Region II-plus is Important for Sporozoite Adhesion to HepG2 Cells

<p>Midgut sporozoites of CS-WT and CS-RIImut (100,000 each) were added to confluent HepG2 cells. Adhesion is shown as the mean number of bound sporozoites in one microscopic field (400× magnification). Results are from three independent experiments.</p

CS-RIImut Oocyst Sporozoites Are Not Released from the Midguts

<div><p>Represented in each graph are the mean numbers of sporozoites per mosquito at different days PI. Each number is calculated based on an average of 20 mosquitoes.</p><p>(A) Numbers of oocyst sporozoites per mosquito from CS-WT– and CS-RIImut–infected mosquitoes. In CS-RIImut, the number of sporozoites increases progressively until day 18 PI, whereas in CS-WT a plateau is reached at day 14.</p><p>(B) Numbers of sporozoites in the hemolymph from CS-WT– and CS-RIImut–infected mosquitoes from the same lot as in (A). CS-WT oocysts release sporozoites into the hemolymph from day 12 to day 18 PI. The peak is around day 18 PI. In contrast, the hemolymph from CS-RIImut contains very few sporozoites.</p><p>(C) In another feeding experiment, the number of hemolymph sporozoites from CS-WT and CS-RIImut infected mosquitoes were calculated from day 14 to day 28 PI. CS-WT releases sporozoites into the hemolymph up to day 28 PI, whereas CS-RIImut does not.</p></div

Ultrastructure and CS Localization of CS-WT and CS-RIImut

<div><p>(A) Immunofluorescence labeling of oocyst sporozoites from CS-WT and CS-RIImut at day 18 PI. Sporozoites were stained with anti-CS antibody and detected by FITC-conjugated anti-IgG antibodies, without prior permeabilization.</p><p>(B) Transmission electron micrographs showing a CS-RIImut oocyst at day 14 PI. The oocyst is surrounded by a capsule and the mosquito basal lamina, and contains sections of sporozoites with normal morphology. Fully developed sporozoites are found within the CS-RIImut oocyst. Sporozoites have homogenous size and morphology. Scale bar represents 1 μm.</p><p>(C) Longitudinal section of the CS-RIImut sporozoite pellicle shows the plasma membrane, inner membrane complex, and an associated microtubule. Scale bar represents 0.5 μm.</p><p>(D) Cross-section of a CS-RIImut sporozoite showing the trimembrane pellicle and subpellicular microtubules. Scale bar represents 0.5 μm.</p><p>(E) Immuno-electron micrographs showing CS localization in the CS-RIImut oocyst. CS protein is predominantly found on the surface of sporozoites and the residual body, and on the inner surface of the capsule. Scale bar represents 1 μm.</p><p>(F) Enlarged picture of the CS-RIImut oocyst capsule. CS protein is detected on the inner surface of the capsule. Scale bar represents 1 μm.</p><p>(G) Western blot analysis of extracts from CS-WT (WT) and CS-RIImut (RII) oocyst sporozoites. Numbers of sporozoites loaded are indicated on the top of each lane. Sporozoites were collected from oocysts in mosquito midguts at days 14 and 18 PI.</p><p>C, capsule; IMC, inner membrane complex; Mo, mosquito tissue; MT, microtubule; Oo, oocyst; PM, plasma membrane; Rb, residual body; SPZ, sporozoite.</p></div

In Vitro Oocyst Sporozoite Release Assay

<p>Release of oocyst sporozoites at 25 °C in vitro in the presence of trypsin (50 μg/ml) at day 14 PI. <i>Y</i>-axis represents released sporozoites as a percentage of total oocyst sporozoites. <i>X</i>-axis indicates the time point when the samples are collected. In the absence of the trypsin, very few sporozoites are released from the midguts. In the presence of the trypsin, oocyst sporozoites from both CS-WT (WT) and CS-RIImut (RII) are released in a time-dependent manner. Compared with CS-WT, CS-RIImut sporozoites are released more slowly.</p

Gene Targeting at the <i>CS</i> Locus of <i>P. berghei</i>

<div><p><b>(</b>A and B<b>)</b> Replacement plasmid pRCS-WT and pRCS-RIImut, wild-type (WT) <i>CS</i> locus and recombinant locus. ORFs are symbolized by boxes. Small black box in the <i>CS</i> ORF indicates the mutation in region II-plus (R290A, K291A, R292A, and K293A). A PstI site is introduced in the mutation site to differentiate CS-RIImut from CS-WT. Thick lines indicate 5′- and 3′-UTRs of <i>DHFR-TS;</i> thin lines, 5′- and 3′UTRs of <i>CS;</i> dashed lines, plasmid vector sequence. B, BamHI; K, KpnI; N, NotI; P, PstI; S, SacI; Xb, XbaI; Xh, XhoI.</p><p>Recombinant CS-WT or CS-RIImut was generated by double crossover occurring between the CS sequences in the KpnI-SacI fragment of plasmid pRCS-WT or pRCS-RIImut and their homologous sequence in the wild-type <i>CS</i> genomic locus. The CS probe used in the genomic Southern hybridization is symbolized by a thick dash-dot line. Restriction fragments of the wild type and of the expected recombinants are shown below the corresponding locus. Locations of primers used for PCR are indicated in (B).</p><p>(C) Schematic structure of the CS protein and sequences of the region II-plus of wild-type and mutant CS.</p><p>(D) Genomic Southern hybridization of the wild-type <i>P. berghei</i> strain (WT), the recombinant lines, control (CS-WT), and mutant (CS-RIImut) parasites upon digestion with XbaI and PstI, using a CS probe.</p><p>(E) PCR amplification with primers CS1 and PB103 of the expressed CS protein at the 5′ recombinant locus in CS-RIImut and CS-WT. The WT is used as a negative control (lack of recombination events). The amplicons (2.3 kb) were subjected to PstI restriction enzyme digestion (two fragments released, 1.6 and 0.7 kb) to examine the presence of mutation in CS-RIImut, which is absent in CS-WT.</p><p>(F) PCR amplification of the 3′ recombinant locus using primers PB106 and CS4.</p></div

UIS2: A Unique Phosphatase Required for the Development of <i>Plasmodium</i> Liver Stages

<div><p><i>Plasmodium</i> salivary sporozoites are the infectious form of the malaria parasite and are dormant inside salivary glands of <i>Anopheles</i> mosquitoes. During dormancy, protein translation is inhibited by the kinase UIS1 that phosphorylates serine 59 in the eukaryotic initiation factor 2α (eIF2α). De-phosphorylation of eIF2α-P is required for the transformation of sporozoites into the liver stage. In mammalian cells, the de-phosphorylation of eIF2α-P is mediated by the protein phosphatase 1 (PP1). Using a series of genetically knockout parasites we showed that in malaria sporozoites, contrary to mammalian cells, the eIF2α-P phosphatase is a member of the PP2C/PPM phosphatase family termed UIS2. We found that eIF2α was highly phosphorylated in <i>uis2</i> conditional knockout sporozoites. These mutant sporozoites maintained the crescent shape after delivery into mammalian host and lost their infectivity. Both <i>uis1</i> and <i>uis2</i> were highly transcribed in the salivary gland sporozoites but <i>uis2</i> expression was inhibited by the Pumilio protein Puf2. The repression of <i>uis2</i> expression was alleviated when sporozoites developed into liver stage. While most eukaryotic phosphatases interact transiently with their substrates, UIS2 stably bound to phosphorylated eIF2α, raising the possibility that high-throughput searches may identify chemicals that disrupt this interaction and prevent malaria infection.</p></div

The N-terminus of UIS2 binds eIF2α-P.

<p>(A) PfeIF2α-P interacts with endogenous UIS2. In these experiments we used the codon-optimized eIF2α of <i>P</i>. <i>falciparum</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref007" target="_blank">7</a>] that shares 89% identity with its <i>P</i>. <i>berghei</i> ortholog. The immobilized GST-PfeIF2α was incubated with the lysates of <i>P</i>. <i>berghei</i> blood stage parasites in the presence or absence of Sal (50 μM) or GA (70 μM). The bound proteins were detected by immunoblot using antibodies against PP1, UIS2, phosphorylated eIF2α, and total eIF2α. Levels of PbeIF2α-P and total PbeIF2α were quantified by densitometry analysis. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.s010" target="_blank">S8 Fig</a>: The mouse anti-PbPP1 antibody recognized the 35 kDa endogenous PP1 from the parasite lysates. (B) PfeIF2α<i>S59D</i> mutant protein interaction with UIS2. The immobilized GST-PfeIF2α wt, <i>S59A</i>, or <i>S59D</i> were incubated with the lysates of <i>P</i>. <i>berghei</i> blood stage parasites, with or without Sal, and UIS2 was detected by immunoblot. (C) Schematic representation of the PbUIS2 coding sequence. UIS2 contains a putative conserved phosphatase domain (PD, 535–1054 amino acids) and <i>Plasmodium</i> specific sequences at N- and C- terminus. (D) The N-terminus of PbUIS2 bound PfeIF2α-P. The PbUIS2 N-ter, PD, and C-ter were fused to GST-tag at their N-terminus and His-tag at their C-terminus, respectively. After 2-step affinity purification, the <i>E</i>. <i>coli</i> expressed fusion proteins were immobilized on glutathione sepharose 4B. After incubation with purified recombinant PfeIF2α-P, the sepharose was washed three times with high-salt NETN buffer (300 mM NaCl, 20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, and 0.5% (v/v) Nonidet P-40). The retained proteins were detected by SDS-PAGE followed by coomassie brilliant blue staining. Lane 1,3,5: GST and His-tagged PbUIS2 N-ter, PD, and C-ter, respectively. Lane 2,4,6: proteins retained on the glutathione sepharose 4B after pull down assays with PfeIF2α-P. Lane 7, PfeIF2α-P control. The pulled down 38 kDa protein in lane 2 was analyzed by mass spectrometry and identified as PfeIF2α-P. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.s011" target="_blank">S9 Fig</a>. (E) Co-immunoprecipitation (IP) of endogenous complex between UIS2 and eIF2α-P. Whole cell extracts from <i>P</i>. <i>berghei</i> blood stage parasites were subjected to immunoprecipitation with anti-eIF2α-P or anti-UIS2 antibodies followed by immunoblot analysis.</p

Characterization of <i>Plasmodium pp1</i> (<i>PBANKA_102830</i>).

<p><i>(A) pp1</i> mRNA levels during the <i>P</i>. <i>berghei</i> life cycle. <i>Pbpp1</i> mRNA levels were analyzed by real-time PCR using cDNAs from the different stages of <i>P</i>. <i>berghei</i>. The <i>arginyl-tRNA synthetase</i> (<i>PbArgRS</i>, <i>PB000094</i>.<i>03</i>.<i>0</i>) was used as internal control. Each value is the mean ± SD of two independent experiments. MG: midgut; SG: salivary gland; Liver: liver stages; BS: asexual blood stages; Gtc: gametocyte. <i>(B) Pbpp1</i> mRNA levels in <i>Pbpp1</i> cKO Ssp were quantified by qPCR. P = 0.041. P value was calculated by t test. Shown are mean ± SD of two independent experiments. <i>(C)</i> Immunoblot analysis of <i>Pbpp1</i> cKO and wt TRAP/FlpL(-) Ssp using anti-PbPP1 serum. CSP was used as control. <i>(D)</i> The liver stage development of <i>Pbpp1</i> cKO Ssp in HepG2 cells was indistinguishable from the wild type TRAP/FlpL(-) Ssp. Hepatic parasites were stained with anti-PbHSP70 and anti-PbPP1 antibodies at 6h, 24h, 36h, 48h, and 55h post-infection. Bar, 10 μm. <i>(E)</i> eIF2α phosphorylation level in <i>Pbpp1</i> cKO sporozoites was indistinguishable from wild type. Five hundred thousand wt TRAP/FlpL(-) or <i>Pbpp1</i> cKO sporozoites were dissected from mosquito salivary glands. Levels of PbeIF2α-P and total PbeIF2α were quantified by densitometry analysis of immunoblots performed with antibodies against anti-eIF2α-P and anti-total eIF2α [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref015" target="_blank">15</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref016" target="_blank">16</a>]. Values are shown below the bands. Results were similar in two independent experiments.</p

Phenotypes of <i>uis2 cKO parasites</i>.

<p>(A) mRNA levels of <i>uis2</i> in the <i>P</i>. <i>bergthei</i> life cycle. <i>Pbuis2</i> (<i>PBANKA_132800</i>) mRNA levels were analyzed by real-time PCR using cDNAs from different stages of <i>P</i>. <i>berghei</i>. mRNA level was normalized to arginyl-tRNA synthetase (<i>PbArgRS</i>, PB000094.03.0). MG: midgut; SG: salivary gland; Liver: liver stages; BS: asexual blood stages; Gtc: gametocytes. Each value is the mean ± SD of two independent experiments. (B) Phosphorylation level of eIF2α was higher in <i>uis2</i> cKO Ssp. Levels of UIS2, PbeIF2α-P and total PbeIF2α from 5X10⁵ Ssp are shown by immunoblots. CSP was used as a control. Values of densitometry analysis are shown below the bands. This experiment was repeated three times and similar results were obtained. (C) The mutant sporozoites invaded HepG2 cells as effectively as wt parasites. Wild-type TRAP/FlpL(-) and <i>uis2</i> cKO Ssp were added to HepG2 cells and fixed 1 h post-infection. The parasites inside and outside the HepG2 cells were quantified by the hepatocyte invasion assay. Each value is the mean ± SD of two independent experiments. <i>(</i><b><i>D</i></b><i>)</i> The mutant parasites maintained the crescent shape in contrast to the round shape of the wt parasites in HepG2 cells. Wt TRAP/FlpL(-) and <i>uis2</i> cKO Ssp were added to HepG2 cells and were detected 48 h post-infection by immunofluorescence using antibodies against the liver stage antigen UIS4. Bars, 10 μm. (E) The development of <i>uis2</i> cKO sporozoites was blocked inside HepG2 cells. <i>P</i>. <i>berghei</i> Ssp infectivity of HepG2 cells was evaluated 48 h post-infection by counting exo-erythrocytic stage (EEF) numbers and measuring18S rRNA level. The left panel shows the mean of EEF numbers ± SD of two independent experiments. The right panel shows liver-stage parasite burden measured by real-time RT-PCR. (F) The development of <i>uis2</i> cKO sporozoites was blocked in the mouse liver. C57BL/6 mice (five per group) were injected intravenously with 1×10⁴ wt TRAP/FlpL(-) or with the same number of <i>uis2</i> cKO Ssp. Liver-stage parasite burden was measured 42 hours post-infection by real-time RT-PCR. Each value is the mean ± SD of two independent experiments.</p

Model for UIS2 function in the <i>Plasmodium</i>.

<p>The eIF2α kinase eIK2 (also called UIS1) and Pumilio protein Puf2 are highly transcribed and translated in the mosquito salivary gland [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref003" target="_blank">3</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref005" target="_blank">5</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref025" target="_blank">25</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref026" target="_blank">26</a>]. The highly transcribed eIF2α phosphatase <i>uis2</i> mRNA binds to Puf2 and the translation of phosphatase UIS2 is likely repressed. The eIF2α of Ssp is highly phosphorylated since the eIF2α kinase eIK2 activity is dominant, leading to translation inhibition and latency of sporozoites in the mosquito salivary glands. When sporozoites are injected into the mammalian host, the repression of UIS2 translation is probably alleviated, the eIF2α phosphatase UIS2 activity is dominant, eIF2α is dephosphorylated, and Ssp transform into the liver stages. Knockout of <i>puf2</i> or <i>eIK2</i> contributes to the dominance of UIS2 and the mutant sporozoites inside of mosquito salivary glands prematurely transform into liver stages [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref004" target="_blank">4</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref023" target="_blank">23</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref028" target="_blank">28</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref029" target="_blank">29</a>].</p