A Unique Virulence Gene Occupies a Principal Position in Immune Evasion by the Malaria Parasite <i>Plasmodium falciparum</i>

<div><p>Mutually exclusive gene expression, whereby only one member of a multi-gene family is selected for activation, is used by the malaria parasite <i>Plasmodium falciparum</i> to escape the human immune system and perpetuate long-term, chronic infections. A family of genes called <i>var</i> encodes the chief antigenic and virulence determinant of <i>P</i>. <i>falciparum</i> malaria. <i>var</i> genes are transcribed in a mutually exclusive manner, with switching between active genes resulting in antigenic variation. While recent work has shed considerable light on the epigenetic basis for <i>var</i> gene activation and silencing, how switching is controlled remains a mystery. In particular, switching seems not to be random, but instead appears to be coordinated to result in timely activation of individual genes leading to sequential waves of antigenically distinct parasite populations. The molecular basis for this apparent coordination is unknown. Here we show that <i>var2csa</i>, an unusual and highly conserved <i>var</i> gene, occupies a unique position within the <i>var</i> gene switching hierarchy. Induction of switching through the destabilization of <i>var</i> specific chromatin using both genetic and chemical methods repeatedly led to the rapid and exclusive activation of <i>var2csa</i>. Additional experiments demonstrated that these represent “true” switching events and not simply de-silencing of the <i>var2csa</i> promoter, and that activation is limited to the unique locus on chromosome 12. Combined with translational repression of <i>var2csa</i> transcripts, frequent “default” switching to this locus and detection of <i>var2csa</i> untranslated transcripts in non-pregnant individuals, these data suggest that <i>var2csa</i> could play a central role in coordinating switching, fulfilling a prediction made by mathematical models derived from population switching patterns. These studies provide the first insights into the mechanisms by which <i>var</i> gene switching is coordinated as well as an example of how a pharmacological agent can disrupt antigenic variation in <i>Plasmodium falciparum</i>.</p></div

Effect of over-expression of PfSET2 dom-neg on <i>var</i> gene expression.

<p>(A) Schematic diagram of the domain structure of PfSET2. The top image shows the conserved domains identified using SMART (Simple Modular Architecture Research Tool, <a href="http://smart.embl-heidelberg.de" target="_blank">http://smart.embl-heidelberg.de</a>). PHD domains are shown as blue polygons while the methyltransferase domain (labeled SET) is shown in red. The SET2 Rpb1 Interacting Region (SRIR) is shown as a white triangle. (B) <i>var</i> gene expression is shown as a pie-chart, with each slice of the pie representing the fraction of the total <i>var</i> mRNA pool transcribed from each <i>var</i> gene. The left chart shows the <i>var</i> gene expression pattern in a population over-expressing firefly luciferase. This is unchanged from the untransfected population and the annotation number of the dominant gene is shown in white text. The chart on the right shows the <i>var</i> gene expression pattern in a population after over-expression of the PfSET2 dominant negative construct for 6 weeks. <i>var2csa</i> (PF3D7_1200600, shown in green) has become the dominant transcript. Individual copy number values for each transcript are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005234#pgen.1005234.s001" target="_blank">S1 Fig</a>.</p

Primer sets used in real-time PCR assays specifically to amplify <i>var2csa</i> from HB3.

<p>The numbers in parentheses are the original primer set # from Salanti et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005234#pgen.1005234.ref043" target="_blank">43</a>].</p><p>Primer sets used in real-time PCR assays specifically to amplify <i>var2csa</i> from HB3.</p

Induction of <i>var2csa</i> in HB3 specifically activates the locus on chromosome 12.

<p>The nucleotide sequences of the two copies of <i>var2csa</i> found within the genome of HB3 are shown. Of note are the polymorphisms (surrounded by red boxes) that easily distinguish the gene found on chromosome 1 from that found on chromosome 12. In particular, two highly polymorphic regions are labeled. The location and sequence of PCR primers (red text) that amplify across these polymorphic regions are shown. Sequencing of this PCR product after induction of <i>var2csa</i> expression by chaetocin treatment or over-expression of PfSET2 dom-neg detected only the locus on chromosome 12.</p

Induction of <i>var2csa</i> expression in response to PfSET2 dom-neg over-expression in a heterogenous population expressing many <i>var</i> genes.

<p>The original population expressed many <i>var</i> genes and there was no single gene that was dominant (left panel). After selection with 10 μg/ml blastidicin leading to over-expression of the PfSET2 dom-neg construct for 4 weeks, <i>var</i> gene expression began to shift to <i>var2csa</i>, resulting in this gene being the most highly expressed gene in the population (middle panel). Similar over-expression of luciferase did not lead to expression of <i>var2csa</i> (right panel), while a different <i>var</i> gene became somewhat highly expressed, indicating that switching can occur. Individual copy number values for each transcript are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005234#pgen.1005234.s005" target="_blank">S5 Fig</a>.</p

Induction of <i>var2csa</i> expression by treatment with a histone methyltransferase inhibitor.

<p>(A) The A3 subclone of the NF54 line of <i>P</i>. <i>falciparum</i> was treated for 2 weeks with sub-IC₅₀ concentrations of various compounds known to inhibit histone modifiers in high eukaryotes. After 2 weeks of exposure, the <i>var</i> gene expression pattern was determined for each population and shown as a pie chart. In all cases the <i>var</i> gene that was originally dominant in this population (PF3D7_0617400, shown in red) remained dominant, however the population exposed to chaetocin displayed a significant proportion that had switched to <i>var2csa</i> (green). (B) Two subclones of NF54 (A3 and a mixed population) were cultured in the presence of chaetocin and their <i>var</i> gene expression patterns determined after varying lengths of time. In both cases, <i>var2csa</i> became the dominant <i>var</i> transcript detectable in the population. Individual copy number values for each transcript are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005234#pgen.1005234.s006" target="_blank">S6 Fig</a>.</p

Calculated IC₅₀ values of BIX-01294, chaetocin, UNC0321, Garcinol, trichostatin A (TSA), and chloroquine (CQ) treated NF54 parasites.

<p>Calculated IC₅₀ values of BIX-01294, chaetocin, UNC0321, Garcinol, trichostatin A (TSA), and chloroquine (CQ) treated NF54 parasites.</p

<i>var2csa</i> expression in response to chaetocin treatment is a true switching event and maintains mutually exclusive expression.

<p>The transgenic parasite line DC-J contains a modified <i>var</i> gene that encodes the drug resistance marker <i>blasticidin-S-deaminase</i> (<i>bsd</i>). Thus upon selection with blasticidin, these parasites are forced to express <i>bsd</i> and consequently silence the rest of the <i>var</i> gene family. DC-J parasites were grown under different conditions and the <i>var</i> gene expression profile determined by Q-RT-PCR and displayed as pie charts. (A) Growth of DC-J in the presence of blasticidin results in stable expression of <i>bsd</i> which remains the dominant <i>var</i> gene transcript detectable. (B) Growth of DC-J in the absence of blasticidin but in the presence of chaetocin results in induction of <i>var2csa</i> expression. <i>var2csa</i> has become the dominant <i>var</i> gene expressed by this population after 6 weeks of exposure to chaetocin. (C) Growth of DC-J parasites in the absence of blasticidin selection results in switching to several other <i>var</i> genes, including <i>var2csa</i>. After 6 weeks of growth in the absence of selection, both <i>var2csa</i> and a third <i>var</i> gene (PF3D7_071200, shown in purple) have become prominently expressed. (D) DC-J grown in the presence of both blasticidin and chaetocin results in stable expression of <i>bsd</i> for 4 weeks, after which the population fails to grow and eventually dies out. (E) Parasite growth charts for populations treated with blasticidin, chaetocin, blasticidin + chaetocin or untreated. Parasitemias were calculated daily by blood smear and three weeks are shown. Populations were allowed to replicate until they reached ~3% parasitemia, at which time the population density was reduced to 0.5% (red arrow) and the culture allowed to continue. By the end of week 5, the population exposed to both blasticidin and chaetocin failed to replicate efficiently (blue arrow) and the population died out by week 6. Individual copy number values for each transcript are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005234#pgen.1005234.s007" target="_blank">S7</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005234#pgen.1005234.s011" target="_blank">S11</a> Figs.</p

Induction of <i>var2csa</i> in the parasite line HB3.

<p>The expression level of <i>var2csa</i> in HB3 was determined by Q-RT-PCR and displayed as relative copy number as compared to seryl-tRNA synthetase. Three separate PCR primer pairs (shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005234#pgen.1005234.t002" target="_blank">Table 2</a>) were used that anneal at different points within the <i>var2csa</i> coding region. (A) mRNA expression levels of <i>var2csa</i> are shown before and after 3 weeks of exposure to over-expression of the PfSET2 dom-neg expression construct. B. mRNA expression levels of <i>var2csa</i> are shown before and after 3 weeks of exposure to chaetocin.</p

Dynamics of <i>var2csa</i> activation in response to PfSET2 dom-neg over-expression.

<p>(A) <i>var</i> gene expression profiles were determined at 2 week intervals after induction of PfSET2 dom-neg over-expression. The total <i>var</i> gene transcript pool is represented as a pie chart, with each slice of the pie representing the fraction of the total <i>var</i> mRNA pool transcribed from each <i>var</i> gene. In this population of parasites, the <i>var</i> gene PF3D7_0617400 (shown in red) was initially dominant, however over time it is replaced by <i>var2csa</i> (shown in green). (B) The <i>var</i> gene pattern in response to two different levels of PfSET2 dom-neg over-expression. Increasing the selection pressure from 10 μg/ml to 20 μg/ml results in even greater dominance of the <i>var2csa</i> message as a portion of the total <i>var</i> mRNA pool. (C) In response to luciferase over-expression, the <i>var</i> gene pattern does not change substantially over 6 months, although activation of <i>var2csa</i> is detectable. Individual copy number values for each transcript are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005234#pgen.1005234.s002" target="_blank">S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005234#pgen.1005234.s003" target="_blank">S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005234#pgen.1005234.s004" target="_blank">S4</a> Figs.</p