HRPII causes vascular leakage <i>in vivo</i>.

<p><b>(A)</b> Scheme of experimental design. Two doses of HRPII or BSA (200 μg) were injected into 4-week old female C57Bl/6 mice at 0 and 24 hours. At 48 hours, fluorescein levels in the cortex (<b>B</b>) and cerebellum (<b>C</b>) of the mice was measured. HRPII treatment was significantly different from control by two-tailed t-test, p = 0.01 (cortex) and p = 0.02 (cerebellum). Data are mean values +/-SEM for 8–16 mice per group accumulated over 3 independent experiments.</p

HRPII-mediated vascular leakag<i>e</i> is blocked by antibody to IL-1β.

<p>Mice were infused as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177142#pone.0177142.g001" target="_blank">Fig 1</a>, with HRPII plus an isotype antibody (Iso, positive control), HRPII plus anti-IL-1β antibody (experimental condition) or anti-IL-1β antibody alone (negative control). Untreated mice (no HRPII, no antibody) served as a further control. Vascular leakage in mice infused with HRPII/isotype is statistically significantly different from mice infused with HRPII/ anti-IL-1β, p = 0.01 (cerebellum) and p = 0.01 (cortex), by two-tailed t-test; p = 0.003 (cerebellum) and p = 0.06 (cortex) by ANOVA one-way variance with significance between HRPII/isotype and HRPII/ anti-IL-1β. Data are mean values +/-SEM for 6–12 mice per group accumulated over 3 independent experiments.</p

HRPII reduces survival time in an experimental cerebral malaria model.

<p>(<b>A</b>) Survival curves of 4-week old female mice infused with 50 μg of BSA or HRPII prior to infection with <i>P</i>. <i>berghei</i> ANKA (10⁵ parasites). Shown are the means for n = 24 to 27 mice pooled from four independent experiments. Curves are significantly different, p = 0.03, by the log-rank (Mantel-Cox) test. Mean time to death for HRPII = 11.5 days and for BSA = 16 days, p = 0.018, by two tailed t-test. (<b>B</b>) Mice displaying cerebral malaria-like symptoms died at low parasitemia by day 10, yet parasitemias between HRPII-infused mice and controls were closely matched on each day. Representative data from one of three experiments shown in panel A, 10 mice per group.</p

HRPII causes vascular leakage <i>in vivo</i>.

<p><b>(A)</b> Scheme of experimental design. Two doses of HRPII or BSA (200 μg) were injected into 4-week old female C57Bl/6 mice at 0 and 24 hours. At 48 hours, fluorescein levels in the cortex (<b>B</b>) and cerebellum (<b>C</b>) of the mice was measured. HRPII treatment was significantly different from control by two-tailed t-test, p = 0.01 (cortex) and p = 0.02 (cerebellum). Data are mean values +/-SEM for 8–16 mice per group accumulated over 3 independent experiments.</p

Pantagruel

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Electron micrographs illustrating the process of cell death observed in oocysts of the ΔRep and ΔNΔRep mutants.

<p><b>A.</b> Low power of a ΔNΔRep oocyst with a degenerating, undifferentiated central cytoplasmic mass. Bar is 10 µm. <b>B.</b> Detail from the degeneration of a ΔNΔRep oocyst similar to that in <b>A</b> showing a dilated nuclear membrane containing a number of nuclei (N) exhibiting peripheral chromatin condensation. Bar is 100 nm. <b>C.</b> Low power of a ΔRep oocyst in which sporozoite formation had occurred but now exhibited features of cell degeneration. Bar is 10 µm. <b>D.</b> Detail of the nuclei observed in an intact ΔNΔRep oocyst showing the absence of any peripheral nuclear condensation. Bar is 100 nm. <b>E.</b> Longitudinal section through a sporozoite showing the nucleus with peripheral chromatin condensation and dilatation of the nuclear membranes. N – nucleus. Bar is 500 nm. <b>F.</b> Low power of a ΔRep oocyst in which there is a cross section of a central mass of degenerating sporozoites (S). Bar is 10 µm. <b>G.</b> Histogram comparing the relative number of immature mature and degenerate oocysts at two time points (12–14 days and 18–21 days) for WT, ΔRep and ΔNΔRep oocysts. (Based on EM examination of multiple midguts from multiple experiments – number of oocysts evaluated: 405 wild type; 236 ΔRep mutant; 165 ΔNΔRep mutant).</p

Electron micrographs showing unusual aspect of inner membrane complex development of the ΔNΔRep mutant.

<p><b>A.</b> Low power of a mid-stage oocyst showing the retracted plasmalemma and areas of IMC invagination into the cytoplasmic mass (arrows). Bar is 1 µm. <b>B.</b> Detail of the surface of an early oocyst showing extensive growth of the IMC (arrows) but no evidence of budding. Bar is 100 nm. <b>C.</b> Detail of a more advanced stage in development showing areas of abnormal IMC/plasmalemma formation and invagination into the cytoplasmic mass of the sporoblasts (arrows). Bar is 100 nm. <b>D.</b> Cross section through two sporozoites showing loss of shape, adhesion, and folding of the plasmalemma of the sporozoites (arrows). R – rhoptry; Mt - microtubule. Bar is 100 nm. <b>E, F.</b> Enlargement of cross sections through ΔNΔRep (E) and WT (F) parasites, showing the relative distance between the plasmalemma of adjacent sporozoites. Note in the ΔNΔRep mutant the plasma membranes appeared tightly adhered (similar to that between the IMC membranes) (<b>E</b>) compared to the significantly wider space observed in the WT (<b>F</b>). I – IMC; Mt - sub-pellicular microtubules; P – plasmalemma. Bar is 100 nm.</p

Generation of CSP repeatless mutants.

<p><b>A.</b> Schematic representation of CSP structure in wild type and mutant parasites ΔRep and ΔNΔRep. Region I is shown as hatched, repeat region as light grey and the TSR domain as dark grey. <b>B.</b> Western blot analysis of wild type (WT), WT-GFP and RCon as control parasites and the two repeat mutants: ΔRep and ΔNΔRep. Lysates from midgut sporozoites or infected midguts were probed using antisera specific for each of the three CSP domains: polyclonal antisera specific for the CSP NH₂-terminus, anti-repeat region (mAb 3D11) and polyclonal antisera specific for the CSP COOH-terminus. Molecular weight markers (kDa) shown on the left of each gel photograph.</p

Light microscopy analysis of cell death in ΔRep oocysts.

<p><b>A.</b> Photographs of representative mosquito midguts on days 14 and 21 post infection. Control and ΔRep parasites express GFP in most oocysts at day 14 post infection whereas by day 21 most of the ΔRep oocysts have lost the GFP fluorescence. Top panel shows a mosquito midgut and lower panel shows representative oocysts at higher magnification with degenerated features and absence of GFP fluorescence due to loss of plasmalemma integrity at day 21 post infection in ΔRep oocysts. <b>B.</b> Quantification of GFP positive and GFP negative oocysts at 14, 16, 18, and 21 days post infection for control and ΔRep oocysts.</p

Electron micrographs of sporogony in WT, ΔRep and ΔNΔRep mutants.

<p>A series of electron micrographs of oocysts illustrating the progressive stages in the sporogonic process undergone by WT, ΔRep, and ΔNΔRep oocysts in the mosquito midgut. The structure of the oocysts at the end of the growth phase was similar for WT (<b>A</b>) and both mutants. (<b>B, C</b>) The initiation of sporozoite formation with retraction of the plasmalemma was also similar (<b>D-F</b>). However, while sporozoite formation continued by a budding process in both WT (<b>G</b>) and the ΔRep mutant (<b>H</b>) there was no budding seen in the ΔNΔRep mutant (<b>I</b>). This budding process continued until the sporozoites were fully formed in the WT (<b>J</b>) and ΔRep (<b>K</b>). In contrast the mature oocyst of the ΔNΔRep mutant contained a tightly adhered mass of sporozoites (<b>L</b>). Bars represent 10 µm.</p