Variant antigen gene expression in malaria - Wiley Online Library

Blackwell Publishing LtdOxford, UKCMICellular Microbiology1462-5814© 2006 The Authors; Journal compilation © 2006 Blackwell Publishing Ltd???20068913711381Review ArticleVariant genes of malariaR. Dzikowski, T. J. Templeton and K. Deitsch

Cellular Microbiology (2006) 8(9), 1371–1381

doi:10.1111/j.1462-5822.2006.00760.x First published online 18 July 2006

Microreview Variant antigen gene expression in malaria Ron Dzikowski, Thomas J. Templeton and Kirk Deitsch* Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA. Summary Pathogens of the genus Plasmodium are unicellular parasites that infect a variety of animals, including reptiles, birds and mammals. All Plasmodium species target host erythrocytes and replicate asexually within this niche. In humans, proliferation within erythrocytes causes disease symptoms ranging from asymtomatic infection to severe disease, including mild to severe febrile and respiratory symptoms, profound anaemia and obstruction of blood flow. The most serious form of human malaria is caused by Plasmodium falciparum, a pathogen that is responsible for several million deaths annually throughout the developing world. Malaria parasites succeed in evading the host immune response to establish long-term, persistent infections, thus increasing the efficiency by which they are transmitted to the mosquito vector. The ability to evade the host immune system, in particular the avoidance of antibody-mediated immunity against parasite-encoded surface proteins, is the result of amplification of extensive repertoires of multicopy, hypervariable gene families that encode infected erythrocyte or merozoite surface proteins. Via switching between antigenically diverse genes within these large families, populations of parasites have the capacity for rapid variation in antigenicity and virulence over the course of an infection. Here we review the amplification and generation of antigenic diversity within the Plasmodium variant gene families, as well as discuss the mechanisms underlying their tightly controlled gene expression and antigenic switching.

Received 24 April, 2006; revised 31 May, 2006; accepted 7 June, 2006. *For correspondence. E-mail [email protected]; Tel. (+1) 212 746 4976; Fax (+1) 212 746 4028. © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd

Malaria, caused by the apicomplexan parasite Plasmodium, continues to be a source of extensive morbidity and mortality among people living in the developing world, and in particular sub-Saharan Africa. Recent studies have estimated that several million African children die each year as a result of infection by the most virulent malaria parasite, Plasmodium falciparum (Snow et al., 2005). In addition to decreasing quality of life, malaria also imposes a heavy economic burden on developing countries (Sachs and Malaney, 2002; Malaney et al., 2004). The rapid emergence of drug resistance in natural parasite populations highlights the need for new intervention strategies that are both effective at treating the disease and economically viable for widespread distribution. Recognition of these needs has led to a recent increase in research interest towards drug and vaccine development. These efforts are underpinned by studies on the basic biology of the parasite, which in turn have benefited from the enormous amount of data that have been made available through multiple Plasmodium genome sequencing projects (Carlton et al., 2002; Gardner et al., 2002). As a result, the field has witnessed great advances in our understanding of parasite virulence and evasion of the host immune response; including the subject of this review, the evolution of large multicopy gene families encoding hypervariable surface antigens that are implicated or, in the case of PfEMP1, demonstrably involved in host–parasite interactions and the pathogenicity of malaria. Plasmodium shares with other protozoan and bacterial pathogens the ability to vary surface protein expression, and as a result alter the profile of antigens that are exposed to the host immune system. The mechanism of antigenic variation and diversity in Plasmodium can be compared with pathogens such as the unicellular protozoans, African trypanosomes (Cross, 1996; Donelson, 2003) and Babesia sp. (al Khedery and Allred, 2006); infectious bacteria such as Borrelia sp. (Barbour and Restrepo, 2000) and Neisseria sp. (Criss et al., 2005); and pathogenic fungi including Candida sp. (De Las et al., 2003). For each organism, the process of antigenic variation involves the variable expression of genes that encode immunodominant surface antigens. These surface antigens frequently play a role in the virulence of the disease, thus linking antigenic variation to pathogenicity.

1372 R. Dzikowski, T. J. Templeton and K. Deitsch The completion of the genome nucleotide sequence for the P. falciparum isolate 3D7 resulted in the identification of several large, multicopy, hypervariable gene families; notably, the var, rif, stevor and Pfmc-2TM genes (Gardner et al., 2002). Recent genome sequencing efforts for additional P. falciparum isolates indicate that within natural populations of parasites the sequence variability within these gene families is virtually limitless. The process of antigenic variation depends on the ability of parasites to tightly regulate the expression of individual genes within these large, hypervariable gene families, thus exposing only a small portion of the parasite’s antigenic repertoire to the host at any given time. As the immune system responds, the parasite is able to switch to the expression of previously silent genes, thus altering antigenic display and concomitant avoidance of immune clearance. Repeated switches in gene expression patterns underlie the waves of parasitaemia typical of persistent P. falciparum infections. In the following sections we review the P. falciparum gene families that encode the variant surface antigens, and discuss recent hypotheses regarding control of their expression. The var multigene family In P. falciparum the most extensively studied variant antigens are the PfEMP1 (P. falciparum erythrocyte membrane protein 1) cytoadhesive proteins that are displayed within electron-dense knobs on the infected erythrocyte surface. These are large proteins, ranging between 200 and 350 kDa, that are expressed by the parasite on the surface of the infected erythrocyte and are encoded by a multicopy gene family that is collectively named var (Su et al., 1995). The PfEMP1 proteins are composed of several conserved structural features: an N-terminal segment (NTS); Duffy Binding Like domains (DBL; α-ε); cysteinerich interdomain regions (CIDR; α-γ); a transmembrane (TM) domain; and a conserved intracellular acidic terminal segment (ATS) (reviewed by Smith et al., 2001). Although all var genes maintain this basic architecture, the amino acid sequence is highly variable when comparing PfEMP1 proteins among paralogues and across parasite isolates, indicating that there exists a virtually unlimited repertoire of PfEMP1 variants within natural populations. This high level of sequence diversity is probably maintained through both gene conversion and recombination events within the family (Deitsch et al., 1999; Freitas-Junior et al., 2000; Flick and Chen, 2004), thus generating new combinations of the structural domains. The P. falciparum genome contains approximately 60 var genes that encode PfEMP1 proteins that exhibit both sequence diversity and differing combinations of DBL and CIDR domains. Because only a single PfEMP1 protein is expressed at a given time, the cytoadherence and

antigenic phenotype of the infected cell can vary dramatically, and as a result the protein is a principal virulence factor of P. falciparum malaria. PfEMP1 has the ability to bind several host endothelial cell surface receptors, including CD36 (cluster determinant 36), ICAM1 (intercellular adhesion molecule 1), TSP (thromobospondin), CR1 (complement receptor 1) and CSA (chondroitin sulphate A) (Smith et al., 2001). This adhesive property allows parasitized erythrocytes to sequester within the deep tissues by binding to the blood vessel walls, thus avoiding passage through and clearance by the spleen. In addition, adhesion of infected cells contributes to the acute clinical pathologies of the infection, including cerebral malaria and severe malaria associated with pregnancy. The link between var gene expression and virulence has been observed in the association of particular var gene expression patterns to clinical manifestations of disease. For example, expression of a specific subset of var genes, called type A genes (Lavstsen et al., 2003), was associated with severe disease in children (Jensen et al., 2004), and a single distinctly structured var gene known as var2csa (PFL0030c and its paralogues in natural populations) was found to be highly expressed in placental isolates (Salanti et al., 2004). Chondroitin sulphate A (CSA) was shown to be the primary placental-specific ligand for infected erythrocytes (Fried and Duffy, 1996); and selection of cultured parasites for the ability to bind CSA resulted in preferential expression of var2csa transcripts (Salanti et al., 2003) and the encoded protein on the erythrocyte surface (Salanti et al., 2004). The role of the var2csa gene in CSA binding was confirmed by the inability of the parasite to recover the CSA binding phenotype following targeted gene disruption of var2csa (Viebig et al., 2005), indicating that this gene is in fact responsible for CSA binding and consistent with its role in placental adhesion. Interestingly, var2csa is one of the few var genes that are conserved across parasite isolates, raising the important consideration of its use as the basis for a syndromespecific vaccine (Smith and Deitsch, 2004). var genes have been shown to be expressed in a mutually exclusive manner at both the mRNA (Chen et al., 1998a; Scherf et al., 1998; Voss et al., 2006) and protein levels (Dzikowski et al., 2006). By limiting expression to a single var gene copy the parasite limits exposure to a single antigen at a time to the host’s immune system. Over time the immune system generates an antibody response against the surface of the infected erythrocytes, thus recognizing the predominantly expressed form of PfEMP1 and consequently selecting for subpopulations of parasites that have arisen via switching expression to different var genes. Seemingly inexhaustible repetitions of this cycle result in long-term persistence of a single infection that is clinically characterized by oscillations of peripheral

© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 8, 1371–1381

Variant genes of malaria 1373 parasitaemia having magnitudes varying from undetectable levels to high parasite burdens (Miller et al., 1994). Annotation of the complete genome sequence of the 3D7 isolate identified 59 intact var genes that are scattered on all chromosomes except 14. The majority are located in subtelomeric regions; however, 22 are found in internal regions of the chromosomes, mainly arranged as clusters of tandemly repeated genes (Gardner et al., 2002). All var genes have a similar structure consisting of a long exon 1 that encodes the variable extracellular portion of the protein; an intron that has a highly conserved nucleotide sequence; and a short exon 2 that codes for a conserved intracellular domain that is thought to anchor the protein within a knob structure at the cytoplasmic face of the erythrocyte membrane (Baruch et al., 1995; Su et al., 1995) (Fig. 1A). Each gene has been shown to possess two separate promoters, one upstream of exon 1 that is responsible for expression of the mRNA and a second promoter within the intron that leads to expression of a non-coding or ‘sterile’ RNA (Calderwood et al., 2003). Sequence analysis of the upstream regulatory regions of all 3D7 isolate var genes identified three main subgroups: UpsA, UpsB and UpsC (Kraemer and Smith, 2003; Lavstsen et al., 2003). UpsA genes are found in subtelomeric regions while those containing UpsC promoters are in the internal regions of the chromosomes. Genes that contain UpsB upstream sequences are found in both subtelomeric and internal locations. Gel mobility shift assays identified

regions of both UpsB and UpsC upstream sequences that were differentially bound by nuclear protein complexes (Voss et al., 2003); however, the specific proteins involved in these binding events have yet to be identified. Interestingly, genes containing the UpsC upstream sequences have recently been shown to also be expressed during gametocyte development (Sharp et al., 2006). The promoters found upstream of the genes and responsible for mRNA transcription are active early in the cell cycle, from 12 to 18 h after invasion, while late-stage parasites actively produce the ‘sterile’ RNAs that are transcribed from the introns (Calderwood et al., 2003; Kyes et al., 2003). The role of the sterile non-coding RNAs in var regulation is still elusive, although it has been speculated that they could be involved in chromatin assembly, as has been shown for non-coding RNAs in other systems (Morey and Avner, 2004). Interestingly, however, recent microarray data could not detect significant expression of either sense or antisense sterile transcripts associated with var2csa silencing (Ralph et al., 2005a), thus more experimental data are needed to elucidate the possible role of these non-coding RNAs in var regulation. The rif multigene family The rif (repetitive interspersed family) and stevor (subtelomeric variable open reading frame) gene families were first characterized as multicopy gene families encoding

A transmembrane domain

rif

pexel/vts signal peptide stevor

promoter DBL domain CIDR domain

Pfmc-2TM

var

B Typical P. falciparum chromosome

subtelomeric domain

internal chromosome gene cluster

subtelomeric domain

var rif/stevor Pfmc-2TM telomeric repeats


Fig. 1. Structure and arrangement of variant antigen genes in P. falciparum. A. The gene structure of each of the four gene families. Note that rifs, stevors and Pfmc-2TM all have a two-exon structure in which the signal peptide is encoded on the small first exon, while the PEXEL/VTS motif responsible for erythrocyte trafficking is found on the second exon. In contrast, while var genes also have a two-exon structure, they do not contain a signal peptide. In addition, var genes have two promoters, the first upstream of exon one and responsible for transcription of the mRNA and the second in the intron, leading to transcription of the ‘sterile’ transcripts. The domain structure of the PfEMP1 protein encoded by var genes is also shown. B. Location and arrangement of the var, rif, stevor and Pfmc-2TM gene families on a typical P. falciparum chromosome. Within the subtelomeric regions, var genes are typically located closest to the telomeric repeats, followed by interspersed rifs and stevors, and lastly by members of the Pfmc-2TM family. These genes are also found as clusters within the central regions of chromosomes 4, 7, 8 and 12.

1374 R. Dzikowski, T. J. Templeton and K. Deitsch potential variant antigens (Cheng et al., 1998; Weber, 1988). The rif genes, with approximately 150 copies identified within the 3D7 genome nucleotide sequence, make up the largest gene family in P. falciparum (Gardner et al., 2002). The protein products of these genes were first identified in experiments in which the surface proteins of infected erythrocytes were radiolabelled and visualized by SDS-PAGE. These proteins were initially correlated with erythrocyte rosetting (the binding of uninfected red blood cells to infected cells) and were termed ‘rosettins’; however, this phenotype was subsequently shown to be mediated by PfEMP1 expression (Rowe et al., 1997; Chen et al., 1998b). The products of the rif multigene family (Fernandez et al., 1999; Kyes et al., 1999) are now termed RIFINs, and are isolate-specific antigens (Helmby et al., 1993) that are localized to the infected erythrocyte surface and compose a significant group of clonally variant polypeptides that are targeted by the human immune response. The rif genes share with two other gene families, stevors and Pfmc-2TM, a gene architecture consisting of a short first exon that encodes a signal peptide sequence, followed by a longer (c. 1 kb) exon that encodes the remainder of the protein (Fig. 1A). The RIFIN proteins also share with STEVOR and Pfmc-2TM proteins a trafficking motif, termed Pexel/VTS (Hiller et al., 2004; Marti et al., 2004), that confers trafficking to the cytoplasm of the infected erythrocyte and is encoded near the 5′ end of the second exon. The RIFIN, STEVOR and Pfmc-2TM proteins are predicted to have two membrane-spanning domains flanking a hypervariable loop, and comprise a superfamily in terms of structure and, perhaps, function. The length of the predicted loop differs between RIFIN (170 aa long), STEVOR (60 aa long; Cheng et al., 1998; Sam-Yellowe et al., 2004) and Pfmc-2TM (less than 20 aa long; Sam-Yellowe et al., 2004) proteins; and the loop contains the predominant sequence variability that exists within the respective gene families. In contrast, the Nterminal regions are highly conserved within each family, as well as the short, positively charged C-terminal regions that follow the second TM domain. The hypervariability within the loop, combined with the presence of a Pexel/ VTS trafficking motif, leads to the logical hypothesis that the two TM domains are targeted to and span the erythrocyte membrane, thus exposing the loop to antibodymediated immune selection. The rif and stevor genes are predominantly found within the subtelomeric regions and are frequently internal or adjacent to var gene clusters. Similar to var genes, they are also found within blocks in more central regions of the chromosomes (Fig. 1B). Northern blot analysis indicates that rifs are transcribed in a narrow time window at the late ring stage (18 h) in parallel to the decrease in var transcription (Kyes et al., 2000), indicating that there could be a functional relationship that underlies their close prox-

imity in the genome. Analyses of transcription in other eukaryotic organisms indicates that gene clusters sometimes function as transcription units in which the genes are co-regulated (Cohen et al., 2000; Caron et al., 2001; Lercher et al., 2002). Gene clusters encoding coexpressed proteins have been identified in the P. falciparum genome (Florens et al., 2002), although specific correlation of var and rif expression has not been confirmed. Radiolabelling of the infected erythrocyte surface of clonal populations of P. falciparum indicated that expressed RIFINs were both variant and subject to switches in gene expression (Fernandez et al., 1999). Studies of immune responses to recombinant RIFINs in individuals living in endemic areas of Africa confirmed that RIFINs are indeed immunogenic. Exposure to RIFINs initiates a naturally acquired immune response that was later correlated with rapid parasite clearance and asymptomatic infection (Abdel-Latif et al., 2002; 2003). Proteomic analysis over the entire P. falciparum life cycle revealed an unexpectedly high level of expression of RIFIN, as well as PfEMP1 and STEVOR proteins, in sporozoites and no observed expression in trophozoites (Florens et al., 2002). In view of the recent discovery of erythrocyte trafficking motifs within these proteins (Hiller et al., 2004; Marti et al., 2004), the expression of these gene families in extraerythrocytic sporozoites should be further investigated to validate a possible role in multiple stages. The stevor multigene family The stevor multigene family (originally named 7h8; Limpaiboon et al., 1991) consists of 30–40 genes, depending on the parasite isolate, that are located within rif-containing subtelomeric gene neighbourhoods in all P. falciparum chromosomes. The STEVOR proteins are slightly smaller (30–40 kDa versus 30–45 kDa) and are more conserved than RIFINs between parasite isolates; however, both gene families share sequence and structural similarities suggesting that they have an ancestral relationship and might share similar or collaborative functions. An early proteomic screen identified STEVOR expression only in sporozoites (Florens et al., 2002); however, recent studies using anti-STEVOR antibodies demonstrated that these genes are transcribed and translated during a tight window at the mid-trophozoite stage (22–32 h after invasion) (Kaviratne et al., 2002) that overlaps the end of the rif transcription window (12–27 h after invasion) (Kyes et al., 2000). In addition to trophozoite stage transcription, stevor genes are expressed in early gametocytes at lower levels (Sharp et al., 2006), and STEVOR protein persists until gametocyte maturation (day 17) (McRobert et al., 2004). Similar to the proteomic analysis, these authors demonstrated that STEVORs are expressed in mosquito salivary gland sporozoites, although mRNA transcripts could not


Variant genes of malaria 1375 be detected. Reverse transcription polymerase chain reaction (RT-PCR) of single micromanipulated trophozoite-infected erythrocytes showed that only a subset of stevor genes are transcribed within a single parasite (Kaviratne et al., 2002). Gametocytes also express a dominant subset of stevors, although the same stevor population is predominant in the progenitor asexual population (Sutherland, 2001; Sharp et al., 2006). These studies indicate that there might be a stage-specific component to the regulation of stevor transcription. In gametocytes, stevor transcripts have been reported to have a diverse transcript structure possibly due to the use of alternative mRNA splicing donor or acceptor sites, resulting in frame shifts and deletions (Sutherland, 2001); thus the predicted polypeptides in gametocytes might be different from those encoded by asexual parasites. Unlike RIFINs and PfEMP1, which are ultimately targeted to the surface of infected erythrocytes, immunofluorescence and immunoelectron microscopy studies using antibodies against STEVORs indicate that they remain within the Maurer’s clefts (MC), colocalizing with MC resident proteins. Maurer’s clefts are a unique feature of infected erythrocytes and consist of a flat vesicular membranous structure within the erythrocyte cytoplasm. They are also found associated with the erythrocyte membrane, as well as the tubovesicular membrane network that extends from the parasitophorous vacuole through the erythrocyte cytoplasm. These structures have been suggested to be involved in the trafficking machinery of proteins from the parasite to the surface of infected cells (Przyborski et al., 2003), and the localization of STEVORs within MC suggests they might function within this organelle. Recently, Przyborski et al. (2005) identified motifs, in addition to the Pexel/VTS, which are required for exporting STEVOR proteins across the parasitophorous vacuole to the MC. This study also showed that STEVORs colocalize with PfEMP1 in the MC membrane, although a significant portion of STEVORs were urea inextractable, suggesting their interaction with the membrane was not solely the result of protein–protein interactions as was the case for PfEMP1 (Papakrivos et al., 2005). The authors therefore suggest that they share similar but not identical trafficking machinery. While STEVORs localized to MC in blood stages, STEVORs expressed in gametocytes did not colocalized with MC (McRobert et al., 2004); moreover, they were present within the infected erythrocyte after MC had disappeared. It is possible that the MC is not the final destination of STEVOR proteins and, similar to PfEMP1, they are trafficked through the MC to the erythrocyte surface. Further ultrastructural studies will likely aid in unveiling the final destination of STEVORs and will also be valuable for studying their function. The function of STEVOR proteins remains enigmatic and several hypotheses have been proposed based on

their predicted structure, cellular localization and expression patterns. The highly variable loop between the two TM domains suggests a role for antigenic variation in the face of immune pressure at the erythrocyte surface. However, their late blood stage expression, after the transcription of PfEMP1 and RIFINs, was suggested to imply a functional role in MC (Blythe et al., 2004). Based on their unique expression during both asexual stages and sporozoites, these authors speculated that they might be a multifunctional protein family. Synthetic peptides from STEVOR putative proteins bind with high affinity to human erythrocytes (Garcia et al., 2005) suggesting a possible role in forming rosettes. Clearly, more data are needed to unveil the function of this protein family and its relation with the other 2TM proteins, in particular RIFIN and Pfmc-2TM. The Pfmc-2TM multigene family As described above, the P. falciparum genome sequence contains a catalogue of 200 two-exon genes which encode putative proteins that have the sum architecture of a signal peptide sequence; Pexel/VTS trafficking motif; and two predicted TM domains within the C-terminal portion of the protein. In addition to the repertoire of rifins, with 130-plus genes, and stevors, with 30-plus genes, the catalogue of 2TM proteins includes several smaller families of paralogous genes (Marti et al., 2005; Templeton and Deitsch, 2005; Sargeant et al., 2006), most notable of which are 13 genes that encode the Pfmc-2TM proteins (Sam-Yellowe et al., 2004; C. Lavazec, S. Sanyal and T.J. Templeton, submitted). The Pfmc-2TM proteins are highly conserved across paralogues and between isolates within the N-terminal, 2TM domains, and short, positively charged C-terminal regions. Similar in theme to the RIFIN and STEVOR proteins, the Pfmc-2TM proteins are highly divergent within a loop between the two TM domains; but in contrast the loop is shorter and encompasses less than 20 amino acids. The profound diversity within this loop, both between paralogues and across isolate boundaries (C. Lavazec, S. Sanyal and T.J. Templeton, submitted), justifies the inclusion of Pfmc-2TM genes as an antigenically variant gene family. Indirect immunofluorescence studies have shown that the Pfmc-2TM proteins are localized to the MC (Sam-Yellowe et al., 2004); however, it has not been determined whether the erythrocyte surface is the ultimate, perhaps functional destination of the Pfmc-2TM proteins. Epigenetic regulation of variant gene transcription: the var gene example Investigations into the molecular basis for transcriptional regulation of the multicopy gene families of P. falciparum have largely centred on the var gene family. In particular,


1376 R. Dzikowski, T. J. Templeton and K. Deitsch several recent papers have begun to address the roles that chromatin structure, subnuclear localization and DNA regulatory elements play in var gene silencing, activation and mutually exclusive expression. While the work described here relates specifically to the var gene family, the concepts developed will likely also apply to the other multicopy gene families found in the malaria genome, such as the 2TM gene family members, of which little is known. The intron as a silencing element Early efforts directed towards understanding var gene silencing identified the conserved var intron as being important in the silencing process. Specifically, in both transient and stable transfections of cultured parasites, constructs carrying a var promoter driving expression of either a luciferase reporter gene or a drug selectable marker were constitutively active unless paired with a var intron on the same plasmid (Deitsch et al., 2001; Calderwood et al., 2003; Gannoun-Zaki et al., 2005). A more recent study determined that the interaction between var promoters and introns that leads to transcriptional silencing can occur regardless of whether the intron is upstream or downstream of the var promoter, but that this interaction requires a strict one-to-one stoichiometry (Frank et al., 2006). It was further shown that the function of the intron as a silencer is dependent on its own promoter activity, a quality reflected in the production of the previously mentioned sterile transcripts, and by the fact that knocking out the promoter activity of the intron impairs its ability to silence a var promoter (Calderwood et al., 2003). These experiments have led to the hypothesis that promoter– promoter interactions between the intron and upstream regulatory regions of each var gene are responsible for the silencing phenomenon. A recent study by Voss et al. (2006) reached a conclusion that differs from the work of Gannoun-Zaki et al. and Frank et al. These authors found that the presence of the intron was not required for a var promoter to assume the silent state, and that silencing was the default state in the transfected var promoter constructs that were tested. However, the presence of the intron did significantly decrease the frequency of activation of the var promoter in their constructs, implying that the intron did contribute to the silencing process. While all three studies detected a role for the intron in var gene silencing, they differ significantly in their conclusions regarding the default transcriptional state of a var promoter. The primary difference in the experimental designs used by Gannoun-Zaki et al. and Frank et al. which indicated that the intron was necessary for silencing, versus that used by Voss et al. which determined only a minor suppressive role, lies in the placement of the selectable marker used for selection

of stably transformed parasites. The constructs employed by Gannoun-Zaki et al. and Frank et al. contained only the promoter activities that are associated with var genes, namely those found in the upstream regulatory region and in the intron, and hypothesized that interactions between these two promoters were responsible for the silencing effect. In contrast, Voss et al. placed an additional promoter, hsp86, driving the bsd selectable marker on their constructs. It is therefore possible that the same promoter–promoter interaction demonstrated for var intron promoters (Calderwood et al., 2003) is occurring between the heterologous (hsp86) promoter and the adjacent episomal var promoter, thus rendering the intron redundant and unnecessary for silencing in this context. Consistent with this hypothesis, we have recently found that heterologous promoters can indeed interact with var promoters when placed in close proximity, leading to silencing in the same S-phase-dependent manner that was described for var introns (A. Eisberg, unpubl. data). Nonetheless, further experiments are required to fully verify and understand the role of var introns in gene silencing. Nuclear positioning Eukaryotic organisms remodel their nuclei into regions of actively expressed euchromatin, often in the centre of the nucleus, and condensed, transcriptionally repressed heterochromatin typically found at the nuclear periphery. Consistent with this, electron microscopy of P. falciparum ultrastructure reveals a similar subdivision of the nucleus (Ralph et al., 2005b), but with the retention of a distinct euchromatic domain within the peripheral heterochromatin rich region (Fig. 2). To determine whether a subnuclear structure plays a role in var gene regulation, Ralph et al. (2005b) determined the subnuclear location of var2csa following selection for active and silent states via panning of infected erythrocytes on CSA. Fluorescence in situ hybridization (FISH) showed that a silenced var2csa gene primarily colocalizes (84% of the time) with telomeric clusters within the perinuclear heterochromatin. However, when selected for activation, while the gene remained within the nuclear periphery, it dissociated from the telomeric clusters. The authors speculate that var gene activation may involve the repositioning of the active locus to a discrete region of euchromatin found within the nuclear periphery. The linkage between heterochromatin silencing and nuclear repositioning was further investigated in two additional studies that employed FISH to localize transcriptionally active and silent genes (Duraisingh et al., 2005; Voss et al., 2006). In the first, Duraisingh et al. observed that an active var2csa gene preferentially colocalized with a transcriptionally active episome (55% of the time) versus


Variant genes of malaria 1377 Fig. 2. Model for the role of nuclear organization, chromatin structure and telomere gene clustering in var gene expression. The basic structure of the nucleus is shown, with dark blue representing areas of pericentric heterochromatin, while light blue symbolizes less condensed euchromatic regions near the centre of the nucleus. Clusters of telomeres are found near the nuclear membrane. var genes have been proposed to be organized into groups at the nuclear periphery when in the silent state, and when activated to move away from the telomere clusters into a small region of euchromatin within the largely heterochromatic outer region of the nucleus. The box on the lower right shows a ‘bouquet’ of silent var genes. Also noted is the production of ‘sterile’ transcripts being expressed from the promoter found within the var intron.

when it was silent (22%), suggesting that transcriptionally active var promoters specifically move away from regions of the nucleus that are rich in condensed heterochromatin and towards subnuclear locations that are transcriptionally competent. In contrast, Voss et al. found no linkage between subnuclear localization and the transcriptional state of chromosomally internal var genes, and instead found that they preferentially colocalized with the telomeric clusters in both the active and silent state. Interestingly, the latter authors also found that active var promoters located on transfected episomes preferentially colocalized with the telomere clusters (c. 65%), while silent episomes did not, thus suggesting a model in which the transcriptionally active region of the nucleus is adjacent to the telomeric clusters. In conclusion, although the subnuclear localization and organization of the chromosomes is likely to influence var gene expression, important questions remain concerning the ultrastructure of the subnuclear regions and molecular mechanisms of var gene regulation.

Chromatin modification Epigenetic regulation of gene expression is generally thought to involve alterations in chromatin structure, in particular the modulation of histone tail acetylation and methylation. P. falciparum possesses a homologue, PfSIR2, of the yeast histone deacetylase SIR2 (silent information regulator 2) protein that is involved in specifying the assembly of a silent chromatin structure by binding to regions that are transcriptionally repressed (Duraisingh et al., 2005; Freitas-Junior et al., 2005). Using immunofluorescence assay (IFA), FISH and chromatin immunoprecipitation assays (ChIP), these authors found that PfSIR2 localizes to the telomeric foci within the electrondense heterochromatic regions at the nuclear periphery, and that PfSIR2 association spreads from the telomeres up to 50 kb towards the centre of the chromosomes, including large portions of the regions that contain the var, rif, stevor and Pfmc-2TM gene families. Using var2csa as a model gene for silencing and activation, they further


1378 R. Dzikowski, T. J. Templeton and K. Deitsch showed that silent subtelomeric var genes are bound by PfSIR2, and that this binding is lost when a var gene is activated, thus correlating PfSIR2 binding to var gene silencing. In contrast to the binding of PfSIR2 within the telomeric foci of the nucleus, the authors observed that acetylated histone H4 was found specifically at active var loci as well as within the euchromatic interior of the nucleus. Additional evidence for the role of PfSIR2 in var gene silencing was provided by phenotypic analysis of a transgenic parasite line in which PfSIR2 was disrupted (3D7/ ∆sir2) (Duraisingh et al., 2005). Microarray experiments comparing gene transcription of 3D7/∆sir2 with wild-type 3D7 detected an increase in expression for a subset of subtelomeric vars and rifs; however, expression of most of the genes in these families remained unchanged. The fact that not all var genes became transcriptionally active in the absence of PfSIR2 demonstrates that while this protein might play a role in silencing a subgroup of var genes, its role is not universal for the entire var gene family. Moreover, ChIP assays did not detect PfSIR2 binding at internal var loci (Freitas-Junior et al., 2005), and these genes were not substantially upregulated in the 3D7/∆sir2 line, indicating that central var genes are regulated in a PfSIR2-independent manner. One possible addition to this complicated regulatory pathway is the presence of a second sir2-like deacetylase that has been identified in the P. falciparum genome sequence. This gene might serve as symbolic reminder that the ‘histone code’ for P. falciparum may not be as simple as it is sometime portrayed, and that elucidation of the details of chromatin modifications will likely shed substantial light on the regulation of expression of multicopy gene families in malaria parasites. Mutually exclusive expression The paradigm of mutually exclusive expression is a key element that underlies the process of antigenic variation and the ability of parasites to evade the host immune response, thereby promoting transmission to additional hosts. Two examples of mono-allelic expression of surface receptors that warrant comparison are the odorant receptors of the mammalian olfactory system and the immunoglobulins in mammalian B-cells (Serizawa et al., 2004; Corcoran, 2005). In both cases the expression of a functional surface protein initiates a negative feedback cascade that maintains the remaining gene family members in a transcriptionally silent state. Thus, in these systems, mutually exclusive expression is ultimately regulated at the level of protein production. In contrast, production of a functional PfEMP1 protein is not needed for mutually exclusive expression of var genes – using transgenic parasite lines in which the PfEMP1 coding region of a var gene was replaced with a drug-selectable marker,

Dzikowski et al. (2006) and Voss et al. (2006) demonstrated that expression is dependent solely on the transcriptional regulatory elements that surround each var locus. Remarkably, in these lines, drug-mediated selection for expression of the transgenic var gene led to silencing of the entire var gene repertoire, thus knocking out PfEMP1 erythrocyte surface expression. Similar to its role in gene silencing, the role of the var intron in mutually exclusive expression is somewhat controversial. In the experiments described by Voss et al. (2006), selection for activation of a recombinant var promoter placed on an episomally replicating plasmid led to silencing of the endogenous var genes even in the absence of an intron, thus leading to the conclusion that the intron is not essential for either var silencing or mutually exclusive expression. In contrast, other studies have suggested a link between the intron, silencing and mutually exclusive expression. Several transgenic parasite lines have been generated in which the interactions between a var promoter and intron have been disrupted (Gannoun-Zaki et al., 2005; Viebig et al., 2005; Frank et al., 2006). In each of these cases, the var promoter became constitutively active, yet did not affect expression of the rest of the var gene family, implying that it was no longer ‘counted’ by the mechanism controlling mutually exclusive expression. Similarly, the var1csa pseudo gene (also called varcommon) has a large deletion within its intron and is actively transcribed in many isolates; yet other var genes are also expressed implying that it remains uncounted (Kyes et al., 2003; Winter et al., 2003). In the episomal studies of Voss et al. it should be noted that the recombinant var promoter is present in multiple copies, making it difficult to pinpoint how many are transcriptionally active. Indeed, selection for activation of the var promoter driving expression of the selectable marker also resulted in a substantial increase in the size of the concatamer, thus implying that more than one promoter may be simultaneously active within the concatamer. Thus, while the exact role of the intron in this process remains unclear, what seems apparent is that constitutively active var promoters that have been disrupted in silencing are also no longer ‘counted’, implying that the transcriptional mechanisms that control var gene silencing and mutually exclusive expression may be somehow linked, although the details remain largely a mystery. Conclusions The amplification of gene families within the P. falciparum genome, coupled with the mechanisms driving antigenic diversity and variation within these surface protein families, insures that essential parasite proteins acting at the host interface do not stimulate a sterilizing host immune response. The parasite devotes large portions of its


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