Molecular characterization of RON9.

<p>(A) Whole cell lysates of <i>Δhxgprt</i> were separated by SDS-PAGE, in non reduced (NR) or reduced (R) conditions, and mAb 2A7 was used to probe the membrane. (B) RON9 and RON9HP were affinity purified with mAb 2A7 from <i>Δhxgprt</i> whole cell lysate. Proteins identification was performed by mass spectrometry and subsequent searches against <i>Toxoplasma</i> database. (C) Western-blot using mAb 2A7, or anti-RON9rec antibodies produced against a TgRON9 recombinant-GST protein or anti-PEST antibodies produced against the TgRON9 PEST-repetition peptide. The three sera recognize the same protein migrating at a high molecular mass. Scale bar = 5 µm. (D) Co-immunostaining of intracellular <i>Δhxgprt</i> parasites with mAb 2A7 and anti-PEST antibodies. (E) Proteins from <i>Δhxgprt</i> lysates were immuno-purified using mAb 2A7, then separated by SDS-PAGE and probed with either mAb 2A7 or anti-PEST antibodies.</p

RON9 and RON10 form a highly stable complex, distinct from the AMA1/RON2/4/5/8 junctional complex.

<p>Immunopurification assays (A, B and D) were performed on lysates of <i>Δhxgprt</i> parasites in the presence of 1% NP40 (A) or 0.6% SDS (B) as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032457#pone.0032457-Besteiro1" target="_blank">[12]</a> with anti-RON9 (IP αRON9) or anti-RON10 (IP αRON10) antibodies. Immuno-purified proteins were detected using the same antibodies, as mentioned on the top of the figure, following SDS-PAGE separation in reduced condition. (C) Whole cell lysates of <i>Δhxgprt</i> were separated by SDS-PAGE, in non reduced (NR) or reduced (R) conditions, and mAb 2A7 (αRON9) and αRON10 (αRON9HP) were used to probe the membrane. The arrows indicate the size of the expected protein for each antibody and its corresponding molecular mass. (D) To verify the identification of the proteins co-immunopurified with mAb 2A7 (IP lanes), Western-blot was simultaneously carried out on a tachyzoites lysate (WB lanes) and revealed with secondary anti-mouse alone, mAb 2A7, anti-RON10, anti-AMA1 or anti-RON4 as mentioned on the top. The major band at 110 kDa indicated with an asterisk corresponds to a protein present in the ascitic fluid that binds to protein G-sepharose and was consistently released during the elution step (except when antibodies were CNBr cross-linked to sepharose, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032457#pone-0032457-g002" target="_blank">Fig. 2B</a>).</p

mAb 2A7 recognizes a rhoptry neck protein.

<p>(A) Co-immunofluorescence-staining on intracellular tachyzoites with the rhoptry bulb marker anti-ROP1, the rhoptry neck marker RON2 and mAb 2A7. Scale bar = 5 µm. (B) Electron microscopy of <i>T. gondii</i> parasites showing the rhoptry neck labeling with mAb 2A7 (black arrow). Scale bar = 0.5 µm.</p

<i>Δron9</i> parasites do not display any defect in replication and invasion <i>in vitro</i>, nor in virulence <i>in vivo</i>.

<p>(A) The intracellular replication of <i>Δron9</i> parasites was compared to that of the parental <i>Δku80</i>, 18 h post-infection. For this, the number of parasites per vacuole was counted after anti-SAG1 labeling of the parasite surface. Values represent means ± standard deviations (SD), n = 3, of 4 independent assays. (B) Invasion assays were carried out on freshly released highly synchronized tachyzoites. Invasion was allowed to take place for 5 min prior cells fixation and IFA processing as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032457#s4" target="_blank">material and methods</a> section. Values represent means ± standard deviations (SD), n = 3, from a representative experiment out of 3 independent assays. (C) Deletion of <i>RON9</i> did not reduce virulence in mice. Twenty tachyzoites of the indicated strains were injected i.p. into Balbc mice (<i>n</i> = 20), and mouse survival was monitored daily for 15 d.</p

RON10 is mis-targeted in <i>Δron9</i> parasites.

<p>To follow RON10HA biosynthesis in time and space, co-localization experiments have been performed on <i>Δron9</i>-R10HA parasites or RON10HA parasites as a control. Anti-HA antibody was used to follow RON10 fate, while anti-RON9 (A) and anti-RON4 (B) were used to label the rhoptry neck, anti-IMC1 antibodies (C) and anti-ISP1 antibodies (D) allowed us to follow the endodyogeny process during cells replication. Anti-proROP4 antibodies allowed detection of pre-rhoptries (E) and co-transfection of DER1-GFP plasmid allowed detection of the ER (F). Scale bars = 5 µm.</p

RON9 is mis-localized in <i>Δron10</i> parasites.

<p>(A) Scheme depicting the <i>RON10</i> disruption strategy. Folllowing a single cross-over event, the <i>RON10</i> wild type locus is replaced by a truncated version in frame with a Ty tag. The arrows represent the primers ML493/ML802 used to verify the 5′ integration at the <i>RON10</i> locus (p5′). (B) The correct vector integration was verified by PCR using primers ML493/ML802 (p5′), and primers ML936/ML937 located in the <i>RON2</i> gene were used as a positive control. (C) Western-blot using anti-RON10 antibodies was performed on <i>Δku80</i> or <i>Δron10</i> parasites, thus confirming the absence of RON10 in the <i>Δron10</i> strain. Anti-Ty antibodies show truncated forms of RON10 in the <i>Δron10</i> parasites compared to the control, and detection of RON4 was used as a loading control. (D) IFAs were performed on <i>Δku80</i> or <i>Δron10</i> parasites to follow RON10 and RON9 localizations. Scale bar = 5 µm.</p

RON9HP is a rhoptry neck protein, renamed RON10.

<p>(A) Western-blot carried on <i>Δku80</i> cell lysate reveals a major band at 140 kDa with anti-RON9HP antibodies. A protein of the same size is recognized with anti-HA antibodies on RON9-HP-HA₃ parasites. (B) Scheme depicting the C-terminal HA tagging of the endogenous copy of <i>RON9HP</i> (TGME49_061750). A C-terminal fragment of <i>RON9HP</i> gene was cloned in frame with an HA epitope in pHA₃-LIC-DHFR vector. Vector linearization followed by <i>Δku80</i> parasites transfection allowed the obtention of a RON9HP-HA₃ population by single homologous recombination event. (C) To verify the correct genomic integration of the vector into the <i>RON9HP</i> locus, PCR reactions were performed on gDNA from the parental <i>Δku80</i> strain or RON9HP-HA₃ parasites using primers ML176 and ML292 depicted by the arrows in (B). PCR allowed amplification of a 2.5 kb product from the recombinant parasites while no amplification was obtained with the parental strain. As a control, amplification of <i>ATG3</i> gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032457#pone.0032457-Besteiro2" target="_blank">[58]</a> was obtained with both gDNA using primers ML339 and ML340. (D) IFAs were carried on RON9HP-HA₃ parasites with anti-HA antibodies and mAb 2A7 or anti-RON2-4 antibodies. Scale bar = 5 µm. (E) Electron microscopy performed on RON9HP-HA₃ parasites labelled with anti-HA antibodies shows staining of the rhoptry neck. Scale bar = 0.5 µm. (F) Proteins from RON10-HA₃ parasites were immuno-purified with mAb 2A7 an run on SDS-PAGE prior to detection using mAb 2A7, anti-PEST or anti-HA antibodies.</p

Generation of <i>Δron9</i> parasites.

<p>(A) Scheme depicting the strategy used to obtain a <i>Δron9</i> strain. The 5′ and 3′ flanking regions (FR) of <i>RON9</i> were cloned on both sides of <i>HXGPRT</i> selection marker, and the vector was linearized with KpnI prior transfection of <i>Δku80</i> parasites. Following a double homologous recombination event, <i>RON9</i> was replaced by <i>HXGPRT</i>. The arrows represent the primers ML503/ML134 and ML136/ML504 used to verify the integration at the 5′ (p5′) and 3′ (p3′) side respectively. (B) PCR reactions to check the vector integration at the RON9 locus were performed on the gDNA of two independent clones of <i>Δron9</i> parasites. gDNA of <i>Δku80</i> strain was used as a control and amplification of the <i>ATG3</i> gene was used as a control of gDNA integrity. As expected, DNA fragments were amplified in the <i>Δron9</i> clones with the integration PCR while no DNA could be amplified from the parental strain. Primers ML339 and ML340 in <i>T. gondii ATG3</i> gene allowed DNA amplification for the 3 gDNAs tested. (C) mAb 2A7 and anti-RON2 antibodies were used in IFA experiments to verify the absence of expression of RON9 in <i>Δron9</i> parasites, compared to the control <i>Δku80</i>. Scale bar = 5 µm. (D) Western-blot using mAb 2A7 was performed on control <i>Δku80</i> and on <i>Δron9</i> parasites, thus confirming the absence of RON9 in the mutant strain. Detection of AMA1 protein was used as a loading control.</p

GPIs of RH and PTG strains induce TNF-α and IL-12p40 secretion by macrophages.

<p>(<b>A</b>) Macrophages were incubated for 24 h with medium alone, or with individual GPIs of the PTG strain extracted from 1×10⁸ parasites and assayed for TNF-α cytokine production. (<b>B</b>) Macrophages were incubated for 24 h with medium alone, or with individual GPIs of the RH (left panel) and the PTG strain (right panel) extracted from 2×10⁸ parasites, respectively, and assayed for IL-12p40 cytokine production. (<b>C</b>) Macrophages were incubated for 24 h with medium alone, or with GPIa (3 mM), a chemically synthesized structure of RH strain GPI III core glycan and assayed for IL-12p40 cytokine production. ^***P<0.0005 PTG GPI II compared to medium control. ND, not determined.</p

Protein-free Glc-GalNAc-substituted GPIs are clustered on extracellular parasites.

<p>Staining after permeabilization of HFF cells infected (72 h p.i.) with RH (upper panel) and PTG (lower panel) strains was performed using mAb T54 E10, recognizing both the EtN-PO₄ and the Glc-GalNAc side-branch epitopes of protein-free GPIs. Filled arrowheads point to intracellular parasites residing inside parasitophorous vacuoles. Unfilled arrowheads point to extracellular parasites. DIC, differential interference contrast.</p