Antigenic Variation and the Genetics and Epigenetics of the PfEMP1 Erythrocyte Surface Antigens in Plasmodium falciparum Malaria

CHAPTER

3 Antigenic Variation and the Genetics and Epigenetics of the PfEMP1 Erythrocyte Surface Antigens in Plasmodium falciparum Malaria David E. Arnot*,†,‡,1 and Anja T. R. Jensen*,†

Contents

I. Introduction: Malaria Immunity, Vaccines, and Antigenic Variation II. The Interaction Between Antigenic Variation and Malaria Pathogenesis III. P. falciparum Genetics and Genomics IV. var Genes and the Structure of PfEMP1 Proteins V. var Gene Transcription and PfEMP1 Antigen Expression VI. Is MEE an Absolute Rule for var Genes? VII. Epigenetic Antigenic Variation VIII. Conclusions Acknowledgments References

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* Centre for Medical Parasitology, Department of International Health, Immunology and Microbiology,

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Faculty of Health Sciences, University of Copenhagen, CSS Oester Farimagsgade 5, Copenhagen K, Denmark Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), CSS Oester Farimagsgade 5, Copenhagen K, Denmark Institute of Immunology and Infection Research, School of Biology, University of Edinburgh, Edinburgh, Scotland, United Kingdom Corresponding author: Centre for Medical Parasitology, Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, CSS Oester Farimagsgade 5, Copenhagen K, Denmark e-mail address: [email protected]

Advances in Applied Microbiology, Volume 74 ISSN 0065-2164, DOI: 10.1016/B978-0-12-387022-3.00007-0

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2011 Elsevier Inc. All rights reserved.

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Abstract

David E. Arnot and Anja T. R. Jensen

How immunity to malaria develops remains one of the great unresolved issues in bio-medicine and resolution of its various paradoxes is likely to be the key to developing effective malaria vaccines. The basic epidemiological observations are; under conditions of intense natural transmission, humans do become immune to P. falciparum malaria, but this is a slow process requiring multiple disease episodes which many, particularly young children, do not survive. Adult survivors are immune to the symptoms of malaria, and unless pregnant, can control the growth of most or all new inoculations. Sterile immunity is not achieved and chronic parasitization of apparently healthy adults is the norm. In this article, we analyse the best understood malaria ‘‘antigenic variation’’ system, that based on Plasmodium falciparum’s PfEMP1-type cytoadhesion antigens, and critically review recent literature on the function and control of this multi-gene family of parasite variable surface antigens.

I. INTRODUCTION: MALARIA IMMUNITY, VACCINES, AND ANTIGENIC VARIATION Malaria remains a severe global public health problem which is unlikely to disappear without the introduction of a highly effective vaccine (Aguas et al., 2008). Many, but not all, malariologists consider this a realistic, albeit long-term, goal because repeated exposure to the bite of infected mosquitoes confers substantial immunity to the disease (Aponte et al., 2007; Doolan et al., 2009). However, despite the accumulation of a great deal of data on immune responses to malaria, an improved understanding of the epidemiology of malaria and development of a partially protective vaccine based on the circumsporozoite antigen (Cohen et al., 2010), it remains uncertain if highly effective vaccination can be achieved. Whether other parasite antigens can improve on the protection seen with current formulations is a subject of much current research. Explicitly or implicitly, malariological thinking is strongly marked by inclination toward one (Cohen et al., 1961), or the other (Brown and Brown, 1965), of two long-established schools of thought. What might be called the ‘‘antigenic variation’’ school attributes the slow acquisition of natural immunity to the need to accumulate immunological memory of many immunodominant variable surface antigens (VSA) expressed by the intraerythrocytic (IE) stages of the microorganism (Bull et al., 1998; Hviid, 2005; Fig. 3.1). This school has tended to view malaria vaccination as a formidably difficult problem due to the antigenic diversity of the malaria parasite, not susceptible to vaccine development by any established approach (Brown and Tanaka, 1975). The ‘‘strain-transcending immunity’’ school has been more optimistic about malaria vaccines because its adherents focus on data indicating that significant immune protection is being achieved after a few infections

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B

A

5 μm

5 μm

FIGURE 3.1 Intraerythrocytic infection by Plasmodium falciparum. (A) The early intraerythrocytic stages of the protozoan malaria parasite P. falciparum, around 4 h after the invasion of a human red blood cell by the invasive merozoite stages. (B) After 48 h of intraerythrocytic growth and development of the parasites, the infected erythrocyte ruptures, releasing 8–20 daughter merozoites. The lower infected red blood cell is in the late stages of development and will shortly lyse, as has occurred in the cell above. Released merozoites will rapidly invade new erythrocytes and unchecked asexual growth causes the complex pathology, morbidity, and mortality, associated with P. falciparum malaria.

(Baird, 1994) or that immune protection can be conferred by transfused antibodies (Bouharoun-Tayoun et al., 1990; Cohen and Butcher, 1971). Both types of observation can support a view that significant immunity is acquired via pan-specific rather than variant-specific immune responses and that relatively simple malaria vaccines are conceivable—with the appropriate protective antigens (Doolan et al., 2009). Between these confines, many nuanced hypotheses have been proposed, although the experimental evaluation of conserved versus variable targets of immunity has been a slow process and this controversy is not close to being resolved. However, the genetics and genomics revolution undoubtedly offers some interesting new possibilities for faster evaluation of the predictions of competing theories of malaria immunity and uncovering the elusive immunological Achilles Heel of Plasmodium falciparum. Access to malaria parasite genome sequence data revealed that all human, simian, and rodent Plasmodium species have evolved large gene families encoding variable, but related protein antigens that vary in expression between isolates (Su et al., 1995). This was solid evidence for the existence, if not the function and importance, of malaria antigenic variation systems. The best characterized of these families are the PfEMP1 (P. falciparum erythrocyte membrane protein 1) proteins encoded by the var genes. A substantial body of evidence now indicates that these encode an immunodominant antigenic variation system of P. falciparum malaria (Marsh and Kinyanjui, 2006; Salanti et al., 2003). We will review the biology, genetics, and emerging principles underlying the control of expression of PfEMP1 genes.

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II. THE INTERACTION BETWEEN ANTIGENIC VARIATION AND MALARIA PATHOGENESIS The seminal experiments demonstrating that recrudescent waves of malaria parasites differ in the antigens present on the Intra-erythrocytic (IE) surface were performed 50 years ago, using Plasmodium knowlesi infections of rhesus monkeys (Brown and Brown, 1965; Brown and Hills, 1974). Proof that the descendants of a single haploid clone can switch between the expression of alternative VSA during the course of an infection was provided a decade later (Barnwell et al., 1983). The discovery of antigenic variation in the P. knowlesi system in the 1960s strongly influenced the interpretation of the course of the therapeutic human malaria infections widely used in neurosyphillis treatment between the 1920s and early 1960s. These induced infections follow a course of recurrent waves of parasitaemia, although peaks decline in severity after the initial wave (Molineaux et al., 2001). The fact that repeated inoculations, with both Plasmodium vivax and P. falciparum, showed ‘‘a complete absence of ‘solid immunity,’ even when homologous strains were concerned’’ ( Jeffery, 1966) supported the view that it was antigenic variation which inhibited the acquisition of ‘‘solid immunity’’ and that changes in the antigenic composition of the parasite population led to the recurrent waves of parasitaemia (Kyes et al., 2001). Linkage between parasite erythrocyte surface proteins and serological antigenic variation was established by detection of variable, high-molecular weight P. falciparum proteins on the IE membrane (Leech et al., 1984) and the discovery of families of genes with features expected of candidate P. falciparum VSAs (Su et al., 1995). The discovery of the var genes provided a genetic basis for surface antigen switches. The realization that var-encoded PfEMP1 proteins bind to host endothelium proteins also provided a link to the pathology associated with IE sequestration (Smith et al., 1995) and further increased interest in the role of PfEMP1 as adhesion antigens. Among many interesting aspects of the role of PfEMP1 proteins as adhesins is their unusual topological distribution on the so-called ‘‘knob protuberances’’ at the IE surface (Aikawa et al., 1983). Antibodies against PfEMP1 domains can also be used to detect specific PfEMP1 antigens on the surface of live P. falciparum-infected erythrocytes (Staalsoe et al., 1999; Fig. 3.2). Antibody responses to PfEMP1 proteins are associated with the development of immunity to malaria (Bull et al., 1998; Khunrae et al., 2010), the most direct evidence for PfEMP1 being a protective antigen deriving from the study of pregnancy-associated malaria (PAM). Increasing levels of protection from the effects of PAM, such as low birth weight, correlate with antibody to the VAR2CSA PfEMP1 (Staalsoe et al., 2004) and these antibodies block the adherence of IE to placental chondroitin sulfate A (CSA), the host receptor for the PfEMP1 VAR2CSA protein (Salanti et al., 2003). That

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FIGURE 3.2 Exported antigens of the parasite at the membrane of human red blood cells infected by P. falciparum. (A) Phase contrast microscopy of infected and uninfected erythrocytes. The dark granule is the optically dense, iron-containing hemozoin, a by-product of the parasite’s digestion of host hemoglobin which is sequestered in the digestive vacuole inside the growing parasite cell. (B) The same three infected and one uninfected erythrocytes, stained with an antibody detecting an exported parasite protein (KHARP) which binds to the submembranous actin–spectrin cytoskeleton of the erythrocyte. The immunofluorescently stained confocal micrograph shows the relatively abundant quantities of this antigen deposited at the periphery of the infected host cells. (C) Immunofluorescent live cell antibody staining (Bengtsson et al., 2008) of the VAR2CSA PfEMP1 (superimposed in the phase contrast image) on the outer surface of a live-stained cell. Fluorescent speckling represents PfEMP1 antigen concentrations at a surface knob structure ( Joergensen et al., 2010).

immunity to PAM is a special case of general malaria immunity, and that surviving childhood malaria involves progressive acquisition of immunity to different PfEMP1 antigens is the position of those who propose that antigenic variation plays a dominant role in malaria pathogenesis and acquired immunity (Chen, 2007; Hviid and Staalsoe, 2004; Kyes et al., 2001). PFEMP1 proteins enable IE to sequester to the host’s vascular endothelium (Smith et al., 1995) and certain binding motifs of PfEMP1 proteins also occur in otherwise unrelated proteins on P. vivax and P. knowlesi merozoites which interact with the red blood cell Duffy antigens (Chitnis and Miller, 1994). PfEMP1 was also quickly recognized to be the candidate VSA required for the role of the serum agglutination antigen discovered in serological studies in malaria endemic areas. African children’s plasma antibodies had a variable capacity to agglutinate diverse samples of parasitized erythrocytes but the capacity of children’s sera to recognize IE increased with the age and malaria infection experience of the child (Marsh and Howard, 1986). Large-scale serological studies in cohorts of African children confirmed this age and transmission intensity-related acquisition of VSAspecific antibodies and its strong correlation with the acquisition of clinical immunity (Bull et al., 1998, 1999, 2000; Nielsen et al., 2002). The finding that an IE’s capacity to be agglutinated by diverse plasma samples was associated with severe disease and young patient age was explained in terms of immune responses being first directed against ‘‘early-encountered’’ VSA, presumably those best adapted to growth in

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that particular host (Bull et al., 2005). This implies that particular PfEMP1 adhesion receptors may be associated with the severe malaria episodes seen in young African children ( Jensen et al., 2004). That P. falciparum infections in individual malaria cases switch VSA expression was shown in the low-intensity transmission setting of Eastern Sudan (Staalsoe et al., 2002) and switching in PfEMP1 expression in experimental human infections has also been detected (Lavstsen et al., 2005; Peters et al., 2002).

III. P. FALCIPARUM GENETICS AND GENOMICS P. falciparum is a eukaryotic protozoan, with nucleus and intracellular organelles, and an obligate parasite of insects and humans. For most of its life cycle, it has a haploid complement of 14 chromosomes, in a single nucleus (Fig. 3.3). Following fusion of haploid ‘‘male’’ and 748

7G8

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Mb –3.13 –2.70 –2.35

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FIGURE 3.3 Pulsed field gel electrophoretic separation of the chromosomes of five P. falciparum clonal isolates. Aside from the small amounts of genetic information in the mitochondrion (6 kb) and the apicoplast (35 kb), the
23 Mb of P. falciparum genomic DNA are contained in the 14 chromosomes observed to be present in all isolates. Even with the limited resolution possible when separating such large (700–3500 kb) DNA molecules, the marked size differences between homologous chromosomes in different clonal isolates are apparent. PfEMP1 genes are present at the telomeres of almost all of these chromosomes, as well as in internal clusters.

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‘‘female’’ gametes taken up in a blood meal from an infected host, there is a brief diploid phase in the mosquito midgut, followed by meiosis and a return to asexual multiplication of haploid forms in an oocyst formed in the mosquito stomach wall. Genome replication occurs repeatedly during sporogony in the mosquito midgut oocysts (producing
104 sporozoites/oocyst), during hepatocytic schizogony in the host liver (producing
104 hepatocytic merozoites), and during the 8- to 20-fold genome amplification occurring during intraerythrocytic replication (Arnot et al., 2011). Chromosomes present in the short-lived diploid zygote represent lineages that have completed 30–40 rounds of mitotic replication between meioses. Mitotic recombination between sister chromatids will not result in new allelic combinations unless mutations occur during growth. The rate of P. falciparum mitotic recombination has not been estimated, but in systems where mitotic recombination can be measured, it is thousands of times less frequent (104–105) than meiotic recombination (Barbera and Petes, 2006). However, relatively frequent accretions and deletions of telomeric DNA sequences occur during in vitro culture (Scherf and Mattei, 1992). Telomeric rearrangement of var genes during asexual growth (Duffy et al., 2009), as well as after meiosis (Freitas-Junior et al., 2000) are also documented. The relative contribution of mitotic and meiotic events to the generation of var gene diversity remains unclear, but the great majority of viable rearrangements of existing allele combinations, contributing to most of the evolution of the P. falciparum genome, are likely to occur at meiosis (Keightley and Otto, 2006; Petes and Pukkila, 1995). A limited number of laboratory genetic crosses between cloned P. falciparum lines have been carried out, which required chimpanzees to receive the sporozoite products of meiosis and develop the initial blood infection (Walliker et al., 1987; Wellems et al., 1990). The analysis of progeny, adapted to in vitro culture from the chimpanzee blood infections and cloned by limiting dilution, indicated that gametes fuse randomly and that meiotic recombination is significantly more frequent (30–50 times) than in higher eukaryotes, 1 cM in P. falciparum corresponding to 15–30 kb (Su et al., 1999). An excess of recombinant over parental genotypes was also found in the progeny of the cross, possibly indicating that during passage through the chimpanzee, or in in vitro culture, recombinant genotypes have been favoured by selection (Walliker et al., 1987). Intragenic recombination has been detected, although its origin and frequency are uncertain (Kerr et al., 1994). In nature, recombination frequencies have been shown to be roughly proportional to malaria transmission intensity (Babiker et al., 1997; Tanabe et al., 2007). P. falciparum chromosomes do not condense at mitosis, possibly because the parasite does not have detectable H1 linker histones (Miao

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et al., 2006). Electron microscopic counting of spindle kinetochores (Prensier and Slomianny, 1986) and pulsed field gel (PFG) electrophoresis (Triglia et al., 1992) indicate that P. falciparum has 14 chromosomes, ranging from 0.6 to 3.5 Mb in size, in a total genome of 23 Mb (Fig. 3.3). P. falciparum has one of the most intensively studied genomes known (Gardner et al., 2002) and more than a dozen genomes have now been sequenced, with varying degrees of ‘‘finishing’’ of the repetitive and A þ T rich sequences of P. falciparum intergenic regions and telomeres. Progress has also been made in using genomic sequencing to clarify parasite population structure and evolutionary history (Mu et al., 2002, 2005). Combining genomic sequence information with other experimental data and genetic crossing results, some general observations on the genetics of P. falciparum can be made (Box 3.1).

BOX 3.1

General features of the genetics of P. Falciparum

1. P. falciparum replicates by mitotic division of a haploid genome in various vector and host cells. Recombination occurs during a brief diploid zygote phase after fusion of haploid gametes present in infected human blood meals, in the mosquito midgut. Reciprocal exchanges between parent genotypes, which segregate according to Mendelian principles, occur at meiosis in the zygote (Su et al., 1999; Walliker et al., 1987). 2. The position of most of the
5300 P. falciparum gene loci on 14 chromosomes is constant between isolates (Carlton, 2007) and there is considerable conservation of chromosome number and locus positions between different Plasmodium species (Carlton et al., 2008). 3. P. falciparum chromosome telomeres contain repetitive sequences and many hypervariable genes encoding polymorphic exported antigens (Templeton, 2009). The presence of variable genes and repetitive DNA at the telomeres explains the discrepancy between isolates having syntenic gene organization but variable PFG karyotypes showing homologous chromosome size differences (Fig. 3.3). 4. Unconventional elements of P. falciparum genetics include frequent ectopic recombination between nonhomologous chromosomes (Freitas-Junior et al., 2000), with nonreciprocal, gene conversionlike duplications of the donor sequence. Ectopic exchanges have been proposed to occur in telomere clusters located at the nuclear envelope (Freitas-Junior et al., 2000; Ralph et al., 2005b) rather than during chromatid pairing on the mitotic spindle.

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IV. VAR GENES AND THE STRUCTURE OF PFEMP1 PROTEINS Almost all 28 P. falciparum telomeres have at least one var gene and additional intrachromosomal clusters of var genes are situated on chromosomes 4, 7, 8, and 12 of the 3D7 isolate (Gardner et al., 2002). Allelic, orthologous relationships between var genes are difficult to discern, with the exception of the var1csa and var2csa PfEMP1 genes, which are the most evolutionarily conserved var genes (Salanti et al., 2002; Sander et al., 2009). The paralogous relationships within gene families has been described as the hallmark of hypervariable malaria VSA, probably deriving from evolutionary expansion of gene families by gene duplication and immunity-driven divergence (Templeton, 2009). All var genes are organized into two exons separated by a 1–2 kb intron (Fig. 3.4). Exon 1 encodes the polymorphic extracellular domains, ending in a transmembrane sequence. Exon II encodes the more conserved
4–500 amino acid intracytoplasmic acidic terminal sequence (ATS). The ATS interacts with other exported proteins such as KHARP, which binds to actin and spectrin and connects PfEMP1 to the cytoskeleton. PfEMP1 gene sequences are essentially a mosaic of building blocks encoding amino acid segments combined into larger regions which loosely correspond to protein structural domains (Smith et al., 2001). This architecture is presumably a result of mutation and selection, followed by recombination between members of an evolving family undergoing gene duplications. Receptor-binding functions are combined with variation in the antigenic profile by tolerating mutations which alter antigenic properties, provided these do not compromise function. Structural analysis of the

Exon I

NTS

DBL1α

CIDR1α

DBL2β

Exon II

DBL3β

DBL4γ

DBL5δ

CIDR2β

TM

ATS

FIGURE 3.4 Schematic structure of a P. falciparum var gene and its encoded PfEMP1 protein. The N-terminal extracellular region and the short transmembrane (TM) sequence are encoded by Exon I and the conserved acidic terminal sequence are encoded by Exon II. The actual number and combinations of domains and domain subtypes vary considerably between the different, highly polymorphic, PfEMP1 molecules. NTS: N-terminal segment; DBL: Duffy binding-like; CIDR: cysteine-rich interdomain region; ATS: acidic terminal segment.

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VAR2CSA PfEMP1 indicates that many amino acid substitutions are compatible with a conserved polypeptide backbone (Higgins, 2008).

V. VAR GENE TRANSCRIPTION AND PFEMP1 ANTIGEN EXPRESSION Intra-erythrocytic malaria parasites go through morphological changes (the ring, trophozoite, and schizont stages), and several mitotic divisions during the 48-h cycle between merozoite invasion and cell lysis and daughter merozoite release (Arnot and Gull, 1998; Arnot et al., 2011). These transitions are reflected in a stage-specific pattern of gene expression which may reflect a ‘‘just in time’’ system where translation immediately follows transcription (Bozdech et al., 2003). In synchronized in vitro cultures, PfEMP1 mRNA first appears around 3-5h post-merozoite invasion (PMI), steady-state var transcription peaks at around 18 h PMI, and declines to undetectable levels by 20–24 h PMI (Dahlba¨ck et al., 2007; Kyes et al., 2000). Export of PfEMP1 protein appears relatively slow, but surface-detectable PfEMP1 appears from 16 to 18 h PMI (Bengtsson et al., 2008; Kriek et al., 2003). Each var gene is associated with two distinct promoter regions, a 50 upstream (ups) promoter-regulating expression of the PfEMP1 mRNA and a second intronic promoter found in the relatively conserved intron (Calderwood et al., 2003). The intronic promoter drives expression of large, noncoding transcript whose role in var regulation is uncertain (Epp et al., 2009). Interestingly, subclasses of var genes show clearly conserved relationships between their ups promoter sequences and their positions on particular chromosomes (Kraemer and Smith, 2003; Lavstsen et al., 2003). Early studies of var transcription indicated that a proportion of the repertoire was transcribed in early ring stages (Smith et al., 1995). However, subsequent studies indicated this relaxed or loose pattern of polygenic transcription narrowed to an expression of a single locus by the trophozoite stages (Scherf et al., 1998). Transcription of many var genes in ring stages was also observed in individual IE using single-cell RT-PCR assays, but again, by the trophozoite stage of development, individual IE concentrated on the expression of one var gene (Chen et al., 1998). These studies detected only one PfEMP1 antigen on the surface of each infected erythrocyte and the proposal of a ‘‘mutually exclusive expression’’ (MEE) hypothesis that individual parasites activate only one gene at a time and that all paralogous var loci are in some way silenced. A variant of this hypothesis, where a dominant transcript is developmentally selected from initially polygenic transcripts, was also considered (Chen et al., 1998; Kyes et al., 2003). Observations that were difficult to accommodate in an MEE framework were that several experiments reported little or no

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detectable tightening of relaxed transcription in trophozoites relative to the early ring stages, in both cultured populations and individual IE (Duffy et al., 2002; Noviyanti et al., 2001; Taylor et al., 2000). The publication of the genome sequence of the 3D7 clone of P. falciparum allowed primers to be designed which allowed quantitative RT-PCR to simultaneously measure transcription of all var genes in a population. This improved on the previous partial views of repertoire activity and analyzing the PfEMP1 genes transcribed in CSA adhesionselected parasites gave the striking result that only one gene, var2csa, was being transcribed at above background levels (Salanti et al., 2003). That repeated CSA adhesion selection establishes dominant var2csa expression was confirmed in several later studies (Dahlba¨ck et al., 2007; Duffy et al., 2005; Gamain et al., 2005). The MEE model was also supported by experiments which combined surveillance of the total var repertoire with transfection and gene knockouts to manipulate the context in which var promoters were placed. Disassociating a var ups promoter from its partner downstream intronic promoter was found to lead to constitutive activation of the unpaired ups promoter and simultaneous expression of two var promoters (Frank et al., 2006). However, forcing transfected P. falciparum cultures to express increasing numbers of episomal upsC-type promoters led to silencing of all the endogenous chromosomal var promoters (Dzikowski and Deitsch, 2008). When parasites were released from episome-mediated endogenous var promoter silencing, they reverted to exclusive expression of one var gene, from several different endogenous chromosomal promoters. Previously silenced var genes appeared to be activated randomly and the episome-mediated silencing episode appeared to have deleted any prior ‘‘memory’’ or epigenetic marking of formerly expressed genes. These experiments were interpreted as indicating the existence of a mechanism in which continuous transcription of a promoter established exclusive expression of that var gene, and that this state could be inherited without recombination in an epigenetic rather than genetic fashion (Dzikowski and Deitsch, 2008).

VI. IS MEE AN ABSOLUTE RULE FOR VAR GENES? While the analysis of transfected var promoters and CSA adhesion selection gave results which supported MEE models of var transcription, mRNA assays (Northern blots, nuclear run-on, and single-cell RT-PCR) nonetheless only sometimes (Chen et al., 1998; Horrocks et al., 2004; Scherf et al., 1998), but not always (Duffy et al., 2002; Noviyanti et al., 2001; Taylor et al., 2000), showed a narrowing of initially polygenic transcription during differentiation of untransfected Intra-erythrocytic P. falciparum. These anomalies were generally overlooked until two recent studies reopened this question.

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That the expression of duplicated copies of the var2csa genes, which appear quite common (Sander et al., 2009), did not to appear to be governed by MEE was reported in a recent publication (Brolin et al., 2009). The linked question of whether both var transcripts are translated and exported to the erythrocyte surface was not analyzed. However ‘‘double expression’’ of two different PFMP1 antigens on a single IE has now been closely investigated and using P. falciparum selected using different, specific anti-PfEMP1 antisera, 3D7 sublines were recently shown to simultaneously express two different var mRNAs and to export both antigens to the surface of the same IE ( Joergensen et al., 2010; PLOS Pathogens). Immunofluorescence microscopy and flow cytometric detection of dual antibody binding was reinforced by RNA FISH detection of two different var transcripts in single individual cells. A functional rationale for the dual expression phenomenon was provided by showing that coexpressors, but not monoexpressors, were able to bind synergistically to two host receptors, CD54/ICAM and CD31/PECAM ( Joergensen et al., 2010; PLOS Pathogens). Whether this type of exception to the MEE type of var gene transcription situation is common and occurs in malaria infections, as well in antibodyselected in vitro P. falciparum cultures remains to be seen. Detecting dual expression depends on identifying individual parasites which are expressing two particular PfEMP1 antigen specificities which can be detected by antibody reagents. Given the enormous diversity of naturally expressed PfEMP1, there is a low probability of detecting dual expressor parasite clones with the small number of characterized antisera currently available. However, if the expression of more than one PfEMP1/IE, rather than the absolute imposition of MEE, is within the capacity of parasites in natural infections, this could be advantageous during the posthepatocytic establishment phase. The first generation posthepatocytic parasite genomes lack ‘‘memory’’ of any var gene transcription and the data indicate that MEE-based silencing of the repertoire requires prior transcription of a var gene (Chookajorn et al., 2007; Dzikowski and Deitsch, 2008). If expression of > 1 PfEMP1 by the first wave of posthepatic IE increases the likelihood of successful sequestration, this will promote clonal survival. ‘‘Successful’’ genes would be marked for expression in their descendent genomes, while unexpressed genes remain unmarked and silenced. From initially loose transcription, progressively more exclusive transcription might then be selected from the most effectively sequestering parasites. An optimally exclusive expression pattern, perhaps unigenic, will emerge. In real infections, considerable heterogeneity of surface PfEMP1 expression by the population is predicted, as variants wax and wane in the face of immunity (Dietz et al., 2006). Although this prediction has not been systematically investigated in natural infections, with specific typing sera, a narrowing of initially loose var gene

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transcription has been observed in the earliest stages of blood stage infections induced in human volunteers (Wang et al., 2009).

VII. EPIGENETIC ANTIGENIC VARIATION Epigenetics, in the sense of heritable changes in phenotype that occur in the absence of mating or genomic rearrangement, has been invoked to explain malaria antigenic variation as transcriptional switching of PfEMP1 expression appears to leave the participant genes in situ and unaltered (Dzikowski and Deitsch, 2008; Scherf et al., 1998). Although many var switches do not involve genetic rearrangements, these have been observed. For example, in P. falciparum clone Dd2, switching from the expression of one chromosome 12 var gene to one of two adjacent var genes was accompanied by the deletion of the formerly expressed locus (Deitsch et al., 1999). In the IT/FCR3 P. falciparum lineage, a switch in expression from the A4 var to the R29 var was accompanied by translocation of the 50 end of the A4 var to another chromosome (Horrocks et al., 2004). In the CS2 line, a var gene on chromosome 4 was translocated into an existing var gene’s intron on chromosome 12, where the translocated gene became active (Duffy et al., 2009). Current data are thus compatible with the possibility that P. falciparum, in an inversion of the Trypanosoma bruceii situation where recombinational switching predominates (Morrison et al., 2005), combines some recombination-based VSA activation beside a predominantly in situ switching system. Epigenetic influences on var transcription include several different observations of expression-associated chromatin modifications and nuclear position effects on transcriptional activation (Box 3.2). As in the broader field of gene regulation, there are controversies about prime causes and secondary effects (Ptashne, 2007). Histone modifications are not known to be heritable and must be reestablished after each DNA synthesis phase. They are also generally associated with the passage of the complex transcription apparatus over a locus, or the absence of such effectors, and can be seen as an effect of gene activation rather than a primary actor. There are indications that soluble transcription factors/ promoter regulatory elements bind to var promoters and that their absence will silence ‘‘unbound’’ promoters (Dzikowski and Deitsch, 2008). This may indicate that some form of self-perpetuating (i.e., epigenetic) positive feedback loop ensures continuous transcription similar to the classical phage lambda repressor switch, which is activated by external, nongenetic influences (Ptashne, 2009). Alternatively, it may be more closely related to other eukaryotic systems where one or a small number of genes in a multigene family are selected, by some poorly understood process, for expression by individual cells, such as the Drosophila odor receptor genes in olfactory neurons (Vosshall and Stocker, 2007).

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BOX 3.2 Epigenetic features of P. Falciparum var gene regulation

1. Trans-acting factors, presumably regulatory proteins, bind var gene promoters (Frank et al., 2006; Voss et al., 2005), and the titration of such factors by transfected episomal var promoters leads to a shutdown of endogenous var transcription (Chookajorn et al., 2007; Dzikowski and Deitsch, 2008). 2. Epigenetic marks, that is, the characteristic histone posttranslational modifications (PTM) associated with downregulated genes, including H3K9me3 and histone hypoacetylation have been found to be associated with inactive var genes (Duraisingh et al., 2005; FreitasJunior et al., 2005). 3. P. falciparum sirtuins, homologous to key components of eukaryotic chromatin modification complexes, have been shown to affect var gene regulation, sirtuin gene deletion tending to relax silencing (Merrick et al., 2010; Tonkin et al., 2009). 4. The positioning of transcriptionally active var genes at particular sites underneath the nuclear envelope membrane has been proposed to influence var gene transcription status (Duraisingh et al., 2005; Ralph et al., 2005b). 5. Noncoding RNA (ncRNA) molecules are transcribed by P. falciparum (Li et al., 2008) but no functioning RNAi regulatory system appears to exit (Baum et al., 2009). Whether or not ncRNA is involved in epigenetic silencing is unclear (Epp et al., 2009; Ralph et al., 2005a).

VIII. CONCLUSIONS Despite considerable progress in the analysis of antigenic variation in human P. falciparum malaria, at present we do not understand how the parasite establishes this system in the early stages of infection or how it breaks an established pattern of var gene expression to express a new PfEMP1 antigen. Both genetic and epigenetic features of the system are apparent but relative rates and causes and effects remain uncertain (Cui and Miao, 2010; Horrocks et al., 2009; Merrick and Duraisingh, 2010). With the establishment and characterization of genetically unmodified parasite isolates that show exceptions to the active promoter counting/MEE situation (Brolin et al., 2009; Joergensen et al 2010; PLOS Pathogens), it may be possible to further analyze what permits and/or limits multiple var gene expression. Although there are many difficulties in analyzing in vivo human infections, more data on var gene expression and switching during clinical P. falciparum malaria are needed.

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ACKNOWLEDGMENTS We thank Dominique Bengtsson and Adam Sander for comments and unpublished micrographs and Thor Theander, Lars Hviid, Ali Salanti, Thomas Lavstsen, Morten Nielsen, Lars Joergensen, Elena Ronander, and Louise Joergensen at the Centre for Medical Parasitology, University of Copenhagen, for many interesting discussions. ‘‘We thank the Niels Bohr Foundation and Danmarks Grundforskningsfonds and the Howard Hughes Medical Foundation for support’’.

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