An upstream open reading frame controls translation of var2csa, a gene implicated in placental malaria

Abstract

Malaria, caused by the parasite Plasmodium falciparum, is responsible for substantial morbidity, mortality and economic losses in tropical regions of the world. Pregnant women are exceptionally vulnerable to severe consequences of the infection, due to the specific adhesion of parasite-infected erythrocytes in the placenta. This adhesion is mediated by a unique variant of PfEMP1, a parasite encoded, hyper-variable antigen placed on the surface of infected cells. This variant, called VAR2CSA, binds to chondroitin sulfate A on syncytiotrophoblasts in the intervillous space of placentas. VAR2CSA appears to only be expressed in the presence of a placenta, suggesting that its expression is actively repressed in men, children or non-pregnant women; however, the mechanism of repression is not understood. Using cultured parasite lines and reporter gene constructs, we show that the gene encoding VAR2CSA contains a small upstream open reading frame that acts to repress translation of the resulting mRNA, revealing a novel form of gene regulation in malaria parasites. The mechanism underlying this translational repression is reversible, allowing high levels of protein translation upon selection, thus potentially enabling parasites to upregulate expression of this variant antigen in the presence of the appropriate host tissue.

Figure 1. The var2csa uORF is a repressive element.

A. Constructs used in transient transfections. Constructs drive expression of either the firefly luciferase reporter gene, Renilla luciferase (in the case of control plasmid HRH) or both (in the case of V2RLH). VLH contains the un-modified promoter and upstream regulatory region from var7b. V2LH contains the un-modified promoter and upstream regulatory region from var2csa. V2LHm is identical to V2LH, except a single base pair mutation has altered the start codon of the upstream open reading (uORF) from ATG to ACG. In V2BLH, the uORF has been replaced with the bsd coding region while in V2RLH the uORF has been replaced by the coding region for Renilla luciferase. In V2ΔLH the entire region upstream of the uORF, including the transcription start site, has been deleted. In SURF the uORF has been shortened to 48 bp by the introduction of a premature stop codon, while in uORFL it has been lengthened to 450 bp by eliminating the endogenous stop codon. ICL contains the intact upstream regulatory region, including the uORF, however the intercistronic region has been duplicated. B. Levels of firefly luciferase expression from each construct shown in A. C. Levels of Renilla luciferase expression. V2RLH supports translation of robust levels of Renilla luciferase indicating that the uORF can be translated. The plasmid HRH, containing the strong hrp3 promoter, was employed as a positive control for Renilla luciferase expression, while V2LH, which does not contain the Renilla luciferase gene, was a negative control. All assays were done simultaneously in triplicate.

Figure 2. Translational repression in stably transformed parasites.

Parasites were stably transfected with either pVLH or pV2BLH (A). V2BLH contains the bsd selectable marker and VLH contains hdhfr for maintaining the episomes in transfected parasites (not shown). Integration into the genome occurred spontaneously and was selected for by alternate growth with or without drug. luciferase mRNA levels were determined using Q-RT-PCR (B) with three different primer pairs (1–3), whose corresponding location on the luciferase ORF is shown in (A). Levels of protein expression were assayed by measuring luciferase activity (C).

Figure 3. Evidence for translational regulation of var2csa in cultured, wildtype parasites.

A. The parasite population NF54 VAR2CSA was selected using anti-VAR2CSA rabbit antibodies. Flow analysis indicates that a majority of the population displays VAR2CSA on the cell surface (red) with a MFI value of 14.2 compared to the background (black) with a MFI value of 5.2 (arbitrary units). Flow analysis of NF54-239 indicates that the majority of the population displays low levels of VAR2CSA on the cell surface with a MFI value of 5.0 compared to the background value of 3.6 (arbitrary units). B. Selection for VAR2CSA expression results in recognition by antibodies predominantly from female sera whereas the NF54-239 line is poorly recognized. C. var transcript analysis shows that var2csa is the dominant transcript in both the NF54 VAR2CSA (Black) and Nf54-239 (white) parasite lines.

Figure 4. Selection for reversal of translational repression by the uORF of var2csa.

A. Constructs used for selection of parasites that translate the second open reading frame of the var2csa gene. The drug selectable marker blasticidin-S-deaminase (BSD) was used to select parasites translating the downstream cistron (equivalent to exon I of var2csa). All three constructs also contain the hdhfr selectable marker for maintaining the episomes in transfected parasites prior to blasticidin selection (not shown). B. A drug resistant population appeared after six days suggesting that a subset of parasites is capable of translating the second ORF. Replacing the uORF with Renilla luciferase (V2RB) led to severely retarded growth in presence of blasticidin. Analysis of episomes recovered from these parasites indicated that they had undergone recombination (not shown).

Figure 5. Growth rates of parasites translating different ORFs.

Constructs are shown in Figure 4. A. Parasites that have been selected for translation of the second open reading frame encoding blasticidin-S-deaminase (V2B+blasticidin) grow at the same rate as parasites not under blasticidin selection (V2B−blasticidin) or those in which translation of the uORF has been disrupted (V2mB+blasticidin). Parasites in which the uORF has been replaced with the coding region of Renilla luciferase (V2RB+blasticidin) grow at a slower rate. B. Parasites actively translating the second ORF encoding blasticidin-S-deaminase continue to display resistance to the drug if grown without selection pressure very briefly. C. When parasites are grown in the absence of blasticidin in the media for 21 days, they rapidly begin to repress translation of the second ORF and revert to the drug sensitive phenotype as demonstrated by slower growth when placed back under drug pressure (V2B+blasticidin). The histogram at the right shows that reversion to drug sensitivity is not due to a switch in promoter activity. Parasites grown without blasticidin pressure (V2B−BSD) for three weeks continue to express bsd mRNA at levels equal to or greater than those grown continuously under blasticidin pressure (V2B+BSD). bsd mRNA levels are shown as copy number normalized to seryl-tRNA synthetase.