Morphological change of the <i>Plasmodium yoelii</i> merozoite after released from red blood cell (RBC).

<p>The major axis (A), minor axis (B), longitudinal cross section area (C), and circularity (D) were measured every 10 sec from RBC rupture to pre-invasion for invasive merozoites (n = 9–12). The average and the error representing one standard deviation were plotted in the line charts. Circularity was calculated using the following formula: Circularity = 4πArea/Perimeter². A value of 1 indicates a perfect circle and the value of 0 indicates an increasingly elongated polygon. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050780#pone.0050780.s001" target="_blank">Table S1</a> for detail values. (E) Time-lapse sequence of merozoite release of <i>P. yoelii</i> 17XL was recorded every 0.1 sec. Arrowhead indicates same invasive merozoite in the sequence and the arrow indicates an attachment of an immature flat elongated oval merozoite. A mature spherical invasive merozoite attached to the RBC and deformed RBC (Pre-invasion) at 180 sec. The bar represents 5 µm.</p

Kinetic difference in red blood cell (RBC) invasion between <i>Plasmodium</i> species.

<p>The median time for each step are shown as a box plot with whiskers from minimum to maximum. The interquartile range shows as box with the median marked as a horizontal line, minimum and maximum from lower and upper quartile represent error bar. <i>P</i> values were determined using the Mann-Whitney U test. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050780#pone.0050780.s001" target="_blank">Table S1</a> for detail values.</p

Time-lapse imaging for the rupture of schizont-infected red blood cells.

<p>Images were captured every 0.1 sec with transmitted light for <i>Plasmodium yoelii</i> 17XL (A), <i>P. yoelii</i> 17X1.1 (B), and <i>Plasmodium falciparum</i> 3D7 line (C). The bars represent 5 µm.</p

Phosphorylatable serines 21 and 743 of TgMyoA are important for iiEgress of <i>Toxoplasma</i> tachyzoites.

<p>A. Schematic representation of TgMyoA with relative location of S21 and S743. B. TgMyoA null mutants were complemented with Flag-tagged wild type TgMyoA or Flag-tagged mutant versions of TgMyoA. Localization of the Flag tagged transgenic TgMyoA was examined in intracellular parasites by IFA using anti-Flag antibody. TgGAP45 is used as a marker for the IMC. Scale bar, 2 μM. C. Western blot analysis of TgMyoA null mutants complemented with either wild type or the mutant TgMyoA was performed using anti-Flag antibody. SAG1 is used as loading control. D. Intracellular parasites of the TgMyoA null strain as well as those complemented with either wild type TgMyoA or S21A, S743A or S(21+743)A mutant TgMyoA were analyzed for egress efficiency by treating with the Ca²⁺ ionophore A23187 for 2 minutes. <i>n</i> = 3. Error bars, SEM. (*<i>P</i> < .05, students <i>t</i> test).</p

TgCDPK3-BirA* fusion protein is targeted to the plasma membrane and is functional.

<p>A. Western blot analysis of the <i>Tgcdpk3</i> mutant strain MBE1.1, or MBE1.1 complemented with either TgCDPK3-HA or TgCDPK3-BirA*-HA (BirA* fusion) using anti-HA antibody. The <i>Toxoplasma</i> protein SAG1 is used as loading control. B. Localization of TgCDPK3 and BirA* fusion protein in the <i>Tgcdpk3</i> mutant strain MBE1.1 was examined in intracellular parasites by IFA using anti-HA antibody. TgGAP45 is a marker for the inner membrane complex. Scale bar, 2 μM. C. Intracellular parasites of the MBE1.1 strain, or MBE1.1 complemented with either TgCDPK3-HA or the BirA* fusion were analyzed for egress efficiency by treating with the Ca²⁺ ionophore A23187 for 2 minutes. <i>n</i> = 3. Error bars, SEM. D. MBE1.1 parasites expressing either TgCDPK3-HA or TgCDPK3-BirA*-HA (BirA* fusion) were treated with biotin for 48 hours and biotinylated proteins were pulled down with streptavidin beads and analyzed by western using streptavidin-HRP antibody. Asterisks indicate proteins uniquely biotinylated in MBE1.1 + TgCDPK3-BirA*-HA parasites.</p

Constitutive phosphorylation of TgMyoA negates the requirement for TgCDPK3 during egress.

<p>A. Schematic representation of TgMyoA with relative location of S20, S21 S743 and S744. IFA (B) and Western blot analysis (C) of Flag tagged TgMyoA in MBE1.1 (<i>Tgcdppk3</i> mutant) parasites complemented with either wild type TgMyoA or SS20-21DD, SS743-744DD or S(20,21,743,744)D mutant TgMyoA was performed using anti-Flag antibody. TgGAP45 is used as a marker for IMC in the IFAs and SAG1 is used as loading control in Western blot. Scale bar, 2 μM. D. Intracellular parasites were analyzed for egress efficiency by treating with the Ca²⁺ ionophore A23187 for 6 minutes. <i>n</i> = 3. Error bars, SEM.</p

TgCDPK3-dependent phosphorylation of TgMyoA <i>in vivo</i> and <i>in vitro</i>.

<p>A. Phosphorylation status of TgMyo-A in MBE1.1 and MBE1.1 + CDPK3-WT parasites was analyzed using Phos-tag gel electrophoresis and Western blot using antibody against TgMyoA. Parasites were manually extracted from host cells and incubated in either intracellular (IC) or extracellular (EC) buffer for 2 minutes. B. Mapping of TgCDPK3 phosphorylation sites on TgMyoA by tiled peptide array analysis using purified recombinant TgCDPK3. Phosphorylation intensity of 15 amino acid length peptides that span full-length TgMyoA and are each shifted by 3 amino acid was detected using MultiGauge version 3.0. The serines and threonines in the two peptides that showed phosphorylation signal more than 100 PSL/mm² are indicated above the corresponding peaks. Fine mapping of TgCDPK3 phosphorylation sites on TgMyoA is shown in C and D. Phosphorylation by recombinant TgCDPK3 was tested on peptides that contained single, double and triple mutations of T14, S20 and S21 residues in the peptide ¹³ATALKKRSSDVDHAVD²⁸ (C) and S743, S744 and S748 residues in the peptide ⁷³⁶AALRLLKSSKLPSEE⁷⁵⁰ (D) <i>n</i> = 3, Error bars, SEM. (*<i>P</i> < .05, students <i>t</i> test).</p

Data collection and refinement statistics.

<p>Values in parentheses are for the highest resolution shell.</p>a<p>R_merge = ∑<i>_hkl</i> ∑<i>_i</i> |I<i>_hkl,i</i> - [I<i>_hkl</i>]|/∑<i>_hkl</i> ∑<i>_i</i> I<i>_hkl,i</i>, where [I<i>_hkl</i>] is the is the average of symmetry related observations of a unique reflection.</p>b<p>R_work = ∑|F_obs-F_calc|/∑F_obs, where F_obs and F_calc are the observed and the calculated structure factors, respectively.</p>c<p>R_free is R using 5% (apo) or 10% (complex) of reflections randomly chosen and omitted from refinement.</p

The sporo- and generic versions of RON2-domain 3 interact only with their respective sporo- and generic AMA1 partners.

<p>A. Molar equivalents of GST, GST-gD3, or GST-sD3 were added to lysates of RHΔ<i>hxgprt</i> and after NP-40 solubilization the material that did not bind to the GST fusions (“flow-through”) or that was pelleted with the fusions (“pull-down”) was resolved by polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies to generic AMA1 or SAG1 as a control for loading and nonspecific pelleting. Parentheses indicate parasite equivalents of the different fractions relative to input (1×). Size markers indicated in kDa. B. GST-pull-down experiments were performed as described in (A) except using RHΔ<i>hxgprt</i> that were transiently expressing sporoAMA1-HA and the sporoAMA1 was detected using antibodies to the HA-epitope tag.</p

SporoAMA1 presents a highly guarded apical groove.

<p>A. Stacked three domain architecture of sporoAMA1 shown in the predicted organization to the <i>T. gondii</i> sporozoite plasma membrane with the three ectodomains indicated as DI in burgundy, DII in green and DIII in blue. Disulfides are shown as yellow sticks. Dotted line indicates extended Pro/Glu rich region between the conserved portion of DIII and the transmembrane domain (grey rectangle) that leads through to the C-terminal domain (grey oval). B. Apical view of apo sporoAMA1 structure, with core structure shown as grey surface and DI surface loops that guard the apical groove shown as burgundy cartoon and semi-transparent surfaces, and the DII loop as a green cartoon and semi-transparent surface. N-linked glycosylation on Asn230 shown as sticks. Numbers indicate surface loops that frame the apical groove. Inset: sporoAMA1 DII loop residues Phe376 and Trp377 (green) are pinned into the apical groove by Pro227 at the tip of loop 2 (burgundy).</p