The effect of Plasmodium falciparum Sir2a histone deacetylase on clonal and longitudinal variation in expression of the var family of virulence genes

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Abstract

Plasmodium falciparum, the most important causative agent of human malaria, undergoes antigenic variation as a means of prolonging infection and ensuring transmission between hosts. Clonal variation is observed in the surface adhesins expressed on infected erythrocytes: primarily in the PfEMP1 adhesin encoded by the large var gene family. The sirtuin PfSIR2A was the first protein discovered to have a major influence on antigenic variation in P. falciparum. In the absence of PfSIR2A, normal silencing of the variantly-expressed var gene family is partially deregulated. To thoroughly investigate the role of PfSIR2A in controlling antigenic variation, multiple independent clones of wildtype and PfSIR2A-knockout (ΔSir2a) parasites were generated. var gene expression was then measured qualitatively, quantitatively and longitudinally over extended periods in culture. ΔSir2a parasites were found to activate about 10 specific var genes in every independent clone analyzed. The activated genes were biased towards the upsA, upsBA and upsE var gene subclasses. The total var transcript level was two to three-fold higher in ΔSir2a parasites than in wildtype parasites and at least one transcript – encoding the pregnancy malaria adhesin VAR2CSA – was successfully translated and expressed on the infected cell surface. In the absence of PfSIR2A, antigenic switching over time was also diminished, although not abolished. This work expands our understanding of clonal antigenic variation in this important human pathogen and demonstrates a central role for PfSIR2A in regulating both the variant expression of specific var gene subsets and the overall quantity of var gene expression.

Introduction

Plasmodium falciparum is the most important cause of human malaria, giving rise to widespread morbidity and approximately 1 million deaths each year. This unicellular eukaryotic parasite lives and replicates inside erythrocytes and is spread between human hosts by a mosquito vector. To avoid the human immune system and sustain chronic infections, P. falciparum has evolved a complex system of antigenic variation, allowing it to persist in the bloodstream for months or years, thus facilitating insect transmission.

Antigenic variation in P. falciparum is best characterized in the major surface antigen expressed on infected erythrocytes, PfEMP1 (Baruch et al., 1995, Smith et al., 1995, Su et al., 1995). This large transmembrane protein mediates adhesion to a variety of host endothelial molecules, sequestering infected cells out of the circulation to avoid splenic clearance. The exposed PfEMP1 protein is, however, a target for host immunity (Bull et al., 1998) and a large family of variant ‘var’ genes encoding antigenically distinct forms of PfEMP1 has therefore evolved. There are 63 genes in the sequenced strain 3D7, with the majority located sub-telomerically and a subset in tandem arrays at chromosome-internal locations (Gardner et al., 2002). var genes have been divided into sub-families according to the similarity of their ‘ups’ upstream promoter regions. UpsB genes are the most telomere-proximal and are transcribed towards the centromere; upsA genes are located sub-telomerically, inside the upsB genes and are transcribed towards the telomere, while upsC genes are chromosome-internal. Intermediate promoter types define upsBA and upsBC var genes. There is also a unique upsE gene encoding the unusual PfEMP1 protein VAR2CSA, which adheres to the placental marker chondroitin sulphate A (CSA) and is likely to play a major role in pregnancy malaria (Salanti et al., 2003, Viebig et al., 2005).

Most evidence suggests that only a single var gene (Scherf et al., 1998, Dzikowski et al., 2006, Voss et al., 2006) or a small subset of them (Duffy et al., 2002, Mok et al., 2007, Mok et al., 2008) is expressed in a single parasite at one time. The expressed gene(s) switches over time, which can correlate with a change in the adhesive phenotype of infected cells (Roberts et al., 1992) and with waves of fever in infected patients, as new parasite populations emerge which are not well controlled by the immune system (Miller et al., 1994). No defined orders for switching between var genes have been discovered in in vitro cultures, suggesting that the process is either highly complex or entirely random. It is also unclear how fast antigenic switching occurs and how widely the switch rate might vary, with published estimates ranging from 2% to 18% per generation (Roberts et al., 1992, Gatton et al., 2003).

The biological mechanism that controls transcriptional switching amongst var genes in P. falciparum has recently been the subject of intensive study. Switches occur without accompanying DNA rearrangements and an expressed gene is maintained semi-stably for multiple generations, implicating an epigenetic mechanism (Scherf et al., 1998). This idea was confirmed when a gene encoding a sirtuin in P. falciparum was genetically disrupted (Duraisingh et al., 2005). Sirtuins are NAD+-dependent (class 3) deacetylase enzymes, named after the canonical Saccharomyces cerevisiae enzyme silencing information regulator 2 (ScSIR2). ScSIR2 is a crucial player in heterochromatin formation and thus in the general silencing of sub-telomeric genes, a phenomenon termed ‘telomere position effect’ in yeast. ScSIR2 deacetylates histone tails, facilitating chromatin condensation and thus gene silencing. In accordance with the S. cerevisiae model, loss of the putative sirtuin PfSIR2A in P. falciparum led to the activation of multiple var genes, particularly in sub-telomeric loci. In a complementary study, P. falciparum chromatin was found to be most condensed in sub-telomeric regions and semi-quantitative chromatin immunoprecipitation showed that histone H4 was less acetylated within a silent sub-telomeric var gene than within an active one. This hypoacetylation correlated with the presence of PfSIR2A on chromatin (Freitas-Junior et al., 2005). The PfSIR2A protein was subsequently shown to be a genuine sirtuin enzyme with deacetylase (and ADP-ribosylase) activity on P. falciparum histones in vitro (Merrick and Duraisingh, 2007). More recently, it has been shown that other histone modifications, notably methylations of histone H3, correlate with var gene expression or silencing (Lopez-Rubio et al., 2007) and a role has been demonstrated for the second sirtuin in P. falciparum, PfSIR2B, in silencing a different subset of var genes (Tonkin et al., 2009).

Thus, var gene regulation appears to center on an epigenetic process which semi-stably silences the majority of the var gene family. PfSIR2A plays an important role in controlling heterochromatinization, at least in sub-telomeric regions. Questions remain, however, as to the precise mechanism by which this enzyme influences antigenic variation, warranting a closer examination of the phenotype of PfSIR2A-knockout (ΔSir2a) parasites. For example, does PfSIR2A normally silence a very specific subset of var genes – in which case each ΔSir2a parasite should up-regulate the same large subset of genes simultaneously – or does it primarily influence switching, in which case there might be rapid switching amongst a preferred gene subset? Alternatively, might PfSIR2A affect both the dynamics of switching and the nature of var expression at any one time? Are there common sequence features in the promoters of the activated var genes, pointing to a specific in-cis mechanism of transcriptional control? Finally, are the var RNAs that are over-expressed in ΔSir2a parasites actually functional, being translated and expressed as a greater quantity and/or variety of PfEMP1 on the infected cell surface?

To answer these questions, the phenotype of ΔSir2a parasites was assessed both qualitatively and quantitatively in terms of var gene expression. Multiple independent clones of wildtype (WT) 3D7 and 3D7ΔSir2a parasites were generated and their var expression profiles were measured and compared. Clones were then followed for a long period of time to assess switching. These experiments yielded, to our knowledge, the first detailed picture of global var expression dynamics, both in 3D7 WT parasites and as affected by the important silencing enzyme PfSIR2A.

Section snippets

Parasite culture

Plasmodium falciparum lines 3D7 (strain obtained from the Walter and Eliza Hall Institute of Medical Research, Australia), 3D7ΔSir2a (Duraisingh et al., 2005) and CS2 (Elliott et al., 2005) were cultured in human O+ erythrocytes at 4% haematocrit in RPMI 1640 supplemented with 0.5% albumax (Invitrogen) and 0.25% sodium bicarbonate, using standard procedures (Trager and Jenson, 1978). For flow cytometry experiments, cultures were switched from albumax to 10% human serum 48 h before collection.

A distinct subset of var genes is over-expressed in multiple independent 3D7ΔSir2a clones

A var over-expression phenotype in 3D7ΔSir2a parasites was initially detected by a semi-quantitative whole genome microarray performed on bulk populations of 3D7ΔSir2a and 3D7 WT (Duraisingh et al., 2005). When the expression levels of all genes in the two populations were plotted against each other, a subset of var genes (as well as some from the sub-telomeric rifin family) were expressed at significantly higher levels in 3D7ΔSir2a than in the control. To confirm this finding in a quantitative

Discussion

This study represents, to our knowledge, the first comprehensive qualitative and quantitative analysis of var gene expression in the commonly-studied 3D7 strain of P. falciparum, together with a detailed description of how PfSIR2A influences var expression in this strain. The PfSIR2A histone deacetylase is the first protein found in P. falciparum to have a major influence on the crucial virulence process of antigenic variation, a more complete understanding of which should allow us to better

Acknowledgements

This work was funded by a Burroughs Wellcome Fund New Investigator in the Pathogenesis of Infectious Diseases Award to M.T.D. and a Charles H. Hood Foundation Postdoctoral Fellowship to C.J.M. The authors thank Dr. Joseph Smith for PfEMP1 antibodies.

References (33)

  • B.A. Biggs et al.

    Subtelomeric chromosome deletions in field isolates of Plasmodium falciparum and their relationship to loss of cytoadherence in vitro

    Proc. Natl. Acad. Sci. USA

    (1989)
  • P.C. Bull et al.

    Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria

    Nat. Med.

    (1998)
  • M.F. Duffy et al.

    Transcription of multiple var genes by individual, trophozoite-stage Plasmodium falciparum cells expressing a chondroitin sulphate A binding phenotype

    Mol. Microbiol.

    (2002)
  • M.F. Duffy et al.

    Broad analysis reveals a consistent pattern of var gene transcription in Plasmodium falciparum repeatedly selected for a defined adhesion phenotype

    Mol. Microbiol.

    (2005)
  • R. Dzikowski et al.

    Mutually exclusive expression of virulence genes by malaria parasites is regulated independently of antigen production

    PLoS Pathog.

    (2006)
  • S.R. Elliott et al.

    Cross-reactive surface epitopes on chondroitin sulfate A-adherent Plasmodium falciparum-infected erythrocytes are associated with transcription of var2csa

    Infect. Immun.

    (2005)
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