been reported for band 3

ovalocytosis (Allen et al., 1999). Future case controls

studies are required to determine the contribution of the

Gerbich-negative phenotype in susceptibility to severe

malaria morbidity.

8. Challenges for the future

The steps of erythrocyte invasion by Plasmodium

merozoites have been defined by videomicroscopy and

ultrastructural analysis. However, these studies cannot

define the biochemical events that allow invasion to

proceed. Some of the questions about erythrocyte invasion

that are unanswered and remain to be explored are as

follows: (i) The protease that prepares the P. chabaudi

erythrocytes for invasion (Breton et al., 1992) and the step

that it involves remain undefined. Is there a similar protease

in P. falciparum that modifies the erythrocyte for invasion?

(ii) Why are the merozoite surface proteins GPI anchored

whereas the apical organellar proteins are transmemebrane

proteins with short cytoplasmic domains? (iii) What is

function of the merozoite surface proteins? (iv) The

function of AMA-1 found in micronemes of Plasmodium

merozoites and T. gondii tachyzoites remains a mystery;

(v) Why are the DBL and RBL families found in different

organelles? (vi) Is signaling involved in release of

organellar proteins or is secretion constitutive? (vii) What

is the molecular basis of junction formation? How does

P. knowlesi form a junction and invade trypsin- and

neuraminidase-treated human Duffy-negative erythrocytes;

(viii) P. yoelii invades reticulocytes through a Duffy-

independent pathway whereas it requires the Duffy blood

group antigen for invasion of mature erythrocytes, what is

the receptor for invasion? (ix) Why did point mutations in

the P. vivax DBL gene not lead to invasion by Duffy-

independent pathways of invasion as the Duffy blood group

antigen was disappearing from West Africa? (x) The

molecular basis for the switch of P. falciparum Dd2 from a

sialic acid-dependent to a sialic acid-independent pathway

is unknown (Dolan et al., 1990); (xi) The basis for the

inability of P. falciparum 7G8 to invade trypsinised

erythrocytes remains to be defined; (xii) The parasite

molecule that binds to glycophorin B remains unidentified

and the basis of the P. falciparum strain Indochina I to

invade trypsinised glycophorin B-negative erythrocytes at

the same rate as into glycophorin B-positive erythrocytes

remains unknown (Gaur et al., 2003).

D. Gaur et al. / International Journal for Parasitology 34 (2004) 1413–1429 1425

These puzzles and many more await investigators who

develop novel ways to study the molecular basis of invasion.

As our toolbox expands, these questions will be answered

and new ones will arise that will require more tools.

Eventually we must understand the molecular basis of

invasion in enough detail to identify the Achilles’ heel of the

parasite for intervention.

Acknowledgements

We wish to thank Dr John Adams for sharing his

unpublished data.

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