Sequestration Properties of Blood Stages of <i>P. falciparum</i> in Humans and <i>P. berghei</i> ANKA in Rodents.

<p>Sequestration Properties of Blood Stages of <i>P. falciparum</i> in Humans and <i>P. berghei</i> ANKA in Rodents.</p

Pbtert deletion and selection of tert- mutants.

<p>(A) Schematic representation of the construct used to delete the <i>tert</i> gene. The construct, containing the <i>Tgdhfr-ts</i> selectable marker (SM) cassette, targets the <i>tert</i> gene at the flanking regions (red) by double cross-over integration. The red arrows indicate primers used for diagnostic PCR to confirm correct disruption of <i>tert</i>. Boxes correspond to lanes on the PCR gels in (B), (D) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108930#pone.0108930.s001" target="_blank">Fig. S1A</a>. (B) Diagnostic PCR of uncloned parasites transfected with a DNA construct to delete the <i>tert</i> gene. Parasites were collected and analysed directly after transfection and selection with pyrimethamine (parent populations). Diagnostic PCR shows the presence of parasites with correct disruption of the <i>tert</i> gene. In all experiments (1065, 1078, 1138, 1207, 1217) the 5′ and 3′ integration fragments (lanes 5′, 3′), as well as the <i>Tgdhfr-ts</i> fragment (lane SM) were amplified. However, all populations contained parasites with a wild type <i>tert</i> gene as shown by amplification of the wild type <i>tert</i> fragment (lane wt). The primer pairs used are shown in (A) and expected fragment sizes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108930#pone.0108930.s004" target="_blank">Table S2</a>. <i>pbs21</i>-specific primers were used as a positive control for all the PCR reactions (“+”). The water control is marked as “-“. (C) Southern analysis of separated chromosomes using the 3′UTR <i>Pbdhfr-ts</i> probe shows only in experiment 1065 and 1217 hybridisation with chromosome 14 on which the <i>tert</i> gene is located. This probe recognizes the endogenous <i>Pbdhfr-ts</i> gene on chromosome 7 in all populations and additional chromosomes in experiments 1078, 1138, 1207 (possible episomal construct signal). (D) Diagnostic PCR of uncloned and propagated parasites transfected with a DNA construct to delete the <i>tert</i> gene. The parent parasite populations of experiment 1065, 1207 and 1217 [see (B)] were propagated in mice (m0  =  mouse 0, m1  =  mouse 1) for another 1–2 weeks. Parasite populations collected were analysed by diagnostic PCR for the presence of parasites with correct disruption of the <i>tert</i> gene [primers same as in (B)]. In all populations no parasites with a disrupted <i>tert</i> gene could be detected by diagnostic PCR after 1 week (1207 all populations) or after two weeks of propagation (1065 uncl.2, 1217 uncl.2 m0 and 1217 uncl.2 m1).</p

Pbtert gene structure (A) and PbTERT (B) and PbTR (C) expression.

<p>(A) The <i>tert</i> gene of <i>P. berghei</i> and homology (percentage identity) of TERT proteins in different <i>Plasmodium</i> species. Sequencing of the gap between two adjacent <i>tert</i> gene models available in PlasmoDB revealed a sequence duplication of 57 nt (19aa). The complete Pb<i>tert</i> gene encodes a protein of 2312aa, which is comparable to the size of other <i>Plasmodium tert</i> genes. (B) Western analysis of PbTERT protein in mixed blood stages. Two bands with a size between 150 and 250 kDa were detected (expected size of the TERT protein is ∼240 kDa). (C) Northern analysis of Telomerase-associated RNA (TR) in different blood stages of <i>P. berghei</i>. RNA was hybridized with a probe recognizing TR (upper panel) (the expected size of TR is 2 kb) and as a loading control with a probe recognizing <i>large subunit ribosomal RNA</i> (expected size 0.8 kb). The “% loading” refers to the quantity of the loading control signal detected for each stage relative to the “late trophozoite” lane which is set as 100%.</p

<i>P. berghei</i> ANKA asexual blood stage development and expression of proteins in mature schizonts.

<p>(A) In vivo and in vitro development of rings, trophozoites, and schizonts during one cycle of synchronized development. In mice, rings and trophozoites do not sequester but schizonts disappear from the peripheral circulation (upper graph). In vitro schizogony takes place between 18 and 24 hours after invasion of the red blood cell (lower graph). The arrow indicates a multiply infected red blood cell containing three trophozoite-stage parasites; above this cell is a 20-hour schizont (graphs adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001032#ppat.1001032-Mons1" target="_blank">[44]</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001032#ppat.1001032-FrankeFayard1" target="_blank">[35]</a>. (B) Live mature schizonts of two transgenic lines expressing two different fluorescently tagged PIR proteins either tagged with GFP (eG; PB200064.00.0) or mCherry (mC; PB200026.00.0). These proteins are exported into the cytoplasm of the erythrocyte nucleus stained with Hoechst (H; blue), red blood cell membrane surface protein stained in mC parasites (TER-FITC; green) (J. Braks and B. Franke-Fayard, unpublished data). (C) Live mature schizonts that express GFP and mCherry in the cytoplasm of the merozoites (J. Braks and B. Franke-Fayard, unpublished data).</p

Imaging of transgenic <i>P. berghei</i> ANKA parasites in brains of mice ex vivo.

<p>Matched sets of experiments with <i>P. berghei</i> ANKA infections in ECM-sensitive mice (i.e., wild-type mice) or knock-out mice (i.e., IP10^−/−). Knock-out mice do not develop cerebral pathology and this corresponds to a strong reduction in irbc accumulation as compared to infections in wild-type mice (adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001032#ppat.1001032-Nie1" target="_blank">[41]</a>). Similar examples of a lack of irbc accumulation can be observed in the brains of mice treated with antibodies against host molecules (e.g., anti-LTβ mAB and anti-CD25 mAB; see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001032#ppat.1001032-Amante1" target="_blank">[42]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001032#ppat.1001032-Randall2" target="_blank">[104]</a>). Parasites express GFP::luciferase fusion protein under the control of the eef1a promoter, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001032#ppat-1001032-box003" target="_blank">Box 3</a>). Also, mice infected with a <i>P. berghei</i> ANKA mutant that has had the gene encoding plasmepsin 4 removed do not develop cerebral complications, and again there is a strong reduction of irbc accumulation in the brain of these infected animals (adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001032#ppat.1001032-Spaccapelo1" target="_blank">[43]</a>).</p

Imaging of transgenic <i>P. berghei</i> ANKA parasites in vivo and ex vivo.

<p>CD36-mediated sequestration of schizonts in adipose tissue and lungs (adapted from PNAS, 2005 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001032#ppat.1001032-FrankeFayard1" target="_blank">[35]</a>). (A, B) Distribution of transgenic <i>P. berghei</i> ANKA parasites, expressing GFP::luciferase fusion protein (<i>ama-1</i> promoter, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001032#ppat-1001032-box003" target="_blank">Box 3</a>). Parasites are visible in lungs, spleen, and adipose tissue in wild-type mice, and principally in the blood circulation and accumulated in the spleen in CD36 knock-out mice. In wild-type mice infected with a non-sequestering K173 line, schizonts are also mainly found in the peripheral blood circulation and accumulated in the spleen (1: adipose tissue; 2: spleen; 3 liver; 4: lungs; 5: heart; 6: kidney; 7: brain). (C) Sequestration of transgenic <i>P. berghei</i> ANKA parasites in microvasculature of adipose tissue (upper panel with under phase contrast and lower panel with GFP-positive schizonts indicated by arrows).</p

<i>P. berghei</i> telomere characterisation.

<p>(A) Determination of telomere length by Telomere Restriction Fragment (TRF) analysis. Left Panel: Southern analysis of separated chromosomes of <i>P. berghei</i> (Pb), <i>P. chabaudi</i> (Pc), <i>P. vinckei</i> (Pv) and <i>P. yoelii</i> (Py) showing hybridization of all chromosomes to a telomere-specific probe. The same probe was used for TRF analysis (middle, right panels). Middle panel: Southern analysis of digested <i>P. yoelii</i> (size control) and <i>P. berghei</i> gDNA probed with the telomeric probe showing the characteristic “smeared” hybridisation pattern in TRF analysis. Right panel: The average telomere length was measured as the highest peak of the signal intensity along the smear. Using the molecular marker (“M”, grey line) as a size reference (relevant marker bands sizes are noted on the graph), the mean telomere length was estimated to be ∼2500 bp and ∼950 bp for <i>P. yoelii</i> (blue line) and <i>P. berghei</i> (red line), respectively. Complete digestion of gDNA was confirmed by hybridisation with a 5′ <i>d-type small unit ribosomal RNA</i> probe. (B) Fluorescence <i>in situ</i> hybridisation with a telomere-specific probe. Fixed late blood stages of <i>P. berghei</i>. The telomeric probe (1.5 kb) was labelled with fluorescein (green). Hoechst (blue) was used for nuclear staining. The size bar is 5 µm.</p

PbPL is involved in merozoite release.

<p>HepG2 cells were infected with wild-type (WT), PbPL-knockout (KO2) and complemented PbPL-KO (CMP2) sporozoites. The percentage of attached hepatocytes containing schizont (S), cytomere (C) and merozoite (M) stage parasites was determined at 54 and 65 hpi. Schizont stages are either negative for the merozoite surface protein MSP1 or display an MSP1 staining only at the parasite plasma membrane without invaginations. Cytomere stages are defined by their MSP1-positive parasite plasma membrane with clear invaginations, while in merozoite-containing hepatocytes, individual merozoites are surrounded by MSP1 staining. Representative MSP1 staining of each parasite stage is shown at the top. For each time point, 50–100 parasites were analyzed. Scale bars = 10 μm. Shown are means +/− SD of three independent experiments. For statistical analysis a one-way ANOVA followed by a Holm-Sidak multiple comparison test was performed (** p < 0.01, *** p < 0.001).</p

PbPL mediates disruption of the PVM.

<p>HepG2 cells expressing GFP (green) were infected with mCherry-expressing wild-type (WT), PbPL-knockout (KO2) and complemented PbPL-KO (CMP2) sporozoites (red). The percentage of merozoite-forming parasites that ruptured the PVM and the time difference between successful formation of merozoites and PVM rupture was measured by quantitative live-cell imaging. The influx of GFP into the PV was used as a measure of PVM rupture. Imaging was started around 55 hpi and lasted for 12 hours. Representative images for WT (A) and PbPL-KO (B) parasites are shown. The upper images show the time point of successful merozoite formation, at which individual merozoites were visible and all larger yet undivided parts of the parasite cytoplasm, typical of the cytomere stage, had disappeared. The lower images show the time point of PVM rupture (GFP influx) in a host cell infected with a WT parasite and the end point of imaging of a host cell infected with a PbPL-KO parasite, in which the PVM did not rupture (the course of events are better visible in the corresponding <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004760#ppat.1004760.s005" target="_blank">S1</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004760#ppat.1004760.s007" target="_blank">S3</a> Movies). Scale bars = 10 μm. C) Time between formation of merozoites and PVM rupture. Each line represents the time difference between successful merozoite formation (beginning of line) and PVM rupture (end of line), as illustrated in A and B, and corresponds to one analyzed parasite. Continuous lines indicate parasites that did not rupture the PVM at all, which were not considered for determination of the average PVM rupture time in E. D) Percentage of merozoite-forming parasites that ruptured the PVM. The percentage of PVM rupture was determined in 3 (KO2, CMP2) or 6 (WT) imaging sessions, in which the number of parasites that successfully developed to merozoites within the first 6 hours of imaging was set to 100% in each experiment. Based on these, the percentage of parasites that successfully ruptured the PVM was calculated. E) Elapsed time from merozoite formation to PVM rupture. In D and E, means +/− SD are shown. Data were acquired in and are representative of 3 (KO2, CMP2) or 6 (WT) imaging experiments, in which a total of 61 WT, 43 KO2 and 37 CMP2 parasites were analyzed. For statistical analysis a one-way ANOVA followed by a Holm-Sidak multiple comparison test was performed (**** p < 0.0001, n.s. = not significant). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004760#ppat.1004760.s005" target="_blank">S1</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004760#ppat.1004760.s007" target="_blank">S3</a> Movies.</p

PbPL does not affect liver stage growth but plays a role in detached cell formation

<p>A) PbPL-knockout (KO2) sporozoites have a similar infectivity as wild-type (WT), and complemented PbPL-KO (CMP2) sporozoites. For determination of sporozoite infectivity, HepG2 cells were infected with 10,000 WT, KO2 or CMP2 sporozoites and the average number of infected host cells per well was quantified 48 hpi in triplicate. Numbers of infected host cells were not statistically different from each other (one-way ANOVA, p = 0.6892). B) PbPL-knockout parasites grow normally in size. HepG2 cells were infected with WT, KO2 and complemented PbPL-KO (CMP1–3) sporozoites. 48 hpi, parasite size (area) was determined by density slicing using ImageJ. For each parasite line, the average size of 50–100 parasites was determined in each of three separate experiments. Parasites did not show a significant difference in size (one-way ANOVA, p = 0.6567). C) PbPL-KO parasites produce fewer detached cells (DCs). DCs in the supernatant were counted at 65 hpi in triplicate and were normalized to the number of infected cells at 48 hpi. D) Detached cells from PbPL-KO parasites show an abnormal morphology. DCs were harvested at 65 hpi and the percentage of cells with an abnormal morphology was determined. DCs with abnormal morphology were defined by merozoites still being clustered in the PV in contrast to merozoites freely distributed in the host cell in DCs with normal morphology. A representative image of DCs with normal and abnormal morphology is shown. Scale bars = 10 μm. For all experiments means +/− SD of three to four independent experiments are shown. For statistical analysis a one-way ANOVA followed by a Holm-Sidak multiple comparison test was performed (** p < 0.01, *** p < 0.001, n.s. = not significant). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004760#ppat.1004760.s004" target="_blank">S4 Fig.</a></p