Background
Plasmodium falciparum is the most virulent form of the malaria species infecting humans, and is responsible for greatest mortality associated with the disease.
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), expressed on the surface of infected red blood cells [
1,
2], plays a key role in the pathogenicity and immune evasion of
P. falciparum [
3]. PfEMP1s, encoded by the
var multigene families, comprising an N terminal segment (NTS), variable numbers of Duffy binding-like domains (DBLα-ε), one or two cysteine-rich, inter-domain regions (CIDRα-γ), a transmembrane (TM) domain, a C2 domain, and a conserved intracellular acidic terminal segment (ATS) [
4,
5]. There are about 60
var genes in the
P. falciparum clone 3D7
, although still under revision now [
6], the most supported view is that each individual parasite expresses only a single
var gene; the others are silenced in a process known as mutually exclusive expression [
7,
8]. Multiple means of genetic regulation mediate this process to protect the limited
var gene repertoire [
9-
14].
Switching among the expression of different
var genes allows parasites to avoid the effects of the acquired immune response generated by the host against PfEMP1 and, thus, to sustain a long-term infection. To date,
in vitro research on
var gene switching has been conducted either in clones or in phenotype-selected parasite lines where a dominant
var gene is expressed. The effects of cellular memory ensure that most daughter parasites will express the same
var gene [
15-
17]. This coordinates gene expression and ensures that the parasite’s repertoire of antigenic types is not rapidly exhausted.
There are three transcription states for a
var gene: active, inactive but capable of being activated, and highly silenced [
18]. The probability that a gene will be turned on or turned off is not associated with chromosomal position nor the type of promoter
per se but rather on the intrinsic properties of each gene [
16]. The initial dominant transcript determines the switch direction, while the ability to switch to particular variant types may depend on the antigenic switching history of the parasite [
18-
20]. Furthermore, switch rates have been suggested to be intrinsic and constant for the same
var gene but different for individual
var genes [
18]. Switch rates have been estimated to be approximately 2% or less per generation in long-term
in vitro cultures, but can reach 16% in hosts infected with the laboratory clone 3D7 [
21,
22]. Recent studies also showed that the switch on/off rate is associated with chromosomal position, with centrally located genes apparently more highly transcribed
in vitro than those in sub-telomeric location [
16,
19,
20,
23,
24], especially those that are short and highly diverse [
25]. As a result of these intrinsic factors, the switching patterns of
var genes are thought to be non-random. Indeed Recker
et al. [
26] revealed a highly structured switching pattern, consisting of an initially dominant transcript that switches via a set of switch-intermediates either to a new dominant transcript or back to the original. Similarly, Enderes
et al. [
20] also suggested the existence of an intrinsic
var gene transcription programme that operates independently of genetic background.
The current understanding of
var gene transcriptional regulation and switching comes from studies conducted on generations of
in vitro long cultured laboratory-adapted parasite lines. Consequently, little is known of the processes operating especially within wild parasites. One of the difficulties with investigating wild isolates is the high sequence diversity observed among
var genes. In addition, there is little genomic overlap of
var genes among different
P. falciparum isolates [
27], making it difficult to determine the sequence of individual
var genes. Despite the high diversity of
var sequences, there are still some common structures among isolates, such as the semi-conserved head structure, consisting of NTS-DBLα-CIDR1 domains, and the single DBLα domain found in nearly each
var gene [
27-
29]. Universal primers and
var group-specific primers have been generated to amplify many DBLα sequences of field isolates and detect the transcription of
var genes in clinical samples [
24,
30-
34]. However, this approach is subject to error from primer bias and the over-estimation of the frequency of minor transcripts [
35]. In addition, it is impossible to compare the transcription of each
var gene quantitatively. To resolve this problem,
var gene specific primer sets are needed (such as those recently designed for 3D7 and HB3) [
25,
36]. It may be possible to place the specific primers in the hypervariable regions of the DBLα domains [
37], while it is first necessary to obtain sequences of the
var gene repertoire of the wild isolates [
27].
In this work, var gene sequences of a wild P. falciparum isolate were obtained by Illumina Solexa sequencing and then to investigate var gene switching in clonal wild P. falciparum isolates and compare these switching patterns with those reported for long cultured laboratory-adapted parasite isolates.
Discussion
Studies on gene expression in the
P. falciparum clone 3D7 have shown that the mutually exclusive expression of virulence genes is used by the parasite to slow the depletion of the limited number of genes contained in the multicopy
var gene family [
7,
8]. However, it is still unknown whether the same strategy is used in other isolates. In this study, a wild isolate was chosen and the transcription levels of the entire
var gene repertoire are quantitative and comparable. The results showed that each of the initial clones exhibited a distinct dominant
var gene and then could even reach 90% of the total signal, which confirm the allelic exclusion of
var genes in this wild isolate.
Previous studies about the clinical
P. falciparum isolates have shown that during the
in vitro adaption of the parasites, the transcription of
var genes changed a lot. The ups A
var genes declined and upsB and upsC
var genes were activated frequently [
24,
34,
37]. The
var genes in this wild isolate could be found as both active and inactive types and most upsA
var genes were silenced and rarely activated. In particular,
var21 was more active in all the four clones and it was clearly recognized by the mutually exclusive expression system as in clone 5H. Thus like
var27 and
var29 that were preferentially expressed in the laboratory strain HB3 [
25],
var21 is the gene preferred by isolate FCYN0906.
The switching of
var genes has been extensively studied in clone 3D7. Horrocks
et al. [
18] first noted that some transitions appear to be disallowed depending on the recent variant antigen expression history of the parasite clone. Similarly, Enderes
et al. [
20] also found that the last active
var locus has an influence on subsequent
var gene activation. In this study, the four clones had different initial dominant
var genes. The following switching patterns and the subsequent activated
var genes seemed to be favoured by each clone, which was in agreement with previous studies.
Previous research has suggested that the rate at which the individual
var genes become transcriptionally activated or silenced (on/off rates) are particular to individual genes and relatively stable over time [
18,
19]. However, the results of this study indicated that the on/off rates of
var genes in the wild isolate were highly variable. As there were also some
var genes with constant on/off rates, it is possible that variable rates also exist among the laboratory strains, but Horrocks happened to investigate
var genes that exhibited constant on/off rates. Recker
et al. [
26] showed that simple differences in switch rates could not explain their switching data, and instead proposed a mechanism of biased switching to explain the very high on rates and very low off rates they observed. Their best model, however, still assumed that the switch rates and switch biases themselves were constant. Although the model is improved continuously [
25,
40], if the switch rates and switch biases of individual
var genes were intrinsic and constant, then the replicates with near-identical initial transcription profiles would be expected to have the same switching pattern, but not for all four clones. In other studies, however,
var genes have been found to switch rapidly once the first gene has been expressed, with subsequent switching occurring at a much lower rate [
22], and switching rates can be much higher in individual clinical isolates [
34] which may be influenced by the physiological states of the patients. The variability of the switch rates of
var genes could also be an explanation of the high on rates and very low off rates generally observed. The advantage of this would be that it ensures that the dominant
var gene is maintained when it is optimal for the infection, but allows it to change rapidly once it is recognized by the immune system. A recent study showed that disrupting PfSET2/RNA pol II interactions in transgenic parasites induced rapid
var gene expression switching [
41]. The ability of the wild isolate to accelerate the
var gene expression switching needs to be further studied.
Frank
et al. [
19] indicated that despite the long-term bias towards expression of
var genes with low off rates, there was no predetermined order of
var gene expression that ensures the generation of heterogeneous
var gene expression patterns. However, in their study, transcription levels were monitored at long intervals, while close attention was only paid to the particular
var genes to which each clone switched. Enderes
et al. [
20] suggests the existence of an intrinsic
var gene transcription process that occurs independently of genetic background. However, in that study, due to epigenetic memory, there were no switches of the dominant
var genes in transgenic parasites and filed parasites during the whole experiment. The same conclusion would be reached if observations had stopped at 30 generations after the division. In fact, continuing culture of the isolates revealed that the following dominant transcripts were diverse and chosen randomly, leading to different patterns. Moreover, when the following dominant
var genes happened to be identical, the two replicates like 6G-C and 6G-D still exhibited the same switching direction.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RY, DMZ and WQP conceived and designed this study. RY and DMZ performed the experiments. RY, BBC, YQZ, YLZ and SYW analysed the data. RY and WQP drafted the manuscript. All authors contributed to the interpretation of the study.