Parasite development in the mosquito.

<p>A) Exflagellation in wild type (WT-GFP) and transgenic Pb<i>^pfpkg</i> parasites. The mean number of exflagellation centres was 7.0 for wild type and 6.8 for Pb<i>^pfpkg</i> parasites. Bar, mean ± SEM (Mann-Whitney U test: ns, not significant, p>0.05 compared to WT-GFP). B) Ookinete conversion in wild type (WT-GFP) and transgenic Pb<i>^pfpkg</i> parasites. Wild type conversion was 63% and Pb<i>^pfpkg</i> conversion was 10%. Data shown as mean ± SEM (Mann-Whitney U test: ***, p<0.001 compared to WT-GFP). C) Gut oocyst numbers in wild type (WT-GFP) and transgenic Pb<i>^pfpkg</i> parasites. Wild type infected guts contained 100 oocysts and Pb<i>^pfpkg</i> infected guts with less than 10 oocysts. Bar, mean ± SEM (Mann-Whitney U test: ***, p<0.001 compared to WT-GFP).</p

Generation of Pb<i>^pfpkg</i> parasites.

<p>A) Schematic diagram of the endogenous <i>pbpkg</i> locus, the targeting construct and the transgenic <i>pb^pfpkg</i> locus. Areas of 5′UTR and 3′UTR cloned into the targeting vector are indicated, S = spacer. Arrows 1–6 indicate binding sites for primers used in diagnostic PCR. Primers 1 and 2 were used to detect 5′ integration. Primers 3 and 4 were used to determine 3′ integration. Primers 5 and 6 bind specifically to the endogenous <i>pbpkg</i> and are used to confirm absence of the endogenous gene in the transgenic line. The area homologous to the probe used in Southern blotting and <i>Bcl</i>I restriction sites used for diagnostic digest are indicated. B) Diagnostic PCR used to determine integration of the targeting construct into the Pb<i>^pfpkg</i> transgenic line. C) Southern blot following <i>Bcl</i>I digest shows integration of the targeting construct as a specific 3.9 kb band and absence of the endogenous 5.1 kb band in the transgenic line (Pb<i>^pfpkg</i>) in comparison to wild type (WT-GFP). D) PFGE of wild type (WT-GFP) and mutant parasite (Pb<i>^pfpkg</i>) confirms integration into the correct chromosome. E) Western blot of asexual blood and ookinete stages confirm expression of PfPKG in the transgenic line (the transgenic PfPKG bands are 25.8% and 21.8% of the PbPKG band in the WT-GFP line in asexual blood and ookinete stages respectively).</p

Pantagruel

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Sequence alignments.

<p>Amino acid sequence alignment of PfCAX with PbCAX. The Clustal W program was used to generate the alignment. The residues highlighted by a bold black line above correspond to transmembrane segment predictions determined with the TMHMM program (<a href="http://www.cbs.dtu.dk/services/TMHMM/" target="_blank">http://www.cbs.dtu.dk/services/TMHMM/</a>). The residues highlighted by a bold green line below correspond to the conserved CAX regions, c-1 and c-2. Green shading denotes residues shown to be essential for Ca²⁺ transport in AtCAX1 and OsCAX1a <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003191#ppat.1003191-Kamiya1" target="_blank">[15]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003191#ppat.1003191-Shigaki3" target="_blank">[16]</a>. Yellow shading denotes the putative mitochondrial targeting motif <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003191#ppat.1003191-Rotmann1" target="_blank">[7]</a>. Grey shading denotes cleaved sequences for mitochondrially imported proteins predicted by MitoProt II – v1.101 (<a href="http://ihg.gsf.de/ihg/mitoprot.html" target="_blank">http://ihg.gsf.de/ihg/mitoprot.html</a>). Red shading denotes phospo-acceptor sites (GeneDB and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003191#ppat.1003191-Treeck1" target="_blank">[17]</a>). CAX sequences are from (accession no.): Pf, <i>Plasmodium falciparum</i> (XP_966025.1) and Pb, <i>Plasmodium berghei</i> (XP_678577.1). <i>Red letters</i>, identical or conserved residues in all sequences; <i>green letters</i>, conserved substitutions; <i>blue letters</i>, semi-conserved substitutions.</p

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

<i>Δpbcax</i> parasite ookinete conversion images.

<p>Immunofluorescence images of <i>in vitro</i> wild-type (WT) and <i>Δpbcax cl9 P. berghei</i> ookinete cultures, 8 and 24 h after gametocyte activation and in the presence of EGTA (10 mM; added directly prior to gametocyte activation). Parasites are immunostained for the female gamete/zygote/ookinete marker P28 (red) and co-stained with the nuclear marker Hoechst 33342 (blue). Development of elongated ookinetes was completely ablated in the <i>Δpbcax cl9</i> line, a phenotype which could be reversed by the removal of extracellular Ca²⁺ using EGTA. The arrow indicates the diffuse DNA staining observed in <i>Δpbcax cl9</i> parasites 24 h after gametocyte activation. Scale bar: 5 µm.</p

<i>Δpbcax</i> parasite phenotype.

<p>(A) Bar graph illustrating exflagellation in wild-type (WT) and <i>Δpbcax cl9</i> parasites. Exflagellation is presented as the numbers of exflagellation centres in 8 fields of view (magnification, ×40). Bars represent the mean ± SEM of 3 independent experiments. (B) Bar graph illustrating ookinete conversion in wild-type (WT or WT GFP) and <i>Δpbcax cl9 and cl5 gfp</i> parasites. The conversion rate is the percentage of P28-positive parasites that had successfully differentiated into elongated ‘banana-shaped’ ookinetes. Bars represent the mean ± SEM of 3–4 independent experiments except for WT GFP, where the bar represents the mean ± range (n = 2). (C) Ookinete conversion after crossing <i>Δpbcax cl9</i> parasites with female-defective <i>nek2</i> and <i>nek4</i> mutants (<i>Δpbnek2/4</i>) and a male-defective <i>map2</i> mutant (<i>Δpbmap2</i>). Wild-type parasites (WT) were used as a control. The conversion rate is the percentage of P28-positive parasites that had successfully differentiated into elongated ‘banana-shaped’ ookinetes. Bars represent the mean ± SEM of 3 independent experiments. (D) Bar graph illustrating the mean ± SEM numbers of oocysts per midgut (20 analysed) of wild-type (WT or WT GFP) and either <i>Δpbcax cl9</i> or <i>cl5 gfp</i> infected mosquitoes in three independent experiments.</p

<i>Δpbcax</i> parasite rescue with EGTA.

<p>Line graph illustrating ookinete conversion, measured at 24 h post-gametocyte activation, in wild-type (WT, open circles: WT GFP, open triangles) and <i>Δpbcax cl9</i> (closed circles) and <i>cl5 gfp</i> (closed triangles) parasites in the presence of 10 mM EGTA added at 0, 0.5, 2 and 3 h post-gametocyte activation. The conversion rate is the percentage of P28-positive parasites that had successfully differentiated into elongated ‘banana-shaped’ ookinetes. Data are expressed as the percentage of wild-type controls. Points represent the mean ± SEM (n = 3) except for WT, where the points represent the mean ± range (n = 2).</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