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

Southern Analysis of pFCEN in the Parasites.

<p>(A) To determine the copy number of pCon (lane a) and pFCEN (lane b), Southern analyses were performed by using two probe DNAs, the <i>gfp</i> and <i>pfsir2A</i> genes. The <i>gfp</i> and <i>pfsir2A</i> probes recognised the plasmids and the single, endogenous genomic <i>pfsir2A</i> copy, respectively. In this analysis, the signal from the <i>pfsir2A</i> gene was used as the internal control. The copy number of each plasmid was determined by comparing the signal intensities of the plasmids with that of the internal control. The asterisks mark three unexpected signals found in the genomic DNA including pCon. In contrast, corresponding signals were not detected in the genomic DNA including pFCEN. Therefore, these signals likely result from various derivatives, which might be irregularly generated from pCon during the replication, suggesting that <i>pfcen5-1.5</i> ensured the accurate replication of the plasmid. (B) The parasites transfected with the pFCEN and the pCon plasmids were maintained in the presence of the drug for 2 months, and their genomic DNA, including the plasmids, were purified. Southern analysis of both plasmids in the parasites was performed using purified genomic DNA and the gfp gene as a probe DNA. In this analysis, the undigested genomic DNA (lane a, pCon, and lane c, pFCEN) and the <i>Bam</i>HI-digested DNA (lane b, pCon, and lane d, pFCEN) were used. The arrows indicate the <i>Bam</i>HI-digested pFCEN and pCon. The upper and lower triangles indicate the closed circular and the super-coiled forms of pFCEN, respectively. Asterisks indicate concatemers of pCon that were detected in the undigested genomic DNA containing pCon.</p

The Growth of the Transgenic Parasite after Transfection with pFCEN.

<p>The transfection of the parasite with pFCEN (red line) and pCon (blue line) were performed 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>. After 48 hours, the drug screening of the transgenic parasites was initiated by supplementing the culture medium with pyrimethamine. The parasitemia of the transgenic parasites was monitored every 48 hours after transfection. The transfection experiments for each plasmid were performed in triplicate.</p

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

AP2-L and related proteins in apicomplexan parasites.

<p>A. Schematic diagrams of <i>P. berghei</i> AP2-L and related proteins are shown. The paralogs in <i>P. berghei</i> are indicated by their gene identifiers in PlasmoDB. The related proteins in other apicomplexan parasites are indicated by the species name and their GenBank accession number. AP2 domains (blue) and another conserved sequence (red) are highlighted by rectangles. Bars indicate the regions shown in B and C. B. Amino acid sequences of a region containing two A domains (highlighted with rectangles) are compared in the AP2-Ls of four <i>Plasmodium</i> species: <i>P. berghei</i> (Pb), <i>P. falciparum</i> (Pf), <i>P. vivax</i> (Pv), and <i>P. knowlesi</i> (Pk). The amino acids conserved across all four species are indicated by asterisks. This region is indicated in A by the left bar. C. Amino acid sequences of another region conserved among the AP2-Ls. This region is indicated in A by the right bar. The amino acids conserved across all four species are indicated by asterisks.</p

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

The expression profile of <i>AP2-L</i> in <i>P. berghei</i> pre-erythrocytic stages.

<p>A. A schematic diagram showing the preparation of transgenic <i>P. berghei</i> expressing GFP-tagged AP2-L. The expression construct was introduced into the 3′ portion of the endogenous <i>AP2-L</i> gene by double cross-over homologous recombination. The results of the Southern blot analysis of wild-type (WT) and transgenic <i>AP2-L</i> parasites (<i>AP2-L ::GFP</i> (−)) are shown on the left. B. Fluorescence images of a salivary gland sporozoite (SG; expressing GFP-tagged AP2-L, extreme left column) and LS parasites (expressing mCherry-tagged AP2-L, other columns). LS parasites were harvested at 12-h intervals following sporozoite inoculation. The LS parasites expressed GFP constitutively in the cytoplasm (upper images). The nuclei were stained with Hoechst 21486 (in sporozoites) or DAPI (in LS parasites). The scale bars represent 10 µm.</p

LS development is arrested during the schizont stage in <i>AP2-L</i>(−) parasites.

<p>A. Sporozoites (2×10⁴) were inoculated into a culture of HepG2 cells and incubated at 37°C. The number of LS parasites was counted 48 hpi after staining with anti-CSP antibody. The data are the mean ± SEM of four measurements. B. The diameters of LS <i>AP2-L</i>(−) 1 and wild-type parasites were compared. When the parasites were not round, the average of the longest and the shortest axis was used as the diameter. The horizontal axis indicates the number of hours post-sporozoite inoculation. The data are the mean ± SEM of 200 measurements. C. Fluorescence images of <i>AP2-L</i>(−) 1 and wild-type (WT) LS parasites at 24 hpi and 48 hpi. The parasites were stained with an anti-CSP antibody. The arrows indicate densely stained portions that were specifically observed in <i>AP2-L</i>(−) LS parasites. D. Representative image of wild-type and <i>AP2L</i>(−) 1 LS parasites at 53 hpi with nuclear staining by DAPI. The scale bars in C and D represent 10 µm. E. Parasite burden in the rat liver was compared between the wild-type and <i>AP2-L</i>(−) parasites. Wild-type or <i>AP2-L</i>(−) salivary gland sporozoites were intravenously injected into rats, and the liver was dissected after perfusion at 24 hpi or 48 hpi. Parasite loads in the liver were determined by measuring <i>P. berghei</i> 18S rRNA using RT-PCR. The rat glyceraldehyde 3-phosphate dehydrogenase gene was used as an internal control to normalize the mean cycle threshold value of the rat 18S rRNA gene. Four rats were used for each condition.</p

Genes whose expression is decreased in <i>AP2-L</i>(−) parasites.

<p>DNA microarray analysis was performed using LS cultures at 24 hpi. Genes whose expression was decreased at least three-fold by depletion of <i>AP2-L</i> are listed. Each gene is indicated by its PlasmoDB gene ID. The functional annotations are essentially in line with PlasmoDB. Signal sequences were predicted with SignalP (<a href="http://www.cbs.dtu.dk/services/SignalP/" target="_blank">http://www.cbs.dtu.dk/services/SignalP/</a>).</p