Centromere Plasmid: A New Genetic Tool for the Study of Plasmodium falciparum

The introduction of transgenes into Plasmodium falciparum, a highly virulent human malaria parasite, has been conducted either by single crossover recombination or by using episomal plasmids. However, these techniques remain insufficient because of the low transfection efficiency and the low frequency of recombination. To improve the genetic manipulation of P. falciparum, we developed the centromere plasmid as a new genetic tool. First, we attempted to clone all of the predicted centromeres from P. falciparum into E. coli cells but failed because of the high A/T contents of these sequences. To overcome this difficulty, we identified the common sequence features of the centromere of Plasmodium spp. and designed a small centromere that retained those features. The centromere plasmid constructed with the small centromere sequence, pFCEN, segregated into daughter parasites with approximately 99% efficiency, resulting in the stable maintenance of this plasmid in P. falciparum even in the absence of drug selection. This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum. In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence. Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions. These unique properties of the pFCEN plasmid will solve the current technical limitations of the genetic manipulation of P. falciparum, and thus, this plasmid will become a standard genetic tool for the study of this parasite

PbAP2-FG2 and PbAP2R-2 function together as a transcriptional repressor complex essential for Plasmodium female development.

Gametocyte development is a critical step in the life cycle of Plasmodium. Despite the number of studies on gametocyte development that have been conducted, the molecular mechanisms regulating this process remain to be fully understood. This study investigates the functional roles of two female-specific transcriptional regulators, PbAP2-FG2 and PbAP2R-2, in P. berghei. Knockout of pbap2-fg2 or pbap2r-2 impairs female gametocyte development, resulting in developmental arrest during ookinete development. ChIP-seq analyses of these two factors indicated their colocalization on the genome, suggesting that they function as a complex. These analyses also revealed that their target genes contained a variety of genes, including both male and female-enriched genes. Moreover, differential expression analyses showed that these target genes were upregulated through the disruption of pbap2-fg2 or pbap2r-2, indicating that these two factors function as a transcriptional repressor complex in female gametocytes. Formation of a complex between PbAP2-FG2 and PbAP2R-2 was confirmed by RIME, a method that combines ChIP and MS analysis. In addition, the analysis identified a chromatin regulator PbMORC as an interaction partner of PbAP2-FG2. Comparative target analysis between PbAP2-FG2 and PbAP2-G demonstrated a significant overlap between their target genes, suggesting that repression of early gametocyte genes activated by PbAP2-G is one of the key roles for this female transcriptional repressor complex. Our results indicate that the PbAP2-FG2-PbAP2R-2 complex-mediated repression of the target genes supports the female differentiation from early gametocytes

Coordinated regulation of gene expression in Plasmodium female gametocytes by two transcription factors

Gametocytes play key roles in the Plasmodium lifecycle. They are essential for sexual reproduction as precursors of the gametes. They also play an essential role in parasite transmission to mosquitoes. Elucidation of the gene regulation at this stage is essential for understanding these two processes at the molecular level and for developing new strategies to break the parasite lifecycle. We identified a novel Plasmodium transcription factor (TF), designated as a partner of AP2-FG or PFG. In this article, we report that this TF regulates the gene expression in female gametocytes in concert with another female-specific TF AP2-FG. Upon the disruption of PFG, majority of female-specific genes were significantly downregulated, and female gametocyte lost the ability to produce ookinetes. ChIP-seq analysis showed that it was located in the same position as AP2-FG, indicating that these two TFs form a complex. ChIP-seq analysis of PFG in AP2-FG-disrupted parasites and ChIP-seq analysis of AP2-FG in PFG-disrupted parasites demonstrated that PFG mediates the binding of AP2-FG to a ten-base motif and that AP2-FG binds another motif, GCTCA, in the absence of PFG. In promoter assays, this five-base motif was identified as another female-specific cis-acting element. Genes under the control of the two forms of AP2-FG, with or without PFG, partly overlapped; however, each form had target preferences. These results suggested that combinations of these two forms generate various expression patterns among the extensive genes expressed in female gametocytes

Identification of an AP2-family Protein That Is Critical for Malaria Liver Stage Development

<div><p>Liver-stage malaria parasites are a promising target for drugs and vaccines against malaria infection. However, little is currently known about gene regulation in this stage. In this study, we used the rodent malaria parasite <em>Plasmodium berghei</em> and showed that an AP2-family transcription factor, designated AP2-L, plays a critical role in the liver-stage development of the parasite. <em>AP2-L</em>-depleted parasites proliferated normally in blood and in mosquitoes. However, the ability of these parasites to infect the liver was approximately 10,000 times lower than that of wild-type parasites. In vitro assays showed that the sporozoites of these parasites invaded hepatocytes normally but that their development stopped in the middle of the liver schizont stage. Expression profiling using transgenic <em>P. berghei</em> showed that fluorescent protein-tagged AP2-L increased rapidly during the liver schizont stage but suddenly disappeared with the formation of the mature liver schizont. DNA microarray analysis showed that the expression of several genes, including those of parasitophorous vacuole membrane proteins, was significantly decreased in the early liver stage of <em>AP2-L</em>-depleted parasites. Investigation of the targets of this transcription factor should greatly promote the exploration of liver-stage antigens and the elucidation of the mechanisms of hepatocyte infection by malaria parasites.</p> </div

Stable Maintenance of pFCEN in the Parasites.

<p>(A) The GFP expression of the parasites transfected with pFCEN and pCon were monitored in the presence of the selective drug (upper panel) and at 8 days after the removal of the drug. The nuclei of the parasites were stained with Hoechst-33258. The scale bars indicate 10 µm. (B) The percentages of GFP-positive parasites in the absence of the drug were calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033326#s4" target="_blank">Materials and Methods</a>. The red and blue lines indicate the percentage of GFP-positive parasites transfected with pFCEN and pCon, respectively.</p

Sequence Properties of the Centromere from Chromosome 5 of <i>P. falciparum</i> and the Plasmid Map of pFCEN.

<p>(A) The sequence analysis of the centromere of chromosome 5 of <i>P. falciparum</i> was performed using Artemis 11 with regard to its length and A/T content. The line at the bottom indicates the centromere, and its length and A/T content are 2170 bp and 97.5%, respectively. The genomic sequence data of chromosome 5 were obtained from PlasmoDB (<a href="http://plasmodb.org/" target="_blank">http://plasmodb.org/</a>). (B) The repetitive region in the centromere of chromosome 5 was identified by the dot matrix analysis using the Dotlet program. The inset box indicates the repetitive region in this centromere. (C) The centromere of chromosome 5 is schematically shown and named <i>pfcen5</i> in this Figure. The line on the top indicates the repetitive and non-repetitive regions. The triangle indicates the 19 bp of the repeat sequence motif, and the number in parentheses is the number of repeats within the repetitive region. The schematic drawing of <i>pfcen5-1.5</i> also is shown. The numbers at the bottom are based on the sequence number of <i>pfcen5</i> and correspond to the beginning and the end of <i>pfcen5-1.5</i>. (D) The pFCEN plasmid is 8018 bp, and <i>pfcen5-1.5</i> is placed downstream of the 3′ UTR of the <i>dhfr-ts</i> gene of <i>P. berghei</i>.</p

Centromere of <i>Plasmodium</i> spp.

<p>Schematic drawings of the centromeres of <i>Plasmodium</i> spp., including <i>P. falciparum</i>, <i>P. vivax</i>, <i>P. berghei</i>, and <i>P. yoelii</i> are shown. Each centromere of <i>P. falciparum</i>, <i>P. berghei</i> and <i>P. yoelii</i> is named after the corresponding chromosome number, and the centromeres of <i>P. vivax</i> are named after the corresponding contig numbers. Triangles indicate the repetitive sequence elements that were identified using the Tandem Repeats Finder program. The numbers in parenthesis show the repeat counts for each repetitive region. Other sequence information is summarised in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033326#pone.0033326.s007" target="_blank">Table S1</a>.</p