Regulators of gametocytogenesis and the sexual stage of the life cycle.

<p>Differentiation of asexual blood stage parasites into gametocytes occurs in the vertebrate host. Ingestion in a mosquito blood meal results in activation of the gametocytes in the lumen of the gut, stimulated by an increase in Ca²⁺, a drop in temperature, and the presence of the mosquito-derived xanthurenic acid, leading to the production of male and female gametes. Fertilization and fusion of these gametes results in zygote formation. Eventual elongation of the zygote leads to the final stage of the sexual cycle, a highly motile ookinete. Genes involved at each stage of the process (as suggested by a number of functional analyses) and time spans for each stage are indicated.</p

Known and novel variant counts at a read-depth of ≥100 that overlapped genes and their flanking regions.

<p>Variants were judged as known by their being listed in dbSNP. Variant counts for those unique to both samples are also shown. The lowest frequency variant detected for each variant type (SNV, insertion, deletion, and substitution, respectively) in each sample was 0.88%, 3.7%, 10.07%, and 4.57% in the tumor, and 7.41%, 3.01%, 10.07%, 2.78% in the node.</p><p>Known and novel variant counts at a read-depth of ≥100 that overlapped genes and their flanking regions.</p

COSMIC mutations called in the primary tumor and axillary lymph node.

<p>Mutations were not present in the normal blood sample. Three mutations were unique to the tumor whilst the node harbored a single unique mutation: a frameshift in the coding sequence of <i>PDS5B</i>, a gene that interacts with the cohesion complex to maintain accurate sister chromatid segregation during mitosis and meiosis and suggested previously as a tumor suppressor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115346#pone.0115346-Denes1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115346#pone.0115346-Kim1" target="_blank">[28]</a>.</p><p>COSMIC mutations called in the primary tumor and axillary lymph node.</p

Total output (Gb) and depth of coverage for each sample.

<p>The amount that was successfully mapped to the reference genome for each sample was >97%, with a mean of 92.7% of each base achieving ≥40x coverage (or 96.4% for the exome fraction).</p><p>Total output (Gb) and depth of coverage for each sample.</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 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

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 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

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