Background
Malaria caused by
Plasmodium falciparum is a global health problem and the majority of severe clinical cases are found among young children and pregnant women.
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a group of variant surface antigens (VSA) expressed on the surface of infected erythrocytes, thus enabling the parasite to sequester in various endothelial tissues of the host and avoid clearance in the spleen [
1,
2]. Several receptors have been identified as mediators of the sequestration, e.g. CD36 [
3], thrombospondin [
4] and ICAM-1, associated with cerebral malaria [
5] and chondroitin sulfate A (CSA), the main receptor in the placenta and associated with pregnancy-associated malaria (PAM) [
6]. PfEMP1 are encoded by the highly diverse
var gene family and each parasite genome harbours ~60 of these genes [
7‐
9]. The
var gene repertoire of the 3D7 parasite [
10] can be sub-grouped into three major groups (A, B and C), two intermediate groups (B/A and B/C), and the
var1csa and
var2csa sub-families based on promoter regions and chromosomal locations [
11,
12].
VSA appear to be the main target of antibodies conferring protective immunity against malaria [
13‐
17]. However, antigenic diversity and
var gene switching have made unambiguous identification of the PfEMP1 molecules that are responsible for specific adhesion phenotypes difficult. A quantitative real-time PCR (Q-RT PCR) method was developed to measure the amount of mRNA being transcribed from 59 different NF54/3D7
var genes simultaneously [
18]. Using this approach, a particular PfEMP1, VAR2CSA, was identified as the parasite ligand likely to be responsible for CSA binding in the placenta [
18,
19]. In another study using the same method, the group A
var genes of 3D7 were associated with severe malaria [
20,
21]. However, correlating expression of
var genes to the phenotype of a parasite population can yield equivocal results, and studies on
var gene transcription have given conflicting results regarding both the number of
var genes being transcribed and the size of these transcripts. Two reverse transcription (RT)-PCR studies suggested that most or all
var genes are transcribed in ring-stage parasites, whereas in mature trophozoites all but one
var gene was silenced. Only the
var gene actively transcribed in late stages would then be translated into protein to confer the phenotype [
22,
23]. Other studies have proposed that multiple full-length
var transcripts are present in ring-stage parasites but not in mature stages, with the exception of the
var1csa gene which was detectable in both trophozoites and later stages [
24,
25]. A third group of studies detected multiple full-length transcripts in ring-stage and mature trophozoite parasites in both clonal populations [
26] and in single cells [
27].
To investigate var gene mRNA levels in general, and var2csa levels in particular, their abundance and quality were determined in two phenotypically different NF54-derived lines using Q-RT PCR throughout the intra-erythrocytic developmental cycle.
Discussion
Transcript levels of
var genes were analyzed throughout asexual parasite development using an improved version of the Q-RT PCR method originally published by Salanti
et al. [
18]. Primer pair coverage was extended to optimize quantitative measurement of mRNA transcribed from 59 full-length
var genes and two pseudogenes in the NF54/3D7 genome. Two phenotypically distinct NF54-derived lines, NF54 (unselected) and NF54VAR2CSA (selected for VAR2CSA expression) showed abundance peaks of dominant and subdominant
var transcripts at ring stage, although the relative abundance of the dominant transcript in each population was higher in ring stage and early trophozoites than in schizonts. This can be explained by the increased uncertainty of measurement when transcript abundance is very low.
Unselected NF54 parasites are clearly a mixture of subpopulations expressing different
var genes (Figure
1). However, after repeated selection, the
var2csa gene has become the overwhelmingly dominant transcript and constitutes >98% of all detected
var transcripts. This pattern of exclusive expression of the
var2csa gene (Figure
1C) supports the general model of mutually exclusive transcription of
var genes. Such a general model was proposed after experiments showed that exclusive transcription of activated group B and C 5' UTR promoters occurred following transfection [
33,
34]. It is likely that a significant proportion of the unselected ring-stage NF54 is exclusively expressing the dominant group B
var transcript. However, this is not as apparent in this line as in the NF54VAR2CSA due to the heterogeneity of this unselected population. While it remains to be proven that
var transcription in circulating parasites in the bloodstream can always be correlated to the binding phenotype of the corresponding trophozoite population, this study supports such a conclusion. Another recent study has shown that the genotypes of the circulating ring-stage parasites represent the types of the mature parasites sequestering in most tissues [
35].
Improved quantification of transcript abundances of each
var gene obtained by Q-RT PCR and use of cross-intron primers also support a revision of the view that
var transcripts are exclusively restricted to the ring stage and that there is promiscuous transcription of many
var genes in ring-stage parasites [
24,
36]. New data, reported here and in other recent publications [
37], indicate that the so-called 'loose' transcription of several
var transcripts per infected erythrocyte results from cross-hybridization artefacts and the background of gene expression from minority populations in cultures which are not expressing the dominant, selected
var gene.
Truncated or unspliced transcripts in parasites have also been reported [
8,
38]. In this study, cross-intron primers for three different
var genes,
var2csa (PFL0030c), a group B gene (PFL0935c) and a group C gene (PFD0615c) all showed splicing of exon I to exon II in both the unselected and VAR2CSA-selected NF54 parasites throughout the whole cycle, indicating that these
var genes are normally expressed as correctly spliced transcripts (Figure
4). The fact that the transcription levels of the four different
var2csa-specific primers were tightly correlated further indicates that the transcripts are not only correctly spliced but also probably full-length at all stages. This is contrary to a conclusion drawn in an earlier study in which a difference between the levels of transcription detected by cross-intron primers and exon I primers of some
var genes using Q-RT PCR were reported [
38]. This discrepancy could be due to the 'amplification bias' of the cross-intron primers, and the difficulties of designing unique primers targeting the exon II. However it is worth noting that promiscuous transcription in any form is incompatible with transfection experiments showing the exclusive expression of a single
var gene regulated at a pre-transcriptional level [
33,
34,
39,
40].
It has also been suggested that there is a differential repression of the various
var gene groups based on their promoters [
41]. The group B genes (subtelomeric) were shown to be transcribed 0–10 hours after invasion, and turned off when the parasites reached later stages, whereas the group C (chromosome-central) transcripts were still detected 4–8 hours later than the subtelomeric genes. This differential transcription pattern was not seen here. All
var genes within group A, B or C in the unselected NF54 population showed the same abundance profile regardless of promoter.
Both
var1csa and
var2csa have unique promoters (group D and E respectively, [
11,
12], and both these genes showed distinctive expression profiles.
var1csa transcription remains anomalous and poorly understood.
var1csa was constitutively transcribed in the unselected NF54 line, a result reported previously for several
P. falciparum isolates, regardless of their receptor binding phenotype [
25,
38,
42]. However,
var1csa transcription was not detected in any stages in NF54VAR2CSA. This observation is most simply explained by a
var1csa-expressing subpopulation of NF54 having been removed by VAR2CSA selection. This interpretation further entails that only one
var gene is transcribed per infected erythrocyte and the
var1csa gene is restrained from transcription by the silencing mechanisms operating on all
var genes that is imposed when another
var gene, in this case
var2csa, is expressed.
The essentially exclusive expression of a single
var gene that is observed in the NF54VAR2CSA parasites, where
var2csa constitutes 98% of total
var gene abundance, has previously only been observed in
P. falciparum cultures transfected with drug-selectable marker genes [
33,
34]. In these experiments, transgenic parasite lines use a
var promoter to drive expression of a drug resistance gene rather than a PfEMP1 protein. Drug selection followed by cloning and analysis of resistant lines indicates that the transfected recombinant promoter is activated in these lines and that all other
var promoters are silenced. There is disagreement as to whether a single active
var promoter is sufficient to silence all other promoters [
34] or whether the system of allelic exclusion requires both the 5'
var promoter and a promoter located downstream in the
var gene intron to interact in some way to silence transcription [
43,
44].
Transcription of
var1csa in the unselected NF54 presumably results from a subpopulation of parasites in which other
var genes are silenced. The relatively constitutive transcription of
var1csa may result from a dysfunction of the transcriptional regulation related to the cell cycle. As
var1csa has an upstream promoter but is truncated at exon I, it can be hypothesized that its 5' UTR promoter, as previously demonstrated [
34] is needed for exclusive transcription of a single
var gene whereas the intron/exon II/3' UTR is needed for the observed wave shaped transcriptional regulation throughout the cell cycle. Yet, the abundance profile of
var1csa during the complete intra-erythrocytic cycle in other isolates than 3D7 and NF54 remains unclear, and more studies are needed to elucidate the role of this
var gene. Furthermore, it is not known whether the apparent lack of other pseudogene-transcribing subpopulations, here represented by PFF0005c, can be related to the difference in the 5' UTR promoter.
var2csa had a pattern of changes in mRNA abundance similar to that of the other
var genes being transcribed in the unselected NF54 line. However, the abundance profile indicated that its transcription peaks earlier in the ring stage than the other
var genes. Since the timing of the cycle of both lines were tightly correlated according to the Giemsa stain assessment, this observation is probably not due to major differences in synchronization. The protein expression of VAR2CSA did not seem to diverge from earlier reports showing that PfEMP1 molecules appear to be present on the infected erythrocyte membrane around 16–18 hours after invasion [
45‐
47]. As expected, the recognition pattern of NF54VAR2CSA by rabbit antibodies raised against recombinant-produced DBL5ε of VAR2CSA was correlated with the recognition of the immune sera of malaria-exposed multi-gravidae women from a malaria endemic area.
Authors' contributions
MD carried out the RNA extractions, cDNA synthesis and all the Q-RT PCR experiments, analysed the data and wrote the manuscript. TL participated in the design of the study, helped with primer design, the Q-RT PCR and to draft the manuscript. AS contributed to the study design and helped to draft the manuscript. LH, DEA and TGT helped to finalise the manuscript. MAN participated in the study design, carried out all the parasite work and flow cytometry experiments and helped to finalise the manuscript. All authors read and approved the final manuscript.