The effect of Plasmodium falciparum Sir2a histone deacetylase on clonal and longitudinal variation in expression of the var family of virulence genes
Graphical abstract
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.
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2014, Current Opinion in MicrobiologyCitation Excerpt :In contrast, nucleosomes at the active var promoter are characterised by H3K9ac and H3K4me2/3 [20] and the variant histones H2A.Z/H2B.Z [12•] (Figure 1). Histone-modifying enzymes known to participate in var regulation include the sirtuin-like HDACs PfSIR2A (required for silencing of the upsA/upsC/upsE var genes and some other subtelomeric gene families including rif [22–25] and PfSIR2B (required mainly for silencing of upsB var genes) [24]. A putative ortholog of the H3K9me-specific HKMT SU(VAR)3-9 (PfSET3/PfHKMT1) has been localised to the nuclear periphery in blood stages but functional characterisation of this factor is still missing [15,26].
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2012, Trends in ParasitologyCitation Excerpt :Although the exact mechanism(s) instructing the formation and maintenance of the P. falciparum H3K9me3/HP1-marked heterochromatin are unknown, more and more players in this machinery are being identified. Several groups have reported the involvement of the NAD+-dependent class III histone deacetylase (HDAC) silent information regulator 2 proteins, PfSir2A and PfSir2B, in heterochromatin maintenance and silencing of antigen gene families [20–22]. PfSir2A was shown to localize to the electron-dense heterochromatic regions in the nuclear periphery and colocalized with Plasmodium telomeric and subtelomeric repeats [20].
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2012, Molecular and Biochemical ParasitologyCitation Excerpt :A number of rifin multigene family members, generally located in close proximity to the affected upsA- and upsE-type var genes, were also found to be similarly affected in the absence of SIR2A expression. Subsequent analysis of independent clones of P. falciparum strain 3D7 lacking expression of SIR2A confirmed a broader spectrum of increased steady state RNA levels (∼2–3 fold up-regulation) of 8–10 var genes including upsBA- upsBC sub-type, with at least one protein product, VAR2CSA (upsE), detectable on the iRBC surface which normally requires selection for iRBC adhesion to chondroitin sulphate A to be detectable in wt parasites) [84]. Similar var sets were expressed by the different parasites lacking SIR2A expression.
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2012, Trends in ParasitologyCitation Excerpt :In Plasmodium there are two Sir2 orthologs, PfSir2A and PfSir2B [47]; these work in concert to regulate var silencing. Plasmodium parasites that lack PfSir2A and B are unable to silence undesired var genes effectively, and transcript levels of var genes are generally elevated [47,48]. Although regulation of var gene expression is seriously compromised, parasites are still able to switch var genes, suggesting that other regulatory mechanisms exist and work in concert with Sir2 [47].