<i>In silico</i> integrative genomic search strategy to identify <i>P. falciparum</i> invasins.

<p>(<b>A</b>) To compile a list of proteins that include invasins, <i>P. falciparum</i> genes with homologues in the tight junction forming <i>T. gondii</i>, <i>P. berghei, P. chabaudi, P. vivax, P. yoelii</i> and <i>P. knowlesi</i> were selected (blue). Orthologues found in the non-tight junction forming <i>C. parvum</i> and <i>C. hominus</i> (pink) were removed from the dataset. Transcriptomic and proteomic data from <i>P. berghei</i> and <i>P. gallinaceum</i> ookinetes (brown) was used to remove proteins involved in motility but not invasion (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046160#pone.0046160.s003" target="_blank">Table S1</a> and Supplemental Experimental Procedures for data sources). (<b>B</b>) The top 50 candidate invasins ranked according to <i>P. falciparum</i> asexual cycle maximum fold change in transcript expression and relative protein abundance in <i>P. falciparum</i> merozoite and sporozoite proteomes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046160#pone.0046160.s004" target="_blank">Table S2</a>). Accession numbers are from PlasmoDB version 8.2. Number of transmembrane domains (TM), presence of a signal peptide (SP) and expression maximum during intra-erythrocytic cycle are listed. Heat diagram demonstrate intra-erythrocytic expression levels, given across 48 hr lifecycle with red representing high relative and green low relative expression. Proteins tagged in this study with an HA epitope are highlighted in yellow, and proteins where tagging was attempted but unsuccessful are highlighted blue.</p

Spatial localisation of different rhoptry proteins before and during merozoite invasion.

<p>(<b>A</b>) IEM of free PfRON2-HA merozoites (pre-invasion) dual labeled with immunogold anti-HA (18 nm) and rhoptry bulb marker RAP1 (12 nm). Scale bar = 0.2 µm. (<b>B</b>) IEM of free PFF0645c-HA merozoites (pre-invasion) dual labeled with immunogold anti-HA (18 nm) and rhoptry bulb marker RAP1 (12 nm). Scale bar = 0.2 µm. (<b>C</b>) Widefield IFA of E64-treated schizonts (to prevent egress – see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046160#s4" target="_blank">Materials and Methods</a>) labeled with anti-PfRh2, anti-PfRON4 and DAPI. Scale bar = 5 µm. (<b>D–G</b>) Independent replicate imaging of merozoites from (<b>D</b>) PfRON2-HA, (<b>E</b>) PfASP-HA (two classes of distribution seen), (<b>F</b>) RAP1 and (<b>G</b>) PFF0645c-HA mid-way through invasion colabeled with anti-PfRON4 and DAPI.</p

RON2 follows the tight junction during <i>P. falciparum</i> and <i>P. berghei</i> merozoite invasion.

<p>(<b>A</b>) Widefield 3D imaging of PfRON2-HA merozoites labeled with anti-HA, anti-PfRON4 (tight junction) and DAPI showing early, mid and late invasion events as well as parasites captured within 10 min post-invasion (<10 min p.i.). Scale bar = 5 µm. (<b>B</b>) IEM of free, invading and post-invasion (<10 min p.i.) PfRON2-HA merozoites labeled with anti-HA (white arrows). Scale bar = 0.2 µm. (<b>C</b>) Widefield 3D imaging of PbRON2-myc merozoites labeled with anti-myc, anti-parasite actin and DAPI captured mid invasion. IFA Scale bars = 5 µm. 3D reconstruction with 0.2 µm grid intervals. (<b>D</b>) 3D SIM of a PfRON2-HA merozoite captured mid way through invasion and labeled with anti-HA, anti-PfRON4 and DAPI. 3D reconstruction with 0.2 µm grid intervals.</p

PFF0645c is released from the rhoptries only after completion of merozoite invasion.

<p>(<b>A</b>) IEM of PFF0645c-HA merozoites pre- and post-erythrocyte invasion labeled with immunogold anti-HA (white arrows). Scale bar = 0.2 µm. (<b>B</b>) Widefield 3D imaging of PFF0645c-HA merozoites labeled with anti-HA, anti-PfRON4 and DAPI showing early, mid and late invasion events. Scale bar = 5 µm. 3D reconstruction with 0.2 µm grid intervals. (<b>C</b>) Widefield 3D imaging of PFF0645c-HA early rings (<10 min post-invasion) labeled with anti-HA, anti-PfRON4 (∼PVM) anti-RAP1 (PV) or anti-MSP1 (plasma membrane) and DAPI. Scale bar = 5 µm. 3D reconstruction with 0.2 µm grid intervals.</p

Cellular localisation of invasins in <i>P. falciparum</i> schizonts and <i>T. gondii</i> tachyzoites.

<p>(<b>A–E</b>) IFA of schizonts from HA tagged <i>P. falciparum</i> parasite lines labeled with anti-PfRON4, to mark the rhoptry neck, DAPI, to mark nuclei and anti-HA. (<b>A</b>) PfRON2 (<b>B</b>) PFF0645c (<b>C</b>) PfASP and (<b>E</b>) PF14_0578. (<b>D</b>) PF14_0375 was also colabeled with MitoTracker® Deep Red, labeling mitochondria, and anti-PfACP, labeling apicoplasts. (<b>F</b>) IFA of intracellular TGME49_115220-YFP tachyzoites (PFF0645c orthologue in <i>T. gondii</i>), colabeled with TgGAP45 (IMC) and TgMIC2 (micronemes). (<b>G</b>) Early intracellular development and extracellular TGME_116540-mCherryHA tachyzoites (PF14_0578 orthologue in <i>T. gondii</i>) colabeled with anti-TgGAP45 (IMC) and anti-TgSAG1 (surface). <i>C. septicum</i> alpha-toxin treatment swells the plasma membrane away from IMC. Scale bar = 5 µm throughout.</p

PfASP shows a dual localisation to both the tight junction and merozoite apex during erythrocyte invasion.

<p>Widefield 3D imaging of ASP-HA merozoites labeled with anti-HA, anti-PfRON4 and DAPI showing early invasion events (<b>A</b>) and two classes of mid/late invasion (<b>B, C</b>) distributions seen during invasion. Scale bar = 5 µm. 3D reconstruction with 0.2 µm grid intervals.</p

PMV conservation and expression.

<p>(A) Structure and size of PMVHA proteins used in this study. Catalytic dyad residues DTG/DSG are shown including Asp to Ala mutations* in red. TM, transmembrane domain. (B) Immunoblot of infected erythrocytes with α-HA antibodies shows expression of PMVHA proteins in <i>P. falciparum</i>. Sizes indicate that the signal peptides were removed (PfPMVHA, 69.1 kDa; PvPMVHA, 60.9 kDa). (C) Immunoblotting of infected erythrocytes with rabbit α-PfPMV antibodies (Rα-PfPMV) validates they are specific for PfPMV. Endogenous PfPMV is the lower band (lanes 1, 3, 4, 5), and the larger band corresponds to 3× HA-tagged PfPMV (lanes 2, 4). Aldolase is a loading control in (B) and (C) and shows slight overloading of some lanes compared to others. (D, Top) Immunofluorescence micrographs show rabbit α-PfPMV antibodies (Rα-PfPMV, green) label PfPMV in the ER. Colocalizations were performed with mouse α-PfPMV antibodies (Mα-PfPMV, red), shown previously to label PMV in the ER <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897-Klemba1" target="_blank">[16]</a>. (Middle) α-HA antibodies (red) label PfPMVHA (Top) and PvPMVHA (Bottom) in the parasite ER. (Bottom) α-HA antibodies (red) label PvPMVHA in the ER, as shown by clocalization with ERC (green). (E) Immunopurified PfPMVHA and PvPMVHA cleave KAHRP peptides containing the PEXEL sequence RTLAQ but not peptides containing point mutations R>K, L>I, or RL>A. Pf and Pv PMVmutHA proteins with catalytic D>A mutations did not cleave the KAHRP RTLAQ peptide. (F) Overexpression of PfPMVmutHA from episomes in <i>P. falciparum</i> 3D7 impairs growth relative to expression of a similar episomal construct encoding a mini PfEMP1HA reporter (miniVarHA). Parasites expressing episomes were selected on 5 nM WR99210 (WR). Two starting inocula were used in triplicate wells, and parasitaemia was determined at 72 h. *<i>p</i><.0001 (<i>t</i> test). Data are mean ± SEM from duplicate experiments.</p

WEHI-916 is lethal to <i>P. falciparum</i> 3D7.

<p>(A) Dose-response curves of <i>P. falciparum</i> 3D7 in the presence of 916, 024, or 025. EC₅₀ values are shown. (B) Parasitemia measured at 72 h (<i>y</i>-axis) following drug treatment at rings (30 min postinvasion) and replacement of the medium with inhibitor-free medium (wash-out) at the time intervals shown (<i>x</i>-axis). (C) Parasitemia at 72 h (<i>y</i>-axis) after replacement of inhibitor-free medium with media containing compounds at the intervals shown (<i>x</i>-axis). Parasitemia was determined by FACS in (A–C) and is relative to DMSO treatment in (B) and (C). Concentrations are as follows: 916, 024, 025 (15 µM); CQ, chloroquine (150 ng/ml); ART, artemisinin (100 ng/ml). Error bars in (A–C) are mean ±SEM from duplicate experiments. (D) Light micrographs of Giemsa-stained parasites 16 and 32 h after drug treatment at early rings (15 µM). 916-treated parasites failed to develop into trophozoites and did not recover. Ring parasites treated with E-64 (10 µM) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897-terKuile1" target="_blank">[22]</a> contained swollen food vacuoles (arrow) due to inhibition of proteases involved in hemoglobin degradation; however, treatment with DMSO, 916, 024, or 025 did not cause swelling. Swelling was quantified using 500 infected cells per condition in duplicate. Scale bar is 6 µm.</p

PMV knockdown or overexpression modulates sensitivity to WEHI-916.

<p>(A) Immunoblot with Rα-PfPMV antibodies shows successful integration of the PMVHA-<i>glmS</i> or -M9 plasmid (M9 is an inactive <i>glmS</i> riboswitch control). The upper band (α-PfPMV blot) in lane 1 (denoted by *) is nonspecific. The same blot is shown below, probed with α-HA antibodies. HSP70 is a loading control. (B) Knockdown of PMV in <i>P. falciparum</i> NF54 following 5 mM GlcN treatment. (Left) 0 h GlcN treatment of trophozoites causes no knockdown. (Center) The 24 h GlcN treatment of trophozoites causes ∼80% knockdown of PMV in subsequent rings compared to “−GlcN.” (Right) 48 h GlcN treatment of trophozoites causes ∼90% knockdown of PMV in subsequent trophozoites compared to “−GlcN.” A small degree of knockdown is seen for M9, indicating GlcN has a minor effect. (C) PMV knockdown by GlcN has no significant effect on parasite growth rate (<i>p</i> = .6250). Trophozoites were treated with 0 mM or 5 mM GlcN and parasitaemia determined 48 h later by flow cytometry. Data are % growth “+GlcN” relative to “−GlcN,” and data are mean ±SEM of a representative of duplicate experiments. (D) PEXEL processing of PfEMP3-GFP in <i>P. falciparum</i> parasites expressing PMVHA-<i>glmS</i> is reduced more by 916 treatment when PMV is knocked down [+GlcN (5 mM for 48 h prior to addition of 916)]. Densitometry shows the ratio of uncleaved to PEXEL-cleaved protein in each lane beneath the blot. Note that PfEMP3-GFP expression is lower in “+GlcN” parasites despite relatively similar HSP70 levels. (E) Dose-response curves of <i>P. falciparum</i> expressing PMVHA-<i>glmS</i> shows parasites have enhanced sensitivity to 916 following PMV knockdown (3.3-fold decrease in EC₅₀). Parasitemia was determined 72 h after addition of 916 to ring parasites with or without PMV knockdown (knockdown ring parasites were obtained by adding 6 mM GlcN to trophozoites for 24 h). GlcN and 916 were maintained in the culture medium throughout. (F) Dose-response curves of <i>P. falciparum</i> overexpressing PvPMVHA or a mini PfEMP1HA reporter (miniVarHA) in the presence of 5 nM WR99210 show parasites have increased resistance to 916 when PMV is overexpressed (1.9-fold increase in EC₅₀).</p

Concentration of actin labelling in the nucleus and around the nuclear periphery.

<p>Widefield IFA of representative <i>P. berghei </i><b>A</b>) ookinetes and <b>B</b>) sporozoites that show pronounced nuclear labelling using rabbit anti-Act_239–253 (Green) surface markers Pbs28 or PbCSP (Red) and DAPI (Blue). Scale bar = 5 µm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032188#pone.0032188.s009" target="_blank">Movie S5</a>. <b>C</b>) Widefield IFA of <i>P. falciparum</i> rings labelled with rabbit anti-Act_239–253 (Red) and DAPI (Blue). <b>D</b>) As <b>C</b> but following 6 hour JAS treatment. <b>E</b>) Two colour widefield IFA using rabbit anti-Act_239–253 (Red), rat anti-ERD2 (Green) and DAPI (Blue) in absence or presence of 1 µM JAS. All scale bars = 5 µm.</p