Promoter regions of Plasmodium vivax are poorly or not recognized by Plasmodium falciparum

Plasmid pE(A)b.luc.^D harbors the intergenic region between the two Plasmodium berghei ef-1α genes, the firefly luciferase gene and the P. berghei dhfr 3' UTR and has been previously described [25]. Excepting for plasmid pPv-msp1, all other plasmids were constructed by digesting pE(A)b.luc.^D with Nde I and/or Hind III, making it blunt and replacing the P. berghei ef-1α intergenic region by the intergenic regions of the P. vivax dhfr (0.733 kbp), msp1 (1.3 kb), vir3 (1.8 kb), vir24 (1.3 kb), and ef-1α (1.4 kp). All intergenic regions from P. vivax were amplified with specific oligonucleotides (Table 1), subcloned into pGEM-T (Promega) or pCR4-TOPO (Invitrogen) and after being released from the vectors with appropriate restriction enzymes all inserts were cloned into pE(A)b.luc.^D. Plasmid pPv-msp1 was constructed by replacing the hrp3 promoter region from plasmid pHLH (kindly donated by Dr. Thomas Wellems) with 1,326 bp of the intergenic region from the P. vivax msp1 gene from the Sal-I strain. The P. falciparum ef-1α intergenic region was PCR amplified, cloned in both orientations in pPCR4-TOPO, digested with Not I, blunted, digested with Spe I and cloned in p0,5A(tetO)5' (Nde I/blunt/Nhe I), creating pPf-EF(A) and pPf-EF(B). p0,5A(tetO)5' is a plasmid derived from pE(A)b.luc.^D with approximately half of the Pb ef-1α promoter region. Plasmids with one or two copies of the EF motif were made by annealing the complementary oligonucleotides EFM01 and EFM01-2 (Table 1), cloning it in pPv-EF(B) (Hind III/blunt) to create plasmid pPv-EF(B)-HEF and then cloning it in pPv-EF(B)-HEF (Nde I/blunt) to create pPv-EF(B)-HNEF. Plasmids with mutated versions of the EF motif were made by site directed mutagenesis of pE(A)b.luc.^D. For all constructs, the thimidines (T) on positions 4 and 6 were replaced by cytosines (C). pE(A)b.luc.^D was hyper methilated, amplified by inverse PCR with oligonucletides mutPbEF1cc-F and mutPbEF1cc-R and transformed into DH5α-T1 cells (Invitrogen) to created pPb-1cc. Plasmid pPb-2cc containing the second EF motif mutated was created following the same methodology and using pE(A)b.luc.^D as a template and oligonucleotides mutPbEF2cc-F and mutPbEF2cc-R (Table 1). To make plasmid pPv-EF(B)-Pb0,2A, the P. vivax ef-1α intergenic region was cloned in pCR4-TOPO, digested with Not I, made blunt, digested with Spe I and cloned into pE(A)b.luc.^D (Hind III/blunt/Spe I). Plasmid pPv-EF(B) was created by digesting pE(A)b.luc.^D with Nde I, making it blunt and digesting it with Kpn I, releasing the luc-dhfr 3' UTR cassette, which was cloned in pPv-EF(B)-Pb0,2A (Spe 1/blunt/Kpn I). pPv-EF(B)-Pb0,2A contains a minimal promoter of circa 0.2 kb from P. berghei. Authenticity of all plasmids was confirmed by DNA sequencing.

The P. falciparum 3D7 clone was continuously cultured in vitro [26] and transiently transfected as described elsewhere [27, 28]. Briefly, 100 μg of each plasmid were used to electroporate 600 μl of uninfected red blood cells and this mix was added to about 10⁷ parasites, which were kept in culture. Parasites were harvested 4 days after eletroporation and luciferase assays performed according to the manufacturer's instructions (Promega).

Erythrocytes were harvested by saponin lysis, parasites washed twice in PBS and pellets ressuspended in 50 μl of 1× lysis buffer (Promega). To 20 μl of lysed parasites, 100 μl of luciferase essay reagent (Promega) were added and luciferase activity measured in the Lumat LB 9507 luminometer (EG and G Berthold) for 45 seconds. Reporter activity values represent the mean of at least three independent experiments done with two or three different DNA preparations. They are expressed in relation to the values of a reference control plasmid indicated in each experiment. Student's t test was used to determine the statistical significance of the data (p value ≤ 0.005).

Sequences of the intergenic region between the two ef-1α genes of six Plasmodium species (Plasmodium knowlesi, Plasmodium reichenowi, Plasmodium yoelii, P. falciparum, P. berghei and P. vivax) were retrieved from PlasmoDB [29]. The Gibbs matrices algorithm [30] of the Regulatory Sequence Analysis tools [31] was used to search conserved motifs in these intergenic regions. This algorithm finds optimized local alignments in related sequences in order to detect short conserved regions or motifs that may not be in the same positions. The matrix length was set from 4 to 20, which allowed the detection of very short and also longer and more complex conserved sequences. Matrices generated, which represent the nucleotide conservation in each position of the motifs, were used to search for the motifs positions, copy number and conservation using the Patser algorithm [32]. The Patser algorithm was applied to each of the matrices generated from the Gibbs analysis and sequences of the intergenic regions. The Alphabet parameter was set to a:t 0.4 c:g 0.1. Sequences of the most conserved motifs were input in the WebLogo program [33] to generate the visual representation of the consensus sequence, which were then compared to the sequence of the motif in the P. vivax intergenic region.

Initially, luciferase reporter plasmids containing 0.733 kB of the 5' upstream region of the P. vivax dhfr-ts gene with circa 49% AT-content (pPv-dhfr), 1.326 kbp of the upstream region of the P. vivax msp 1 gene with circa 59% AT-content (pPv-msp1), and 1.818 kbp of the upstream region of vir 3 gene with circa 81% AT-content (pPv-vir3), were constructed (Figure 1A). Surprisingly, transient transfections of these recombinant plasmids into P. falciparum produced luciferase activity values similar to the background where the luminescence of the substrate alone or of the plasmid p-.luc^D, which has luciferase, P. berghei 3'UTR, but no promoter, was measured and used as negative controls. This contrasts with parasites transfected with plasmid pE(A)b.luc.^D which contains a P. berghei promoter and had been previously shown to give high luciferase values in transient transfections assays in P. berghei and P. falciparum [23, 25]. This plasmid was used as reference and positive control plasmid throughout this study (Figure 1B).

To guarantee that all cis acting regulatory elements within P. vivax promoter regions were included in these transient transfection assays, a new plasmid termed pPv-vir24 containing the entire 1.3 kb intergenic region between vir23 and vir24 genes [34] was constructed (Figure 2A). Transient transfection of pPv-vir24 into P. falciparum produced luciferase activity levels significantly above background, suggesting this P. vivax intergenic region contains cis regulatory elements capable to recruit the P. falciparum transcriptional machinery (Figure 2B). However, the reporter activity was about 1% of that observed with the positive control (pE(A)b.luc.^D) indicating poor recognition of these cis regulatory elements.

To provide further evidences of this observation, reporter plasmids with the entire intergenic regions of the ef-1α genes of P. falciparum, P. berghei and P. vivax were transfected into P. falciparum. These genes are orthologs in Plasmodium containing two copies each per haploid genome in opposite orientations. In addition, the ef-1α intergenic region from P. berghei had already been functionally characterized and shown to have promoter activity in both orientations in P. berghei and in P. falciparum [23, 25]. As shown in Figure 2, recombinant plasmids harboring the entire intergenic regions of the ef-1α genes from P. falciparum and P. berghei reported high and comparable luciferase values in either orientation. In contrast, plasmids containing the intergenic region of the ef-1α genes of P. vivax, cloned in both orientations, reported luc values relative to pE(A)b.luc.^D that were 0.8% (pPv-EF(A)), which is not significantly different from the negative control, and 2.7% (pPv-EF(B)), which is significantly above background (Figure 2B). Together, these results indicate that cis regulatory elements within promoter regions of P. vivax are poorly or not recognized by the transcriptional machinery of P. falciparum.

An in silico approach was undertaken to identify divergent elements in the P. vivax ef-1α intergenic region. The ef-1α intergenic regions of six Plasmodium species (P. berghei, P. falciparum, P. knowlesi, P. reichenowi, P. yoelii and P. vivax) were searched for conserved motifs using the Gibbs sampling and Patser algorithms [30, 32]. Some short motifs, conserved in all Plasmodium species were found. These included homopolymeric poly(dA)poly(dT) tracts, poly(dAT) tracts and also GC rich motifs (data not shown). Using a matrix length of 27, the motif TGG [G/T]G [C/T]T [A/T] [A/T]GAGGGGTGA [A/G]C [A/C] [G/T]TTAAA was found. This sequence is conserved in the intergenic region of P. berghei, P. falciparum, P. reichenowi and P. yoelii, but not in P. vivax and P. knowlesi, where the central part of the motif is conserved (GAGGGGTG), but the extremities are degenerate. This data indicated that P. vivax and its evolutionary related species P. knowlesi could share motifs distinct from the species with AT rich genomes. When the matrix length was set to six, a motif (GCATAT) identical among all Plasmodium species excepting P. vivax (GCANAN) was found. The Patser algorithm was used to determine its position, conservation and copy number in the six ef-1α intergenic regions. The motif is present in two to five copies and its position relative to the start codon of the two ef-1α genes is maintained in pairs of phylogenetically closely related parasites P. falciparum/P. reichenowi and P. berghei/P. yoelii, but not in P. vivax/P. knowlesi (Figure 3A). This motif, referred to here as the EF motif, has no similarities to known eukaryotic transcription-factor binding sites.

Since the highly conserved EF motif is degenerate only in P. vivax, two complementary approaches were performed to demonstrate that it is active in gene expression. Firstly, two recombinant plasmids containing one or two copies of the EF motif in the P. vivax ef-1α intergenic region, were generated (Figure 3B). pPv-EF(B)-HEF, having one copy of the EF motif, significantly reduced the promoter strength to 65% whereas pPv-EF(B)-HNEF, having two copies of the EF motif in different strands, restored reporter activity close to values of the original plasmid pPv-EF(B) (Figure 3C). Secondly, the EF motif in the P. berghei ef-1α promoter was mutated to create a sequence similar to the motif of P. vivax. To do so, thymidines (T) on positions 4 and 6 of the first or the second copy of the EF motif in plasmid pE(A)b.luc.^D were mutated to cytidines (C) (Figure 4A). Interestingly, luciferase activity significantly dropped to 56% and 55% relative to activity detected with the original plasmid (Figure 4B). Together, this data suggests that both copies of the EF motif are important for promoter activity and that this motif is active in gene expression in Plasmodium.