TgCAX localisation.

<p>(A) Immunofluorescence assay of intracellular <i>Toxoplasma</i> tachyzoites transiently transfected with <i>TgCAX-Ty</i> expressed under the control of the tubulin promotor (upper panels) and when expressed stably (lower panels). Parasites are immunostained with the surface marker GAP45 (red). (B) TgCAX transiently transfected tachyzoites detected by anti-TgCAX antibodies and co-stained with anti-Ty antibodies and the nuclear marker DAPI (upper panels) or with GAP45 antibodies (lower panels). (C) TgCAX transiently expressed in tachyzoites co-localised with 2 mitochondrial markers, anti-HSP70 antibodies <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003191#ppat.1003191-Pino1" target="_blank">[26]</a> (upper panels) or transiently co-transfected SPTPSOD2GFP (SP: signal peptide, TP: transit peptide, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003191#ppat.1003191-Pino2" target="_blank">[27]</a>) (lower panels). Scale bars: 2 µm.</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

PbCAX-GFP localisation.

<p>High resolution deconvolution microscopy images of a live female gametocyte shortly after activation and a female gamete/zygote and an ookinete 24 h post activation expressing PbCAX-GFP and co-stained with MitoTracker Red CMXRos (10 ng/ml) and Hoechst 33342. Scale bar: 5 µm.</p

Essentiality of TgCAX in <i>T. gondii</i> tachyzoites.

<p>(A) Plaque assays were performed by incubating host cells with ΔKU80 and ΔCAX parasite strains for 7 days. (B) For the intracellular growth assay, ΔKU80 (open bars) and ΔCAX (closed bars) parasites were grown in host cells for 24 h and then the number of parasites per vacuole was determined. Bars represent the mean ± SEM of 3 independent experiments (each with 2 replicates). (C) For the ionophore-induced egress assay, parasites were cultured for 30 h before treatment with DMSO (open bars) or Ca²⁺-ionophore A23187 (3 µM; closed bars) for 5 min. The results are expressed as a percentage of ruptured vacuoles. Bars represent the mean ± SEM of at least 2 independent experiments.</p

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

<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

Ca²⁺ tolerance of yeast mediated by PfCAX.

<p>(A) RT-PCR analysis of <i>pfcax</i> and <i>spfcax</i> expression in yeast compared with yeast transformed with the empty vector control. (B) Saturated liquid cultures of K665 (<i>pmc1 vcx1</i>) yeast transformed with <i>pfcax</i> in piHGpd, N-terminally truncated s<i>pfcax</i> in piHGpd, <i>scrcax1</i> in piHGpd and empty vector alone were serially diluted to the cell densities as indicated, then spotted onto selection medium lacking histidine (SD –His) and YPD medium containing 50 mM CaCl₂. Yeast growth at 30°C is shown after 4 days. A representative experiment is shown. (C) K665 yeast transformed with the various plasmids described in (B) were diluted to a cell density of 0.5 <i>A</i>₆₀₀ nm and inoculated into YPD medium containing concentrations of CaCl₂ from 25 to 150 mM. Yeast cell density was determined by absorbance measurements at 600 nm following growth shaking at 30°C for 16 h. Bars represent the mean ± SEM of 4 independent experiments (each with 8–12 replicates). Vector only, open bars; sCrCAX, closed bars; PfCAX, horizontally lined bars; sPfCAX, diagonally lined bars.</p

Ca²⁺/H⁺ exchange activity of PfCAX.

<p>ΔpH-dependent uptake of 10 µM ⁴⁵Ca²⁺ into vacuolar-enriched membrane vesicles isolated from K665 (<i>pmc1 vcx1</i>) yeast transformed with empty vector (piHGpd) (A), <i>scrcax1</i> (B), <i>pfcax</i> (C), and <i>spfcax</i> (D), measured over a 22 min time course. Transport measurements were determined in the presence of 0.1 mM NaN₃, 10 mM KCl, 1 mM ATP, 1 mM MgSO₄ and 0.2 mM orthovanadate (a Ca²⁺-ATPase inhibitor). ⁴⁵Ca²⁺ uptake in the absence (open symbols) or presence (closed symbols) of 5 µM of the protonophore CCCP is shown. The Ca²⁺ ionophore A23187 was added at 12 min at a concentration of 5 µM to dissipate any vesicle-loaded ⁴⁵Ca²⁺, as indicated by arrows. Points represent the mean ± SEM of 4–5 independent experiments (where not shown errors lie within the symbols).</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

Geometric mean titres, seropositivity rates, and proportions of seroresponders to rVSVΔG-ZEBOV-GP measured by ZEBOV PsVNA50 in adults.

<p>Geometric mean titres, seropositivity rates, and proportions of seroresponders to rVSVΔG-ZEBOV-GP measured by ZEBOV PsVNA50 in adults.</p