Erythrocyte invasion by the malarial merozoite: Recent advances

EXPERIMENTAL

PARASITOLOGY

69, 94-99 (1989)

MINIREVIEW Erythrocyte

Invasion

by the Malarial

of Biochemical b3UCINS,

Experimental

Parasitology,

The Rockefeller

University,

New York, New York l&l?I,

U.S.A.

M. E. 1989. Erythrocyte invasion by the malarial merozoite: Recent advances. parasitology

ERYTHROCYTE

69, 94-99.

RECEPTORS

The malarial merozoite has the unique ability to distinguish between erythrocytes of its host and nonhost erythrocytes. The basis of this selectivity is the recognition by the parasite of specific receptors on the erythrocyte surface. Only for three species of Plasmodium, P. falciparum, P. vivax, and P. knowlesi, have the receptors been defined in any detail. For P. falciparum the receptor has been defined as the family of sialic acid-rich glycoproteins, the glycophorins; the terminal sialic acid residues being critical for this interaction. For P. vivax and P. knowlesi, the major receptor is the Duffy glycoprotein. The properties of the receptors are listed in Table 1. Experimental evidence for the presence of receptors is similar for each species; the respective parasite does not invade erythrocytes which lack the receptor, lost either by genetic mutation or enzymatic cleavage (Miller et al. 1975, 1976; Perkins 1981; Pasvol et al. 1982). Second, antibodies to specific domains of the receptor block invasion (Facer 1983; Barnwell et al. 1989; Holt et al. 1988). Third, in the case of P. falciparum, it has been shown that specific tryptic peptides of glycophorin A block invasion and glycophorin A binds to isolated merozoites (Perkins 1984). However, for each species of Plasmodium there is evidence for invasion independent of the major receptors. For P. falciparum, some isolates are fully dependent on sialic acid for invasion, while others invade, with lower 94 001~4894/89 $3.00 0 1989 by Academic Press, Inc. Au rights of reproduction in any form reserved.

cOp~ri&I

Recent Advances

E. PERKINS

MARGARET Laboratory

Merotoite:

efficiency, cells lacking sialic acid (Perkins and Holt 1988) or glycophorin A and B (Hadley et al. 1988). Since sialic acid independent invasion is much less efficient, we think of the alternate receptor as being of secondary importance. Furthermore, P. fafciparum can invade, also with low efflciency, mouse erythrocytes. Mouse erythrocytes contain a glycophorin-like molecule but the sialic acid residues are greatly modified from that in human erythrocytes. Similarly, P. knowlesi, which normally invade Duffy-positive rhesus erythrocytes will, however, invade chymotrypsin-treated rhesus erythrocytes, which lack Duffy glycoprotein, indicating that they can interact with a secondary receptor. There is also preliminary evidence that P. vivax can invade via a pathway independent of the Duffy glycoprotein. The molecular basis of strain variability of receptor binding is not understood and its significance will be discussed below. The receptor system for rodent malarias, P. berghei, P. yoelli, and P. chabaudii, remains largely unexplored, although its investigation should certainly yield important information on the evolution of species specificity of invasion. PARASITE RECEPTOR-BINDING PROTEINS (RBP)

Host cell recognition by many pathogens, including viruses and bacteria, often involves specific binding to receptors on the host cell surface. Host receptor molecules recently identified include the CD4

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TABLE I Properties of Erythrocyte Receptors for P. falciparum, P. Knowlesi, and P. Vivax P. falciparum

P. knowlesi

P. vivax

Major receptor

Glycophorin A, B, C (GPA, GPB, GPC)

Duffy glycoprotein (FYI

Duffy Glycoprotein

MW of receptor

90 kDa (GPA), 40 kDa (GE), 20 kDa (GPW

35-45 kDa

3545 kDa

Evidence for secondary receptor

Siahc acid independent invasion in some strains Human, aotus, saimiri, mouse

Chymotrypsin insensitive site on rhesus erythrocytes Human. rhesus.

Not characterized

Human, aotus, saimiri

Human (Fya, b), rhesus, saimiri aotus

Erythrocytes invaded (natural host underlined) Erythrocytes with correct receptor

antigen for HIV virus, the CR2 antigen for EpsteisBarr virus, and the I-CAM protein for rhinovirus. In many cases a virus molecule which binds directly to the host receptor has been identified, with the implication that this protein is responsible for host cell recognition. Host receptor recognition in the merozoite appears to be similar. Parasite proteins that bind to the correct host erythrocyte in a receptor dependent manner have been defined for P. falciparum, P. vivax, and P. knowlesi. Recent data on the proteins, which collectively can be referred to as RBP, are summarized in Table II. In P. falciparum, a sialic acid dependent binding protein has been identified as Pf200, also known as

-’Human. saimiri. aotus

saimiri~

Human, rhesus, saimiri, aotus

gp195, the major merozoite surface antigen (Perkins and Rocco 1988). A 175~kDaprotein that binds to sialic acid has also been defined and is currently being characterized. (Camus and Hadley 1985).Pl200 of all isolates of P. falciparum tested bind to erythrocytes in a sialic dependent manner, regardless of whether the strain is fully or partially dependent on sialic acid and glycophorin for invasion (Perkins and Rocco 1988).Thus in isolates of P. falciparum that invade sialic acid-deficient erythrocytes, a protein other than Pl200 must be responsible for binding to the erythrocyte surface. Such a protein has not been detected in the assay system used which depends on the addition of soluble proteins to intact eryth-

TABLE II Properties of Parasite Proteins Binding to Erythrocyte Receptors: P. falciparum, P. knowlesi, and P. vivax P.

falciparum

P. knowlesi

Receptorbinding protein:

Pram (gp 195)

175kDa

135kDa

Speciticity of binding Erythrocyte binding Location of receptor-binding protein AntibOdy inhibition

Siatic acid

Siabc acid

Duffy glycoprotein

Human, saimiri

Human, aotus nd

Human,aoms, saimiri, rhesus nd

Merozoite suhce glycoproteia Anti-glycophorin

nd

Anti-DuRy

P.

155kDa nd Aotus, rhesus, saimiri nd nd

vivax

135kDa Duftj~glycoprotein Human, aotus Apical Anti-Duffy

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rocytes. The sialic acid-independent erythrocyte binding protein may not be a soluble protein. Pf200 only binds to erythrocytes invaded by P. falciparum, although all erythrocytes contain glycophorin-like molecules. The difference between cells susceptable to P. falciparum and those resistant to invasion may be that the former contain unmodified sialic acid, whereas resistant erythrocytes, such as rhesus cell, contain acetylated sialic acid. However, P. falciparum does invade mouse erythrocytes (Klotz et al. 1987), which have as their major sialic acid species 9-O-acetylated neuraminic acid, but this invasion appears to be independent of Pf200. A most interesting result is that P. falciparum rhoptry proteins of 140,130, and 110kDa bind to mouse erythrocytes (Perkins and Rocco 1988)and that the properties of binding correlate strongly with the properties of P. falciparum invasion into these cells (T. Y. Sam-Yellowe and M. E. Perkins, manuscript submitted). Furthermore, only those strains of P. fafciparum that invade human erythrocytes independently of sialic acid (7G8 isolate) invade mouse erythrocytes efficiently and this correlates with an increased rate of binding of the rhoptry proteins in this strain as compared to a noninvading strain (FCR-3). For P. vivax it has been demonstrated that the Duffy receptor-binding protein is a polypeptide of 135 kDa (Wertheimer and Bamwell, manuscript submitted). Antibodies against this protein locate it to the apical end of the merozoite. For P. knowlesi, two Duffy-binding proteins of 155 and 135 kDa have been identified (Haynes et al. 1988; Miller et al. 1988). The polyclonal antibody raised against the P. knowlesi 135-kDa protein immunoprecipitates its counterpart from P. vivax, indicating that the two Duffy-binding proteins from both species are immunologically as well as functionally related (Wertheimer and Bamwell, manuscript submitted). However, there are important differences, as the 135-kDa P. vivax

protein does not bind to rhesus erythrocytes, whereas that of P. knowlesi does. This is consistent with the differential invasion of rhesus cells by these two species (Table I). In addition to the 135 and 155kDa RBP, a 204-kDa protein of P. vivax and a 230-kDa protein of P. knowlesi selectively bind to erythrocytes, although the binding in each case is not dependent on the presence of the Duffy glycoprotein. ROLEOFRHOPTRYPROTEINS

Merozoite invasion into erythrocytes is usually considered to be a multistep process which involves several sequential events: receptor binding, tight junction formation, erythrocyte membrane invagination, vacuole formation, and closure. However, we do not know in biochemical terms how any of these signals are transmitted. Subsequent to erythrocyte binding, the merozoite assumes an orientation such that its apical end is aligned with the erythrocyte surface. At the apical end of the parasite are the rhoptry organelles, a pair of electron dense structures known to contain several proteins. The rhoptry organelle almost certainly plays a critical role in the steps of invasion following receptor binding, including reorientation and invagination of the erythrocyte membrane to form a vacuole. Erythrocyte attachment and junction formation appear, from electron microscopy studies, to occur independently of rhoptry movement and secretion. In P. falciparum, monoclonal antibodies that react with the rhoptry organelle have defined a number of protein components. The rhoptry proteins appear to be organized in complexes; there is a high MW complex consisting of proteins of 140, 130, and 110 kDa and a low MW complex consisting of an 80and a 41-kDa protein. An additional rhoptry protein of 225 kDa has also been identified. Monoclonal antibodies against most of these proteins reduce or inhibit merozoite invasion significantly, suggesting that the rhoptry proteins are accessible on the ex-

MINIREVIEW:RECENTADVANCES

ternal surface of the merozoite prior to erythrocyte membrane invagination. It has been demonstrated by immunofluorescence that the 1IO-kDa rhoptry protein is secreted into the host erythrocyte membrane during invasion (Sam-Yellowe et al. 1988). What triggers the secretion of rhoptry contents is not known. Using a biochemical assay, it has recently been found that the complex of 140,130, and 1IO&Da rhoptry proteins bind to human erythrocyte membranes pretreated with glutaraldehyde, which presumably exposesan interior site, not exposed in intact erythrocytes. Treatment of erythrocytes with trypsin, chymotrypsin, and soluble glycophorin does not affect the binding of the rhoptry proteins, indicating they are binding to a site unrelated to both the external domain of band 3 and glycophorin. Now that the major rhoptry proteins have been identified, interest is focused on the function of these components in merozoite invasion. ROLEOFPARASITE ENZYMES IN INVASION

Knowledge of the protein structure of the erythrocyte membrane and the underlying cytoskeleton implies that a radical reorganization of these components must occur during vacuole formation and closure. One possible mechanism of reorganization is that the parasite secretesan enzyme or protease which initiates localized enzymatic cleavage, resulting in the disassociation of the cytoskeleton components from the membrane. Indeed several parasite enzymes have been identified, some of which, because of their location, represent possible candidates for a role in merozoite invasion. A neutral endopeptidase of 70 kDa has been identified in P. berghei and antibodies to the protein locate it to the apical end of merozoites (Bernard and Schrevel 1987).The enzyme appears to have a close analog in P. falciparum and is inhibited by leupeptin which also inhibits invasion. In addition, an 83-kDa protein which is pro-

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cessedto a 76-kDa protein is activated as a serine protease following cleavage with phospholipase C (Braun-Breton et al. 1988).

The target of the apical enzymes could also be components of the merozoite surface coat, or the rhoptries. The formation of the tight junction between the erythrocyte and the invading merozoite appears to involve an accumulation of the merozoite surface coat at the zone of contact. As the parasite enters the erythrocyte, its surface coat is removed from the anterior end and is eventually released at the posterior end. Removal of the surface coat must involve selective enzymatic cleavage of the major surface components (e.g., Pf 200). Cleavage could be initiated by a protease or phospholipase located at the apical end of the merozoite. It has recently been shown that merozoite invasion is accompanied by a marked dephosphorylation of erythrocyte membrane proteins, spectrin, band 3, band 4.1, and ankyrin, suggesting that the parasite activates a phosphatase on entry or perhaps secretes a phosphatase into the erythrocyte membrane during invasion (Murray and Perkins 1989). An alternate consideration is that the parasite proteins secreted into the membrane are hydrophobic and render the membrane more fluid or more deformable. A detailed knowledge of the structure of the rhoptry proteins will be useful in distinguishing their possible roles. It has been suggested by Hadley et al. (1986) and others that the ability of the merozoite to invade erythrocytes is related to the host membrane deformability or rigidity. They have proposed that antibodies to erythrocyte receptors block invasion by rendering the membrane less deformable rather than the specific blocking of the receptors per se (Hadley et al. 1986).Results from a recent comprehensive study by Gratzer and colleagues (Rangachari et al. 1989) argue strongly against this hypothesis. In a series of elegant studies in which the erythrocyte membrane was

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rendered less deformable by a number of treatments, no correlation with reduction in invasion was found, except at very high concentrations of reagents. Erythrocytes treated with N-ethyl maleimide, chlorpromazine, and lectins, which, by various chemical and physical mechanisms, resulted in significant increases in membrane rigidity, did not reduce invasion significantly. Furthermore, several different types of abnormal cells, including elliptocytes, which are less deformable, supported invasion normally. Thus it would appear that a less deformable erythrocyte membrane does not affect merozoite invasion directly. CONCLUSION

The more we examine the biochemical events that underly the merozoite invasion process, the more complex and variable it appears to be. It has been known for many years that different species of plasmodia invade selectively different erythrocytes and interact with different host receptors. In the case of P. falciparum, the receptor-binding specificity also varies between strains. In addition, it would appear that a binding site on the 140, 130, and IlO-kDa rhoptry complex varies between strains. At this point we can only speculate as to the significance of the variation in receptor binding and rhoptry protein binding. One explanation is that variants of the antigenic proteins involved in invasion have been selected in response to immune pressure, and that new or modified proteins have been able to interact with the erythrocyte membrane. An alternate hypothesis is that the P. falciparum species is composed of different evolutionary lineages. Available molecular analysis suggests that P. falciparum is most closely related to the rodent malaria species. It is possible that some strains of P. fafciparum diverged from rodent strains more than others and largely lost the ability to interact with mouse erythrocytes. Other strains of P. falciparum, which can still invade mouse erythrocytes (and sialic acid

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INVASION

deficient human erythrocytes), may have diverged less and retained some of the capabilities of interacting with the ancestral host cell. ACKNOWLEDGMENT I thank Dr. John Bamwell and Sam Wertheimer for communication of results prior to publication and for critical reading of the manuscript. REFERENCES J. W., NICHOLS, M. E., AND RUBINSTEIN, P. 1989. In vitro evaluation of the role of the Duffy blood group in erythrocyte invasion by Plasmodium vivax. The Journal of Experimental Medicine 169, 1795-1802. BERNARD, F., AND SCHREVEL, J. 1987. Purification of a Plasmodium berghei neutral endopeptidase and its localization in merozoite. Molecular and Biochemical Parasitology 26, 167-174. BARNWELL,

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SAM-YELLOWE, T. Y., SHIO, H., AND PERKINS, M. E. 1988. Secretion of Plasmodium falciparum rhoptry protein into the plasma membrane of host erythrocytes. The Journal of Cell Biology 106, 15071513. Received 24 April 1989; accepted 8 May 1989