Proteolytic Maturation and Localization of M2AP Is Dependent on Expression of MIC2

<div><p>(A) Western blot of whole cell lysates showing steady-state expression levels. Blots were probed as indicated. Proform M2AP (proM2AP); mature M2AP (mM2AP).</p><p>(B) Expression and localization of M2AP in intracellular (IC) and extracellular (EC) parasites. Top panels: dual staining of M2AP (red) with AMA1 (green) in IC parasites showing mislocalization of M2AP in the PV of Δ<i>mic2e/mic2i</i> + ATc parasites. Bottom panels: immunostaining of M2AP (red) and GRA4 (green) in EC parasites shows a progressive increase in M2AP expression in dense granules, particularly in Δ<i>mic2e/mic2i</i> + ATc parasites. Arrows indicate the apical poles of parasites and an arrowhead indicates the PV.</p></div

Invasion Phenotypes Associated with Reduced MIC2 Expression

<div><p>(A<b>)</b> Illustration of the red-green invasion assay based on differential immunolabeling. Invading parasites (step 2) were counted as green.</p><p>(B) Quantification of the red-green invasion assay: red bars, attached extracellular parasites; green bars, invading and invaded parasites. A single asterisk indicates a statistically significant difference compared to tTA-dhfr; double asterisk indicates statistical difference compared to <i>mic2e/mic2i</i> + ATc (two-tailed Student's <i>t</i>-test). BAPTA-AM-treated parasites were included as a positive control for an attachment/invasion defect. Data are mean values ± s.e.m. of four separate experiments, each with three replicates and counting eight randomly selected fields per well.</p><p>(C) Correlation and linear regression of the percentage of MIC2 expression in cell lysates (left Y-axis and black line) and the percentage of MIC2 secretion (right Y-axis and red dashed line) with the numbers of invaded parasites.</p><p>(D<b>)</b> Attachment to glutaraldehyde-fixed host cells. An asterisk indicates that attachment was significantly lower than tTA-dhfr (<i>p</i> < 0.002, two-tailed Student's <i>t</i>-test). Data were compiled from three separate experiments, counting six fields per well per clone.</p><p>(E) Time-course invasion of tTA-dhfr and Δ<i>mic2e/mic2i</i> parasites ± ATc over an 8 h period. Data represent five individual experiments with three replicates within each experiment.</p></div

Mice Infected with MIC2-Deficient Parasites Produce Lower Levels of Inflammatory Cytokines

<p>Mice were infected with Δ<i>mic2e/mic2i</i> ± ATc and treated with or without ATc in drinking water. Serum was collected from three mice on days 4 (A), 6 (B), and 8 (C) post-infection. Cytokine levels were determined by a quantitative cytokine protein microarray analysis.</p

Δ<i>mic2e/mic2i</i> + ATc Parasites Fail to Reach High Tissue Levels and Are Cleared

<p>Parasite tissue burden of mice during infection with Δ<i>mic2e/mic2i</i> treated with and without ATc. Organs from three mice were isolated at each time point and analyzed by rtqPCR using parasite specific primers. The normal time-till-death Δ<i>mic2e/mic2i</i> without ATc is indicated by a “†.”</p

Gliding Phenotypes by Static Assay and Live Video Microscopy

<div><p>(A) Assessment of gliding motility by trail deposition. Top panels: DMSO is used as a solvent control. Scale bar, 15 μm. Bottom panel: UVT153753 (Enh) enhancer-treated parasites. Arrowheads indicate non-circular trails and arrows denote circular trails.</p><p>(B) Quantification of non-circular and circular trails is presented on the left half of the graph, UVT153753- or DMSO-treated Δ<i>mic2e/mic2i</i> ± ATc parasites are represented on the right half. Results are mean ± s.e.m of at least three experiments. Black bars, % non-circular gliding; grey hatched bars, % circular glide.</p><p>(C) Maximum projection images created from frames 1–60 (1 min videos taken at 1 frame per s). Red arrows, circular glide; red arrowheads, helical glide; closed black arrowhead, non-productive “gliding” parasite; open black arrowhead, twirling parasite.</p><p>(D) Quantification of types of movement in live gliding parasites; error bars represent standard deviation. An asterisk indicates a statistically significant difference between tTA-dhfr and Δ<i>mic2e/mic2i</i>+ ATc parasites (<i>p</i> < 0.02).</p><p>(E) Immunofluorescent images of anti-tubulin-stained tachyzoites showing helical and straight cytoskeletons.</p><p>(F) Bar graph represents enumeration of at least 85 tachyzoite cytoskeletons. Enumeration was performed in a blinded fashion.</p></div

MIC2-Depleted Parasites Are Avirulent in Mice and Confer Protective Immunity to Reinfection

<div><p>(A) 5 × 10⁴ tachyzoites of tTA-dhfr or Δ<i>mic2e/mic2i</i> ± ATc were intraperitoneally injected into four BALB/c mice in each group.</p><p>(B) 5-fold increases in infection dosage with Δ<i>mic2e/mic2i</i> +ATc; six mice were infected in each group.</p><p>(C) Six mice infected with Δ<i>mic2e/mic2i</i> + ATc were challenged with 150 tachyzoites of RH at day 16 post-infection. A group of control mice were infected with RH at day 16.</p></div

Conditional Expression of MIC2

<div><p>(A) Schematic diagram showing the strains in the study, including the parental strain (tTA-dhfr), and the <i>mic2e/mic2i</i> strain with both endogenous <i>(mic2e)</i> and induced <i>(mic2i)</i> copies of mic2. A knockout of <i>mic2e</i> leaves the regulatable <i>mic2i</i> in <i>Δmic2e/mic2i.</i></p><p>(B) Illustration of intracellular structures including the micronemes, dense granules, Golgi, PV, PV membrane, and host and parasite nuclei in a four-parasite vacuole.</p><p>(C) Expression and localization of MIC2 (green) in intracellular parasites with another micronemal marker MIC5 (red). Arrows indicate the apical pole of parasites. Scale bar, 5 μm.</p><p>(D) Western blot analyses showing MIC2 steady-state expression (left blot) and secretion (right blot) levels. Asterisk indicates the full-length myc-tagged MIC2, which is secreted in <i>mic2e/mic2i</i> and Δ<i>mic2e/mic2i</i> parasites based on probing with a myc antibody (unpublished data). Blots were probed with anti-MIC2 6D10 (top blots) and mouse anti-GRA1 (bottom blots), a dense granule protein, to normalize loading in all lanes. The bar graphs represent the relative percentages of MIC2 expressed in each strain and treatment compared to the reference tTA-dhfr level (100%) quantified from direct chemiluminescent imaging. Results are mean ± s.e.m, <i>n</i> = 3.</p></div

A Novel High Throughput Invasion Screen Identifies Host Actin Regulators Required for Efficient Cell Entry by <i>Toxoplasma gondii</i>

<div><p><i>Toxoplasma gondii</i> critically relies on cell invasion as a survival strategy to evade immune clearance during infection. Although it was widely thought that <i>Toxoplasma</i> entry is parasite directed and that the host cell is largely a passive victim, recent studies have suggested that host components such as microfilaments and microtubules indeed contribute to entry. Hence to identify additional host factors, we performed a high-throughput siRNA screen of a human siRNA library targeting druggable proteins using a novel inducible luciferase based invasion assay. The top 100 hits from the primary screen that showed the strongest decreases in invasion were subjected to confirmation by secondary screening, revealing 24 proteins that are potentially involved in <i>Toxoplasma</i> entry into host cells. Interestingly, 6 of the hits appear to affect parasite invasion by modifying host cell actin dynamics, resulting in increased deposition of F-actin at the periphery of the cell. These findings support the emerging notion that host actin dynamics are important for <i>Toxoplasma</i> invasion along with identifying several novel host factors that potentially participate in parasite entry.</p></div

Results of primary screen of an siRNA library targeting human druggable proteins to identify host factors involved in <i>Toxoplasma</i> invasion.

<p>A. 2,742 genes were screened in triplicate replica plates using U-Luc parasites. The values obtained with NT siRNA controls were set at 100% invasion and the values for target genes were calculated relative to the NT siRNA. The black dotted line represents the mean of the non-targeting siRNA control and the red-dotted lines indicate the 3 standard deviations (SD) boundaries of the mean. Each green dot represents the mean percentage invasion from triplicate samples for one target gene. Only those that showed >90% host cell viability after silencing are shown. B. Table summarizing overall screen. A total of 2,742 genes were subjected to primary screen. Of these 300 genes showed more than >3SD inhibition or enhancement of invasion and >90% host cell viability. The top 100 genes showing inhibition were subjected to secondary screen by luciferase assay and 81 of these were thereby validated. When these genes were subjected to high-content red-green assay, 24 of these showed significant inhibition of parasite invasion.</p

A schematic showing the location of 24 validated host-factors that appear to play roles in host cell entry by <i>Toxoplasma</i>.

<p>Arrows indicate enhancement of expression or activity whereas T-lines indicate inhibition of expression or activity. Dashed lines indicate hypothetical interactions. Relationships are based on the findings herein or from the literature. See text for additional descriptions.</p