- Email: [email protected]
Antigenic variation in Plasmodium falciparum: mechanisms and consequences
420
Antigenic variation and consequences Chris I Newbold In the past variation switching expression variant Malaria
year,
have
the major been
advances
concerned
in malaria
with
of variant antigen genes, of regions of the major gene families have Genome Project.
in PIasmodium
been
antigenic
the transcription
and
and the functional variant antigen. Also,
discovered
as a result
new of the
Addresses Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 QDS, UK; e-mail: [email protected] Current
Opinion
in Microbiology
1999,
2:420-425
http://biomednet.com/elecref/1369527400200420 0 Elsevier Abbreviations CIDR CSA DBL AEMP-1 prbc rbc VSA
Science
Ltd ISSN
cysteine-rich Chondroitin Duffy-binding Plasmodium
1369-5274
interdomain sulphate A like falciparum
region
erythrocyte
membrane
protein-l
parasitised red blood cell red blood cell variant surface antigen
Introduction During growth and multiplication in their mammalian hosts, malaria parasites spend the majority of their time within erythrocytes. Here they differentiate from the newly invaded ring form through a trophozoite to a schizont containing the replicated daughter merozoites. These will emerge from the infected cell and invade new red cells, thus continuing the cycle. The erythrocyte does not differentiate, has no internal protein synthesis or trafficking machinery, and does not express class I or class II major histocompatibility complex (MHC) molecules on its surface. In other words, it would seem to be an ideal home for an organism that needs to be present within its host for extended periods in order to be transmitted to its obligate insect vector, the mosquito, to survive. Why then have mechanisms evolved whereby proteins synthesised by the parasite are able to cross the parasite plasma membrane and the parasitophorus vacuole membrane, and then be inserted into the red cell plasma membrane where they are visible to the host immune system? The rationale for the evolutionary drive to export proteins to the infected red blood cell (rbc) surface must be that they perform a vital function. In the case of the human malaria Ptasmodium falciparm, the parasite that is responsible for nearly all malaria-specific deaths, the answer would appear to be clear. In this species, parasites only circulate in the blood for the first half of the erythrocytic growth cycle. At approximately 18 hours post-invasion, variant surface antigen (VSA) appears on the rbc and mediates adhesion to a variety of ligands on host endothelium [ 1,2]. This prevents the
falciparum:
mechanisms
parasitised rbc (prbc) passing through the spleen where they might be destroyed by non-specific clearance mechanisms [3]. Two consequences arise from this: first, the VSA is forced to vary to avoid host immune recognition, and second, the variability of the adhesion specificity associated with the antigenic variation of the VSA family means that certain VSAs have undesirable properties. Therefore, different VSAs can bind to different endothelial receptors [4]. In some cases (perhaps when ICAMis the receptor involved) adhesion occurs preferentially in the brain potentially leading to the life threatening syndrome of cerebral malaria [5-81. In other cases, VSAs can bind to uninfected red cells to form erythrocyte rosettes; a phenotype that has also been associated with severe disease [9-l 11. VSAs, whatever their primary function, are important virulence factors, and an understanding of both antigenic variation and adhesion at the molecular level is, therefore, important for both malaria immunity and pathogenesis. Such proteins were first studied serologically by Brown and colleagues [ 12,131 in the primate malaria E knowL~~z’in which VSAs were shown to undergo antigenic variation as a means of immune evasion. Since then phenotypic homologues of these antigens have been found in rodent, other primate and human malaria species, and also shown to undergo true clonal antigenic variation [4,14-181. In this review some of the recent developments in the function, genomic organisation and control of expression of VSAs in malaria are discussed.
Features
of antigenic
variation
in malaria
In vitro studies with clones of P fahpamm indicate that the rate of antigenic switching away from a given variant is as high as 2% per generation [4]. Frequencies of a similar magnitude have been observed in viva in the rodent malaria J? chabaudz’ [ 191. In both cases there appears to be a range of individual variant switch frequencies with lo-z/generation representing the upper limit. No data currently exist about switching rates in humans in viva but mechanisms must exist whereby expression is tightly controlled at both the organism and population level; for example, expression of all variants at the same time would not be an efficient evasion strategy. A further common feature of VSA expression in several parasite species is that it can be reversibly modulated in viva. Passage of cloned organisms through splenectomised animals results in the appearance of parasite populations that no longer express VSA on the rbc surface [3,1.5,17,20,2 11. Re-introduction of these parasites into intact animals results in the reappearance of VSA at the rbc surface in a manner by which VSA gradually reappears in the whole population. This is, therefore, not
Antigenic
Figure
variation
in Plasmodium
falciparum
Newbold
421
1
Schematic representation of the var gene and encoded protein. The grey shaded boxes represent DBLXIDR domains. The boxes with horizontal lines represent inter-DBL domains; the hatched box represents the transmembrane domain; and the diagonal striped box represents the cytoplasmic tail.
5’ exon
~
-8.14kb
p.
Current
a selective event but signifies active signalling between host and parasite. l%o data currently exist on how this signailing might occur.
antigens
of /? falciparum
Where studied, the variant antigens of different malaria species can be described as a family of polymorphic high molecular weight, trypsin sensitive, surface exposed and ‘liiton X-100 insoluble membrane proteins [Z&23]. Molecules with these characteristics in I? fakipclmm were collectively termed t’lmmodium fuhpnnm erythrocyte membrane protein-l (PfEMP-1). ‘l’he CUE genes encoding these proteins have a characteristic structure (Figure 1) consisting of two exons with a total size of h-13 kb. The 5’ exon codes for a large polymorphic extracellular domain and the shorter 3’ exon codes for a more conserved acidic cytoplasmic tail [24,2.5]. ‘I’he 5 exon contains one to four copies of a motif originally identified in other malaria proteins involved in ligand binding. ‘l’hese were termed Duffy-binding like (DHL) domains after the first of these discovered in I? aiatcx was shown to be responsible for binding to the Duffy blood group essential for rbc invasion by merozoites in this species [26,27]. Several malaria proteins containing these DHI, motifs have now been identified and are clearly recognisable as a superfamily by virtue of the conserved spacing of cysteine residues and the presence of characteristic hydrophobic amino acids at certain positions, but there is little primary sequence conservation between them. Heterologous expression experiments with members of this family other than ~07 have demonstrated that these domains alone have receptor-binding activity 128,29]. ‘I‘hus, the ONI- family encodes proteins with all of the expected properties of PWMP-1 in that the predicted proteins were large, polymorphic, and with regions capable of exhibiting ligand-binding activity expected to be involved in binding to endothelial cells and rbcs [30]. However, experiments involving the selection of antigenic variants of I?,fokipnmm in V&O in Saimiri monkeys suggest that other proteins might vary in their expression under immune pressure and induce specific antibodies [31,32’]. In addition, the genome sequencing project has revealed novel
I” Mlcroblalogy
variant multi-gene families (see below) (see the review TE Wellems era/., this issue pp 415419).
Genomic The variant
Opmn
organisation
1
by
of war genes
there are about 50 copies of z~r with at least one gene present at each telomere. In addition, there ate internal clusters of r?nr genes on chromosomes 4, 7, 8 and 12 ([33,34]; H ‘Fdylor, S Kyes, D Harris, CI Newbold, unpublished data). The telomeric copies are more closely related to each other within a given genotype than they are to the internal clusters, whereas the internal clusters are more similar between genotypes [33-351. This would suggest that recombination at telomeric sites is common in these genes. One recent proposal suggests that UCZTrepertoires would tend not to be overlapping between genotypes if PfEMP-1 is a major target of protective immunity [36]. The overall diversity in field parasite populations is, however, very large indeed as revealed both by P(:R using degenerate DBL-specific primers or by the rare serological cross-reactivity between different antigenic variants [37,38]. In R fakipartim,
Additional with var
variant
gene families
associated
An additional feature of the telomeres revealed by sequencing is a relatively conserved structure that extends well beyond the telomeric and sub-telomeric repeats (1;igure 2). ‘I’he first transcriptional unit to be encountered is a atlrgene. This is followed by a cluster of variable numbers of three gene families ([39”]; The Sanger Centre, http://www.sanger.ac.uk/): the r-if genes, the STEVOR genes, and sequences that have the features of ZM- 3’ exon pseudogenes that appear to form part of a separate multigene family [40]. The rifand STKVOR families contain two exons and encode small predicted transmembrdne proteins [39”,41”] The short 5’ exon encodes a predicted signal sequence. Although they share some homology at the 5’ end and are homologous in the predicted short cytoptasmic tail, the riffamity is much more numerous (perhaps 200 copies/genome) and much more polymorphic than the STEVOR family. The STEVOR genes are transcribed in asexual parasites [41”]. We have shown that the rifgenes are transcribed in asexual parasites at about 24 hours post invasion into proteins termed rifins
422
Host-microbe
Figure
2
interactions:
-
fungi/viruses/parasites
-+-
-
-
RlQnnlll
Centromere
b-
Sub-telomeric
-.m
I
repeats
’
i?llDUl if gene
var gene
B
var exon
2 pseudogene
B
Telomere
IT@3
STEVOR
gene
-
Direction
of transcription Current
Schematic
diagram
of typical
f. falciparum
telomere
based
on available
sequences
[39”], are exported to the infected rbc surface,and, like var, are clonally variant (S Kyes, A Rowe, N Kriek, CI Newbold, unpublished data). Clearly, the transcriptional relationships between ZXZ~; Tif and STEVOR genes at the sametelomere will be of some interest. No convincing data yet exist on what the function of these sequencesmight be but it is becoming clear that the functions of and processesunderlying antigenic variation in malaria are much more complex than had been previously thought.
Transcription
and switching
of WV genes
tilar genes can be transcribed from both telomeric and internal locations [24,34,35,42”] early after invasion for approximately 20-24 hours (S Kyes, CI Newbold, unpublished data). The high rate of switching between vargenes observed Znvitro complicates the analysisof the switching mechanism because cultures rapidly become heterogeneous with regard to the var genes that they express. Nevertheless, several laboratories have devised strategies to obtain cultures monomorphic with respect to oar expression by selection with monoclonal antibody [43’], by adhesioncharacteristics [42”] or by biochemical properties [44]. Individual switch events can then be monitored in such cultures. Unlike many other systemsin which clonal antigenic variation occurs, in P falciparum switches between genes are not commonly accompanied by gene duplication into an expression site by DNA rearrangements or changes in the methylation pattern near to the expressed var gene [30,42”]. There are examples of DNA rearrangements associated with an antigenic switch but these are either deletion events involving irreversible loss of var coding sequence [45”] or an asyet uncharacterised event involving an in vivo switch of a var gene in a squirrel monkey [46]. In all other caseswhere specific switches have been examined in R fakiparxm, no DNA rearrangements were detectable. Expression of var genes seemsto be controlled at the level of transcriptional initiation since, in trophozoites, run-on experiments detected the transcription of only a single var gene [42”]. However, experiments in which an upstream region from an expressed var gene has been fused to a reporter gene and
from
chromosomes
2 and
Opimon m Microbiology
3.
tested for expression by transformation into a parasite expressing an homologousor heterologous var gene reveal identical expression of the reporter in both transformed parasites. Immediate upstream sequences(-2 kb), therefore, do not appear to control expression levels ([45”]; S Kyes, Y Wu, CI Newbold, unpublished data). Using monomorphic lines or single cell reverse transcriptase (RT)-PCR, two groups have demonstrated that even in organismsthat have only one var gene product on their surface and transcribe only a single gene in trophozoites, multiple var transcripts are present in ring stage parasites gene [42”,47”]. It was proposed that one way in which antigenic switching might be regulated was by the transcription of all var genes and the selective degradation of those gene products not destined for expression [48]. Our own data show that in monomorphic ring stage parasites, where many transcripts can be detected by RT-PCR at the 5’ end of the gene, only one full length mRNA messageis detectable by Northern analysis (H Taylor, S Kyes, D Harris, CI Newbold, unpublished data). Thus, if selective degradation occursit must be 3’-5’. Such a mechanism would, however, necessitate some form of imprinting to carry the information from cycle to cycle. Alternatively, these data might reflect relaxed transcription of many genesearly in the asexual cycle. Low level transcripts from other life-cycle stagescan certainly be detected in asexual parasites[42”]. Current opinion, therefore, favours an epigenetic mechanism perhaps associatedwith longer range changesin DNA structure. Recently, the sequence of a variant antigen from a second malaria species, p knowlesi, has been reported [49”]. Although it bears no primary sequence relationship to the var genes of II fakiparmm and has multiple exons, it does have features in common: a repeated structure of cysteine rich domains, and the gene sequenced appearsto be subtelomeric. In this case, there was a change in restriction pattern at the 3’ end of the coding sequence of the expressed gene. It remains possible that since the gene is close to the telomere and only a single switch was examined the rearrangement is not directly connected to the switch. However, this interesting observation, if repeated
Antigenic variation in
with other switch events, might shed some light on the mechanism of antigenic switching in Ii knozz&~i and perhaps in F!fahpamm.
Structure/function gene products
relationships
of var
In intact prbc, PfEMP-1 is associated with parasite induced structures known as knobs, which are formed by the interaction of parasite encoded proteins with the erythrocyte cytoskeleton. Here PfEMP-1 can mediate adhesion of the prbc to a variety of endothelial cell or matrix proteins including (:D36, I<:AM-1. thrombospondin, VCAM, E-selectin, P-selectin, CD31 and Chondroitin sulphate A ((%A) [SO-53,54’,5.5,56]. In general, all parasites bind to CD36 and thrombospondin, but binding to other receptors is a variable property of individual isolates. ‘I’he type of interaction is also receptor specific in that under flow prbc arc immobilised on CD36 but roll on ICAM1.571. Interestingly this behaviour is also modulated by other parasite encoded components because knock-out of a gene essential for knob formation almost abolishes adhesion to CD36 under flow, but does not affect binding to CD36 in static assays [%I. Uearly the way in which PfEMP-1 interacts with different receptors will be an important modulator of endothelial binding in different tissues and, therefore, of pathogenesis. In lines that form rosettes, PfEhlP-1 appears to interact with both CR1 and glycosaminoglycans (GAGS) on the rbc surface [.W,hO]. Heterologous expression studies using fragments of zw genes have to date identified two functional regions that mediate adhesion. Firstly, the non-DBL cysteine-rich interdomain region (CIDR, see Figure l), which is always immediately next to the amino-terminal DBL domain, has been shown to mediate binding to CID36 in several parasite lines expressing different uar genes. [43’,61]. Since the vast majority of parasites do adhere to (2Lj36 and the (2IDR is a common feature, this is likely to be a universal phenomenon in all parasites other than those that bind to (XA. Secondly, in two different rosetting clones binding to uninfected rbcs has been shown to be a function of the amino-terminal I)BI, domain [60,62]. As yet, binding to the other identified receptors has not yet been attributed to isolated uar domains but this work continues.
Why antigenic
variation
in malaria?
Work in several host-parasite systems suggests that antibody responses to WAS are part of the host-protective immune response [13.21,63,64]. It, therefore, seems obvious that antigenic variation has arisen as a means of immune evasion. Nevertheless, this hypothesis neglects to explain why evolution has driven the parasite to insert proteins into the host cell membrane in the first place. The proposal that in ~‘fnlriparzlm, binding to endothelial cells is essential to avoid splenic clearance seems compelling; however, WAS have been detected in all malaria species where they have been sought, and yet only a handful of
Plasmodium
falciparum
Newbold
423
these exhibit specific adhesive properties. This argues that WA expression predates endothelial binding in evolution. Moreover, in several species it has been noted that infection with parasite lines that fail to express VSA leads to lower parasitaemias, and that immunisation with such parasites is more effective than with the homologous antigenic variant [21,65,66]. It thus seems likely that VSAs have evolved primarily to have an immunomodulatory role. In this regard it is interesting that l?fa/a$amm lines that express PfEMP-1 inhibit the activation of and antigen presentation by dendritic cells (671. This promises to be a fertile area for further research.
Conclusions In the four years since variant antigen genes were first identified in malaria, much has been accomplished. Structure-function relationships have been identified for two regions by heterologous expression. Initial work has eliminated a number of potential switching mechanisms and pointed the way for further studies. New variant antigen families have been identified. The primary function of WZT gene products is not unequivocally established but recent data shed new light on these proteins as potential immunomodulators. Future work will need to concentrate on the role of longer range DNA effects on the control of gene expression, the inter-relationships at the transcriptional level between the various multi-gene families, and further functional characterisation of their encoded proteins. A proper understanding of the encoded proteins will be essential if new therapies are to be developed to alleviate pathology caused by specific variant antigen expression.
Acknowledgements
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42. ..
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45. ..
Deitsch KW, Pinal Ad, Wellems TE: lntracluster var transcription switches in the antigenic variation of Plesmodium falciparum. Biochem Parasitol1999, in press. A first class paper that examines in detail the mechamsms underlying gene switching and transcriptional control. 46.
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47. *
Chen 0, Fernandez V, Sundstrom A, Schlichtherle M, Datta S, Hagblom P, Wahlgren M: Developmental selection of var gene expression in Plasmodium falciparum. Nature 1998, 394:392-395. Single cell RT-PCR is used here to examine the transcription of var genes in a clonal parasite culture in which only a single variant antigen is expressed at the red blood cell surface. It concludes that in ring stages many genes are transcribed but in trophozoites only a stngle transcript is detectable. It goes on to discuss the impkcations of these findings. See also [42*-l
l
in Plasmodium
falciparum
Newbold
425
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Crabb BS, Cooke BM, Reeder JC, Waller RF, Caruana SR, Davern KM, Wickham ME, Brown GV, Coppel RL, Cowman AF: Taraeted gene disruotion shows that knobs enable malariainf&ted red cells to’cytoadhere under physiological shear stress. Cell 1997, 69:287-296.
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Le Scanf C, Carey B, Bonnefoy S. Fandeur T, Mercereau-Puijalon 0: A modification in restriction pattern of the Plasmodium falciparum PfSO multigene family associated with a specific antigenic variation switch in the Palo Alto line. Behring lnsr Mitt 1997, 16-24.
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Baruch DI, Ma XC, Singh HB, Bi X, Pasloske BL, Howard RJ: Identification of a region of PfEMPl that mediates adherence of Plasmodium falciparum infected erythrocytes to CD36: conserved function with variant sequence. Blood 1997, 90:3766-3775.
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Al-Khedery B, Barnwell JW, Galinski MR: Antigenic variation in malaria: a 3’ genomic alteration associated with the expression of a /? knowlesivariant antigen. MO/ Cell 1999, 3:i 31-I 41, /? knowlesi is the species in which antlgenic variation was first described for malaria and this paper identifies the genes involved. In contrast to the data obtained to date for f. falciparum, these authors identify a DNA rearrangement asscoiated with the 3’ end of the expressed gene 50.
51.
Barnwell JW, Asch AS, Nachman RL, Yamaya M, Alkawa M, lngravallo P: A human 88 kD membrane glycoprotein (CD36) functions in vitro as a receptor for a cytoadherence ligand on Plasmodium falciparum-infected erythrocytes. J C/in /west 1989, 04:765-772.
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Lowe BS, Kortok M, Molyneux CS, Newbold Cl, Marsh antigens on the infected red cell surface are targets acquired immunity to malaria. Nat Med 1998,4:358-360.
K: for
falciparum
in
Antigenic variation and consequences Chris I Newbold In the past variation switching expression variant Malaria
year,
have
the major been
advances
concerned
in malaria
with
of variant antigen genes, of regions of the major gene families have Genome Project.
in PIasmodium
been
antigenic
the transcription
and
and the functional variant antigen. Also,
discovered
as a result
new of the
Addresses Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 QDS, UK; e-mail: [email protected] Current
Opinion
in Microbiology
1999,
2:420-425
http://biomednet.com/elecref/1369527400200420 0 Elsevier Abbreviations CIDR CSA DBL AEMP-1 prbc rbc VSA
Science
Ltd ISSN
cysteine-rich Chondroitin Duffy-binding Plasmodium
1369-5274
interdomain sulphate A like falciparum
region
erythrocyte
membrane
protein-l
parasitised red blood cell red blood cell variant surface antigen
Introduction During growth and multiplication in their mammalian hosts, malaria parasites spend the majority of their time within erythrocytes. Here they differentiate from the newly invaded ring form through a trophozoite to a schizont containing the replicated daughter merozoites. These will emerge from the infected cell and invade new red cells, thus continuing the cycle. The erythrocyte does not differentiate, has no internal protein synthesis or trafficking machinery, and does not express class I or class II major histocompatibility complex (MHC) molecules on its surface. In other words, it would seem to be an ideal home for an organism that needs to be present within its host for extended periods in order to be transmitted to its obligate insect vector, the mosquito, to survive. Why then have mechanisms evolved whereby proteins synthesised by the parasite are able to cross the parasite plasma membrane and the parasitophorus vacuole membrane, and then be inserted into the red cell plasma membrane where they are visible to the host immune system? The rationale for the evolutionary drive to export proteins to the infected red blood cell (rbc) surface must be that they perform a vital function. In the case of the human malaria Ptasmodium falciparm, the parasite that is responsible for nearly all malaria-specific deaths, the answer would appear to be clear. In this species, parasites only circulate in the blood for the first half of the erythrocytic growth cycle. At approximately 18 hours post-invasion, variant surface antigen (VSA) appears on the rbc and mediates adhesion to a variety of ligands on host endothelium [ 1,2]. This prevents the
falciparum:
mechanisms
parasitised rbc (prbc) passing through the spleen where they might be destroyed by non-specific clearance mechanisms [3]. Two consequences arise from this: first, the VSA is forced to vary to avoid host immune recognition, and second, the variability of the adhesion specificity associated with the antigenic variation of the VSA family means that certain VSAs have undesirable properties. Therefore, different VSAs can bind to different endothelial receptors [4]. In some cases (perhaps when ICAMis the receptor involved) adhesion occurs preferentially in the brain potentially leading to the life threatening syndrome of cerebral malaria [5-81. In other cases, VSAs can bind to uninfected red cells to form erythrocyte rosettes; a phenotype that has also been associated with severe disease [9-l 11. VSAs, whatever their primary function, are important virulence factors, and an understanding of both antigenic variation and adhesion at the molecular level is, therefore, important for both malaria immunity and pathogenesis. Such proteins were first studied serologically by Brown and colleagues [ 12,131 in the primate malaria E knowL~~z’in which VSAs were shown to undergo antigenic variation as a means of immune evasion. Since then phenotypic homologues of these antigens have been found in rodent, other primate and human malaria species, and also shown to undergo true clonal antigenic variation [4,14-181. In this review some of the recent developments in the function, genomic organisation and control of expression of VSAs in malaria are discussed.
Features
of antigenic
variation
in malaria
In vitro studies with clones of P fahpamm indicate that the rate of antigenic switching away from a given variant is as high as 2% per generation [4]. Frequencies of a similar magnitude have been observed in viva in the rodent malaria J? chabaudz’ [ 191. In both cases there appears to be a range of individual variant switch frequencies with lo-z/generation representing the upper limit. No data currently exist about switching rates in humans in viva but mechanisms must exist whereby expression is tightly controlled at both the organism and population level; for example, expression of all variants at the same time would not be an efficient evasion strategy. A further common feature of VSA expression in several parasite species is that it can be reversibly modulated in viva. Passage of cloned organisms through splenectomised animals results in the appearance of parasite populations that no longer express VSA on the rbc surface [3,1.5,17,20,2 11. Re-introduction of these parasites into intact animals results in the reappearance of VSA at the rbc surface in a manner by which VSA gradually reappears in the whole population. This is, therefore, not
Antigenic
Figure
variation
in Plasmodium
falciparum
Newbold
421
1
Schematic representation of the var gene and encoded protein. The grey shaded boxes represent DBLXIDR domains. The boxes with horizontal lines represent inter-DBL domains; the hatched box represents the transmembrane domain; and the diagonal striped box represents the cytoplasmic tail.
5’ exon
~
-8.14kb
p.
Current
a selective event but signifies active signalling between host and parasite. l%o data currently exist on how this signailing might occur.
antigens
of /? falciparum
Where studied, the variant antigens of different malaria species can be described as a family of polymorphic high molecular weight, trypsin sensitive, surface exposed and ‘liiton X-100 insoluble membrane proteins [Z&23]. Molecules with these characteristics in I? fakipclmm were collectively termed t’lmmodium fuhpnnm erythrocyte membrane protein-l (PfEMP-1). ‘l’he CUE genes encoding these proteins have a characteristic structure (Figure 1) consisting of two exons with a total size of h-13 kb. The 5’ exon codes for a large polymorphic extracellular domain and the shorter 3’ exon codes for a more conserved acidic cytoplasmic tail [24,2.5]. ‘I’he 5 exon contains one to four copies of a motif originally identified in other malaria proteins involved in ligand binding. ‘l’hese were termed Duffy-binding like (DHL) domains after the first of these discovered in I? aiatcx was shown to be responsible for binding to the Duffy blood group essential for rbc invasion by merozoites in this species [26,27]. Several malaria proteins containing these DHI, motifs have now been identified and are clearly recognisable as a superfamily by virtue of the conserved spacing of cysteine residues and the presence of characteristic hydrophobic amino acids at certain positions, but there is little primary sequence conservation between them. Heterologous expression experiments with members of this family other than ~07 have demonstrated that these domains alone have receptor-binding activity 128,29]. ‘I‘hus, the ONI- family encodes proteins with all of the expected properties of PWMP-1 in that the predicted proteins were large, polymorphic, and with regions capable of exhibiting ligand-binding activity expected to be involved in binding to endothelial cells and rbcs [30]. However, experiments involving the selection of antigenic variants of I?,fokipnmm in V&O in Saimiri monkeys suggest that other proteins might vary in their expression under immune pressure and induce specific antibodies [31,32’]. In addition, the genome sequencing project has revealed novel
I” Mlcroblalogy
variant multi-gene families (see below) (see the review TE Wellems era/., this issue pp 415419).
Genomic The variant
Opmn
organisation
1
by
of war genes
there are about 50 copies of z~r with at least one gene present at each telomere. In addition, there ate internal clusters of r?nr genes on chromosomes 4, 7, 8 and 12 ([33,34]; H ‘Fdylor, S Kyes, D Harris, CI Newbold, unpublished data). The telomeric copies are more closely related to each other within a given genotype than they are to the internal clusters, whereas the internal clusters are more similar between genotypes [33-351. This would suggest that recombination at telomeric sites is common in these genes. One recent proposal suggests that UCZTrepertoires would tend not to be overlapping between genotypes if PfEMP-1 is a major target of protective immunity [36]. The overall diversity in field parasite populations is, however, very large indeed as revealed both by P(:R using degenerate DBL-specific primers or by the rare serological cross-reactivity between different antigenic variants [37,38]. In R fakipartim,
Additional with var
variant
gene families
associated
An additional feature of the telomeres revealed by sequencing is a relatively conserved structure that extends well beyond the telomeric and sub-telomeric repeats (1;igure 2). ‘I’he first transcriptional unit to be encountered is a atlrgene. This is followed by a cluster of variable numbers of three gene families ([39”]; The Sanger Centre, http://www.sanger.ac.uk/): the r-if genes, the STEVOR genes, and sequences that have the features of ZM- 3’ exon pseudogenes that appear to form part of a separate multigene family [40]. The rifand STKVOR families contain two exons and encode small predicted transmembrdne proteins [39”,41”] The short 5’ exon encodes a predicted signal sequence. Although they share some homology at the 5’ end and are homologous in the predicted short cytoptasmic tail, the riffamity is much more numerous (perhaps 200 copies/genome) and much more polymorphic than the STEVOR family. The STEVOR genes are transcribed in asexual parasites [41”]. We have shown that the rifgenes are transcribed in asexual parasites at about 24 hours post invasion into proteins termed rifins
422
Host-microbe
Figure
2
interactions:
-
fungi/viruses/parasites
-+-
-
-
RlQnnlll
Centromere
b-
Sub-telomeric
-.m
I
repeats
’
i?llDUl if gene
var gene
B
var exon
2 pseudogene
B
Telomere
IT@3
STEVOR
gene
-
Direction
of transcription Current
Schematic
diagram
of typical
f. falciparum
telomere
based
on available
sequences
[39”], are exported to the infected rbc surface,and, like var, are clonally variant (S Kyes, A Rowe, N Kriek, CI Newbold, unpublished data). Clearly, the transcriptional relationships between ZXZ~; Tif and STEVOR genes at the sametelomere will be of some interest. No convincing data yet exist on what the function of these sequencesmight be but it is becoming clear that the functions of and processesunderlying antigenic variation in malaria are much more complex than had been previously thought.
Transcription
and switching
of WV genes
tilar genes can be transcribed from both telomeric and internal locations [24,34,35,42”] early after invasion for approximately 20-24 hours (S Kyes, CI Newbold, unpublished data). The high rate of switching between vargenes observed Znvitro complicates the analysisof the switching mechanism because cultures rapidly become heterogeneous with regard to the var genes that they express. Nevertheless, several laboratories have devised strategies to obtain cultures monomorphic with respect to oar expression by selection with monoclonal antibody [43’], by adhesioncharacteristics [42”] or by biochemical properties [44]. Individual switch events can then be monitored in such cultures. Unlike many other systemsin which clonal antigenic variation occurs, in P falciparum switches between genes are not commonly accompanied by gene duplication into an expression site by DNA rearrangements or changes in the methylation pattern near to the expressed var gene [30,42”]. There are examples of DNA rearrangements associated with an antigenic switch but these are either deletion events involving irreversible loss of var coding sequence [45”] or an asyet uncharacterised event involving an in vivo switch of a var gene in a squirrel monkey [46]. In all other caseswhere specific switches have been examined in R fakiparxm, no DNA rearrangements were detectable. Expression of var genes seemsto be controlled at the level of transcriptional initiation since, in trophozoites, run-on experiments detected the transcription of only a single var gene [42”]. However, experiments in which an upstream region from an expressed var gene has been fused to a reporter gene and
from
chromosomes
2 and
Opimon m Microbiology
3.
tested for expression by transformation into a parasite expressing an homologousor heterologous var gene reveal identical expression of the reporter in both transformed parasites. Immediate upstream sequences(-2 kb), therefore, do not appear to control expression levels ([45”]; S Kyes, Y Wu, CI Newbold, unpublished data). Using monomorphic lines or single cell reverse transcriptase (RT)-PCR, two groups have demonstrated that even in organismsthat have only one var gene product on their surface and transcribe only a single gene in trophozoites, multiple var transcripts are present in ring stage parasites gene [42”,47”]. It was proposed that one way in which antigenic switching might be regulated was by the transcription of all var genes and the selective degradation of those gene products not destined for expression [48]. Our own data show that in monomorphic ring stage parasites, where many transcripts can be detected by RT-PCR at the 5’ end of the gene, only one full length mRNA messageis detectable by Northern analysis (H Taylor, S Kyes, D Harris, CI Newbold, unpublished data). Thus, if selective degradation occursit must be 3’-5’. Such a mechanism would, however, necessitate some form of imprinting to carry the information from cycle to cycle. Alternatively, these data might reflect relaxed transcription of many genesearly in the asexual cycle. Low level transcripts from other life-cycle stagescan certainly be detected in asexual parasites[42”]. Current opinion, therefore, favours an epigenetic mechanism perhaps associatedwith longer range changesin DNA structure. Recently, the sequence of a variant antigen from a second malaria species, p knowlesi, has been reported [49”]. Although it bears no primary sequence relationship to the var genes of II fakiparmm and has multiple exons, it does have features in common: a repeated structure of cysteine rich domains, and the gene sequenced appearsto be subtelomeric. In this case, there was a change in restriction pattern at the 3’ end of the coding sequence of the expressed gene. It remains possible that since the gene is close to the telomere and only a single switch was examined the rearrangement is not directly connected to the switch. However, this interesting observation, if repeated
Antigenic variation in
with other switch events, might shed some light on the mechanism of antigenic switching in Ii knozz&~i and perhaps in F!fahpamm.
Structure/function gene products
relationships
of var
In intact prbc, PfEMP-1 is associated with parasite induced structures known as knobs, which are formed by the interaction of parasite encoded proteins with the erythrocyte cytoskeleton. Here PfEMP-1 can mediate adhesion of the prbc to a variety of endothelial cell or matrix proteins including (:D36, I<:AM-1. thrombospondin, VCAM, E-selectin, P-selectin, CD31 and Chondroitin sulphate A ((%A) [SO-53,54’,5.5,56]. In general, all parasites bind to CD36 and thrombospondin, but binding to other receptors is a variable property of individual isolates. ‘I’he type of interaction is also receptor specific in that under flow prbc arc immobilised on CD36 but roll on ICAM1.571. Interestingly this behaviour is also modulated by other parasite encoded components because knock-out of a gene essential for knob formation almost abolishes adhesion to CD36 under flow, but does not affect binding to CD36 in static assays [%I. Uearly the way in which PfEMP-1 interacts with different receptors will be an important modulator of endothelial binding in different tissues and, therefore, of pathogenesis. In lines that form rosettes, PfEhlP-1 appears to interact with both CR1 and glycosaminoglycans (GAGS) on the rbc surface [.W,hO]. Heterologous expression studies using fragments of zw genes have to date identified two functional regions that mediate adhesion. Firstly, the non-DBL cysteine-rich interdomain region (CIDR, see Figure l), which is always immediately next to the amino-terminal DBL domain, has been shown to mediate binding to CID36 in several parasite lines expressing different uar genes. [43’,61]. Since the vast majority of parasites do adhere to (2Lj36 and the (2IDR is a common feature, this is likely to be a universal phenomenon in all parasites other than those that bind to (XA. Secondly, in two different rosetting clones binding to uninfected rbcs has been shown to be a function of the amino-terminal I)BI, domain [60,62]. As yet, binding to the other identified receptors has not yet been attributed to isolated uar domains but this work continues.
Why antigenic
variation
in malaria?
Work in several host-parasite systems suggests that antibody responses to WAS are part of the host-protective immune response [13.21,63,64]. It, therefore, seems obvious that antigenic variation has arisen as a means of immune evasion. Nevertheless, this hypothesis neglects to explain why evolution has driven the parasite to insert proteins into the host cell membrane in the first place. The proposal that in ~‘fnlriparzlm, binding to endothelial cells is essential to avoid splenic clearance seems compelling; however, WAS have been detected in all malaria species where they have been sought, and yet only a handful of
Plasmodium
falciparum
Newbold
423
these exhibit specific adhesive properties. This argues that WA expression predates endothelial binding in evolution. Moreover, in several species it has been noted that infection with parasite lines that fail to express VSA leads to lower parasitaemias, and that immunisation with such parasites is more effective than with the homologous antigenic variant [21,65,66]. It thus seems likely that VSAs have evolved primarily to have an immunomodulatory role. In this regard it is interesting that l?fa/a$amm lines that express PfEMP-1 inhibit the activation of and antigen presentation by dendritic cells (671. This promises to be a fertile area for further research.
Conclusions In the four years since variant antigen genes were first identified in malaria, much has been accomplished. Structure-function relationships have been identified for two regions by heterologous expression. Initial work has eliminated a number of potential switching mechanisms and pointed the way for further studies. New variant antigen families have been identified. The primary function of WZT gene products is not unequivocally established but recent data shed new light on these proteins as potential immunomodulators. Future work will need to concentrate on the role of longer range DNA effects on the control of gene expression, the inter-relationships at the transcriptional level between the various multi-gene families, and further functional characterisation of their encoded proteins. A proper understanding of the encoded proteins will be essential if new therapies are to be developed to alleviate pathology caused by specific variant antigen expression.
Acknowledgements
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42. ..
Scherf A, Hernandez-Rwas R, Buffet P, Bottius E, Benatar C, Pouvelle B, Gysin J, Lanzer M: Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during i&a-erythrocytic development in Plasmodium falciparum. EM60 J 1998, 17:5418-5426. This excellent paper describes the selection of several parasite lines that are monomorphic with regard to var gene expression and carries out an extensive analysis of the events involved in var gene switching. The overall conclusion is that epigentic mechanisms are most likely to be involved in the regulation of var genes 43. .
Smith JD, Kyes S, Craig AG, Fagan T, Hudson-Taylor D, Miller LH, Baruch DI, Newbold Cl: Analysis of adhesive domains from the A4VAR Plasmodium falciperum erythrocyte membrane protein-i identifies a CD36 binding domain. MO/ Biochem Parasitoll998, 97:133-148. Using a defined antigenic variant expressing a single vargene from parasites able to adhere to ICAMand CD36, this paper attempts several var heterologous expression strategies to map the &ding sites ior these receptors. The CD36 site was confirmed as being in the CIDR from this parasite but the ICAMbinding site was not located. 44.
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Chen 0, Fernandez V, Sundstrom A, Schlichtherle M, Datta S, Hagblom P, Wahlgren M: Developmental selection of var gene expression in Plasmodium falciparum. Nature 1998, 394:392-395. Single cell RT-PCR is used here to examine the transcription of var genes in a clonal parasite culture in which only a single variant antigen is expressed at the red blood cell surface. It concludes that in ring stages many genes are transcribed but in trophozoites only a stngle transcript is detectable. It goes on to discuss the impkcations of these findings. See also [42*-l
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K: for
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in