Differential, Positional-Dependent Transcriptional Response of Antigenic Variation (var) Genes to Biological Stress in Plasmodium falciparum

1% of the genes of the human malaria causing agent Plasmodium falciparum belong to the heterogeneous var gene family which encodes P. falciparum erythrocyte membrane protein 1 (PFEMP1). This protein mediates part of the pathogenesis of the disease by causing adherence of infected erythrocytes (IE) to the host endothelium. At any given time, only one copy of the family is expressed on the IE surface. The cues which regulate the allelic exclusion of these genes are not known. We show the existence of a differential expression pattern of these genes upon exposure to biological stress in relation to their positional placement on the chromosome – expression of centrally located var genes is induced while sub-telomeric copies of the family are repressed - this phenomenon orchestrated by the histone deacetylase pfsir2. Moreover, stress was found to cause a switch in the pattern of the expressed var genes thus acting as a regulatory cue. By using pharmacological compounds which putatively affect pfsir2 activity, distinct changes of var gene expression patterns were achieved which may have therapeutic ramifications. As disease severity is partly associated with expression of particular var gene subtypes, manipulation of the IE environment may serve as a mechanism to direct transcription towards less virulent genes

Protein Kinase C θ Deficiency Increases Resistance of C57BL/6J Mice to Plasmodium berghei Infection-Induced Cerebral Malaria▿

Protein kinase C θ (PKCθ) functions as a core component of the immunological synapse and serves as a key protein in the integrated T-cell antigen receptor (TCR)/CD28-induced signaling cascade leading to T-cell activation. However, the involvement of PKCθ in host-mediated immune responses to pathogens has not been thoroughly investigated. We tested the consequences of PKCθ ablation on the host response to infection by Plasmodium berghei ANKA (PbA). We found that both PKCθ+/+ and PKCθ−/− C57BL/6J mice are susceptible to infection with PbA. However, despite a similar parasite burden, PKCθ+/+ mice had an earlier onset of neurological signs, characteristics of experimental cerebral malaria (ECM), resulting in an earlier death. These mice suffered from an early and pronounced splenomegaly with a concomitant increase in the total number of CD4+ splenic T cells. In contrast, a large proportion of PbA-infected PKCθ−/− mice overcame the acute phase characterized by neurological symptoms and survived longer than PKCθ+/+ mice. The partial resistance of PKCθ−/− mice to ECM was associated with an impaired production of Th1-type cytokines, including gamma interferon and tumor necrosis factor alpha/lymphotoxin-α, which are known to exacerbate symptoms leading to ECM. In addition, PbA infection-induced LFA-1 expression in CD8+ T cells was suppressed in PKCθ-deficient T cells, suggesting a diminished ability to adhere to endothelial cells and sequester in brain microvasculature, which may explain the decrease in neurological symptoms. These data implicate PKCθ in CD4+ Th1+ and CD8+ T-cell-mediated immune responses during PbA infection that contribute to the development of ECM

The relative change in expression of the <i>ups A, B</i>, and <i>C var</i> subtypes upon exposure to stress in NF-54 parasites.

<p>Synchronized ring stage parasites were exposed to a 4 hour pulse of stress inducing conditions (either 10 µM of <i>tert</i>-butylhydroperoxide or cultivation in the presence of 50% of the normal glucose concentration). cDNA prepared from RNA isolated from these parasites was analyzed utilizing qPCR with <i>ups</i>-specific primers. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006991#s3" target="_blank">Results</a> are expressed as fold change in comparison to an unexposed control (Relative Quantification, RQ). Error bars represent mean ± SEM.</p

Analysis of expression of exported non-<i>var</i> genes upon exposure to 10 µM tBHP in NF-54 parasites.

<p>Stress induction, cDNA production and qPCR analysis were performed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006991#pone-0006991-g001" target="_blank">Fig. 1</a>. Error bars represent mean of triplicates ± SEM.</p

Fold change of <i>individual var</i> genes upon exposure to oxidative stress plotted against expression levels.

<p>The data was plotted as logarithmic values of RQ versus the base-line logarithmic level of expression of each of the <i>var</i> genes. Marks above the horizontal line represent genes whose expression is induced and those below the line represent genes whose expression is reduced. Panels A, B and C show the breakdown to the respective <i>ups</i> subtypes.</p

Analysis of expression of <i>var</i> and non-<i>var</i> exported genes upon exposure to oxidative stress in genetically modified parasites.

<p>A. The relative change in expression of the <i>ups A, B, and C var</i> subtypes upon exposure to 10 µM <i>t</i>BHP in Δ<i>pfsir2</i> parasites in relation to unexposed Δ<i>pfsir2</i> parasites. B. Analysis of expression of exported non-<i>var</i> genes upon exposure to oxidative stress in Δ<i>pfsir2</i> parasites. C. Analysis of expression of h<i>dhfr</i> – the gene under control of an <i>upsC</i> element in the 3D7/upsC transgenic line – upon exposure to 10 µM tBHP. Stress induction, cDNA production and qPCR analysis were performed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006991#pone-0006991-g001" target="_blank">Fig. 1</a>. Error bars represent mean of triplicates ± SEM.</p

The effect of <i>pfsir2</i> inactivation on <i>var</i> gene expression.

<p>A. Change of expression of <i>ups</i> subtypes in NAC exposed wild-type culture in relation to an unexposed culture. Synchronized ring stage parasites were exposed to 5 µM of NAC for 8 hours. B. Relative expression of <i>ups</i> subtypes in the <i>pfsir2</i> knock-out line in relation to the wild-type line under normal culturing conditions. Note that <i>y</i> axis is in logarithmic scale. cDNA production and qPCR analysis were performed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006991#pone-0006991-g001" target="_blank">Fig. 1</a>.</p

The effect of resveratrol on <i>var</i> gene expression.

<p>A. Analysis of expression of <i>var</i> subtypes upon exposure to RV in wild-type parasites. Synchronized ring stage parasites were exposed for 8 hours to various concentrations of RV. B. Analysis of expression of <i>var</i> subtypes upon exposure to 10 µM RV in wild-type and Δpfsir2 parasites. Synchronized ring stage parasites were exposed for 8 hours to 10 µM RV. cDNA production and qPCR analysis were performed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006991#pone-0006991-g001" target="_blank">Fig. 1</a>.</p