Male and female <i>pat</i> null mutant gametes suffer egress defects.

<p>(A) Quantification of exflagellation phenotypes in male gametes of wild type, Δ<i>pat</i> and <i>P</i>. <i>berghei</i> complementation parasites. Wild type microgametes form 8 individual flagella, while <i>pat</i> null mutants remain intraerythrocytic. Scale bar: 5 μm. (B) DIC images of wild type and Δ<i>pat</i> microgametes. Scale bar: 5 μm. (C) SEM image of Δ<i>pat</i> intraerythrocytic microgamete. (D) Trapped microgametes have undergone nuclear division and formed flagella as evidenced by tubulin staining. Scale bar: 5 μm. (E) IFA time-course of female wild type and Δ<i>pat</i> gametocytes before (-) and 14 to 30 minutes post activation. PVM (green) is stained with anti-SEP1; the RBCM (red) with anti-Ter119. Wildtype cells egress 14 minutes after activation was induced while Δ<i>pat</i> parasites remain still trapped 30 minutes post activation. Scale bar: 5 μm. (F) TEM images of female wild type and Δ<i>pat</i> gametocytes before (top row) and after activation (bottom row). Scale bar: 1 μm. Insets 200% enlarged. n, nucleus.</p

PAT is required for G377 osmiophilic body secretion.

<p>(A) G377 secretion assay approach. (B) G377::mCherry cannot be detected in egress supernatants of <i>pat</i> null mutants. (C) In <i>pat</i> null mutants G377::mCherry is not secreted into the PV space. HSP70 was used as a saponin lysis control.</p

Expression of <i>pat</i> under the <i>ccp</i> promoter leads to the development of immotile and infection-deficient sporozoites.

<p>(A) Oocyst numbers of wildtype and <i>pat</i>^{<i>ccp</i>::<i>PP</i>} collected on days 12–14 after mosquito infection. (B) Infection rates of sporozoites from midgut (MGS), hemocoel (HLS) and salivary glands (SGS) infected with either wildtype (WT) or <i>pat</i>^{<i>ccp</i>::<i>PP</i>} show reduced salivary gland infections in the mutant. (C) Quantification of speed of sporozoites. (D) DIC live cell imaging of sporozoites. Arrow indicates direction of movement. Scale bar: 5μm. (E) Table summarising number of infected mice and respective prepatency periods after i.v. and bite-back (bb) infections. Data mean ± SEM.</p

PAT is expressed in transmission stage parasites and redundant for asexual development but essential for mosquito infection.

<p>(A) Predicted membrane topology of <i>P</i>. <i>berghei</i> PAT; the cartoon includes a C-terminal GFP tag. (B) Live cell imaging of <i>pat</i>::<i>gfp</i> parasites. In gametocytes and ookinetes PAT::GFP is distributed throughout the cell in a speckled, intracellular pattern, while it localizes to the plasma membrane (arrowheads) and micronemes (asterisks) in sporozoites. (C) Mice infected with either 5000 (5k) or 100 <i>pat</i> blood stage parasites develop parasitemia similar to wildtype. (D) Summary table of blood stage infection with prepatency period of parasites as determined in Giemsa-stained smears. (E) Oocyst numbers of wildtype and the following mutants: <i>pat; pat</i>::<i>gfp; pat;pat</i>^<i>PB</i>::<i>mcherry;pat; pat</i>^<i>PF3D7</i>::<i>mcherry;</i> parasites were quantified on days 12–14 after mosquito infection. Data mean ± SEM.</p

<i>P</i>. <i>berghei</i> PAT localizes to osmiophilic bodies (OB) but is not required for OB formation or trafficking.

<p>(A) PAT::GFP (model drawn to scale and indicating transmembrane domains in grey) traffics to the parasite plasma membrane upon activation in females. Scale bar: 5 μm. (B) PAT::GFP expression in exflagellating microgametes. Scale bar: 5 μm. (C) Female gametocyte expressing PAT::GFP and G377::mCherry. Co-localization was analyzed on white line using ImageJ. Scale bar: 5 μm. (D) Female gametocyte expressing PAT::GFP and PPLP2::mCherry. Co-localization was analyzed on white line using ImageJ. Scale bar: 5 μm. (E) G377::mCherry expression is unaltered in non-activated wildtype (WT) and Δ<i>pat</i> parasites. Scale bar: 5 μm. (F) G377::mCherry expression levels and trafficking of G377+ vesicles to the plasma membrane proceeds normal in activated wildtype (WT) and Δ<i>pat</i> parasites. Scale bar: 5 μm. (G) PPLP2::mCherry expression is unaltered in non-activated wildtype (WT) and Δ<i>pat</i> parasites. Scale bar: 5 μm. (H) PPLP2::mCherry expression levels and trafficking of G377+ vesicles to the plasma membrane proceeds normal in activated wildtype (WT) and Δ<i>pat</i> parasites. Scale bar: 5 μm.</p

PAT is required for microneme secretion.

<p>(A) Localization of SS::GFP::TRAP in wildtype background sporozoites. (B) Localization of SS::GFP::TRAP in <i>pat</i>^{<i>ccp</i>.<i>PP</i>} background sporozoites. (C) Quantification of SS::GFP::TRAP fluorescence intensity in wildtype and mutant. (D) Quantification of speed of sporozoites. (E) Quantification of gliding motility of wildtype sporozoites activated with pluronic acid (F) Model of microneme secretion assay developed for TRAP detection by Western blot. (G) Western blot of 25k sporozoites of SS::GFP::TRAP in wildtype and promoter swap mutant using anti-GFP antibodies. Scale bars: 5μm. Data mean ± SEM.</p

Analysis of the MMS RIT-seq screen.

<p><b>A, B.</b> Scatter plots showing the ratio of mapped RNAi target-specific reads for every gene (grey dots) in the RNAi-induced, MMS-treated population relative to the RNAi-induced, untreated population (MMS+/MMS-); gene location within the 11 megabase chromosomes is shown and dotted lines indicate 2-fold increase and decrease in MMS+/MMS- ratio. Genes are highlighted with roles in (A) homologous recombination (HR, red), mismatch repair (MMR, blue) and nucleotide excision repair (NER, green), or in (B) intraflagellar transport (IFT, red), mitochondrial replication (Mito rep, blue) and encoding histones (green). <b>C.</b> A pie chart of the distribution of all genes displaying an MMS+/MMS- ratio of less than 0.5, excluding 44 genes predicted to be VSGs. Hypothetical and hypothetical unlikely denotes genes for which there are currently no homology-predicted functions. Unknown denotes genes with homology-predicted functions that cannot be readily associated with the response to MMS damage. Finally, genes in seven classes of predicted functions with putative roles in responding to MMS are detailed. <b>D.</b> GO terms, within two headings, which show significantly increased frequency in the MMS+/MMS- <0.5 gene set relative to the whole GO gene set (IDs and further analysis are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.s003" target="_blank">S3 Table</a>).</p

Schematic outline of the whole genome <i>T</i>. <i>brucei</i> MMS RIT-seq screen.

<p>A whole genome tetracycline (Tet) inducible RNAi library was established in BSF <i>T</i>. <i>brucei</i> cells as a pool, within which random RNAi fragments target potentially all genes and provide unique identifiers. Cells were induced by Tet addition (+) for a total of 5 days, during which cells targeting RNAi against important genes (red, green, blue) are lost from or reduced in the population. In parallel, Tet+ cells were grown in the presence of methyl methanesulphonate (MMS, 0.0003%), which was added 1 day after RNAi induction. Cells carrying an RNAi target for a gene necessary for repair of MMS damage (purple) are specifically lost or depleted in the Tet+, MMS+ population relative to the Tet+, MMS- population. PCR was used to amplify all RNAi target fragments after five days of RNAi with or without exposure to MMS; the amplicons were sequenced and mapped to the genome. Read depth mapping is shown schematically for a gene whose RNAi causes loss of fitness without MMS (red), and for a gene whose RNAi causes loss of fitness only after MMS exposure (purple).</p

AUK2 displays dynamic nuclear localisation.

<p><b>A.</b> Western blot of whole cell extracts from wild type (WT) <i>T</i>. <i>brucei</i> and from two clones in which the <i>AUK2</i> ORF has been C-terminally fused to a tag encoding 12 myc epitopes (<i>AUK2</i>+/-::12myc). The blot was probed with anti-myc and anti-EF1α antiserum (as a loading control); a size marker is shown. <b>B.</b> Representative images of <i>AUK2</i>+/-::12myc cells from each cell cycle stage (denoted by N-K ratio). Anti-myc antiserum was used to visualise myc tagged AUK2 (green) and nDNA and kDNA were stained with DAPI (magenta); DC imaging shows cell shape; scale bars = 5 μm. <b>C.</b> Super resolution images of AUK2-12myc localisation. Only in the merged images are DAPI (blue) and anti-myc signals (green) shown in colour. Graphs show fluorescence intensity (arbitrary units; AU) over distance plotted for both the DAPI (blue) and anti-myc (green) signals. The white box represents the area from which the fluorescence intensity was measured; scale bar = 5 μm. <b>D.</b> 3D reconstruction of AUK2-12myc localisation in a 1N1K or 1N2K cell.</p

Loss of AUK2 results in aberrant cell and nuclear morphology.

<p><b>A.</b> Wild type (WT427), <i>AUK2</i>+/- and <i>auk2</i>-/- cells (clones CL1 and CL2) with morphology that deviated from the typical BSF cell shape were classified into three categories: rounded, clumps or aberrant. Each category is shown as a percentage of the total number of cells with morphological defects; >200 cells were counted for this analysis, which was conducted in triplicate. Error bars represent SEM, and * denotes a significant difference (p<0.005; Mann Whitney U test) in the percentage of rounded cells seen in WT <i>T</i>. <i>brucei</i>. <b>B.</b> Representative images of rounded, clumped or aberrant <i>auk2</i>-/-cells; in each case, the upper image shows DNA stained with DAPI (blue), while the lower image shows a merge of differential contrast and staining with anti-KMX-1 antiserum (to visualise βtubulin; magenta). Scale bar = 5μm. <b>C.</b> Representative examples of nuclei in <i>auk2</i>-/- mutants visualised by transmission electron microscopy. Boxes show higher magnifications of an unusual arrangement of nuclear membranes (lower left), or a nuclear ‘bleb’ (lower right). Scale bar sizes are indicated.</p