4 research outputs found
siRNAs against CDA1 Antagonize Compound 1 Inhibition of Parasite Growth
<div><p>(A) HFF cells transfected with the siRNA SmartPool against CDA1 and co-treated with Compound 1 prior to parasite infection allow normal parasite growth (black boxes) and BAG1 expression (dark bars). Compare growth in untreated controls (black circles) with growth (grey boxes) and BAG1 expression (light bars) in Compound 1–treated cells.</p><p>(B) Concurrent with the restoration of normal parasite growth and BAG1 expression, we observed the loss of Compound 1–induced CDA1 mRNAs as measured by RT-PCR of total RNA at 24, 48 following transfection with siRNA and Compound 1 pretreatment. Lamin A/C controls demonstrate siRNA-mediated knockdown of individual genes. Transfection with siRNAs against Lamin A/C, with nonspecific siRNAs or with LipofectAMINE alone, was unable to antagonize Compound 1 inhibition of parasite growth or induction of BAG1 expression (results not shown). RT-PCR primers specific to GAPDH were used as a control for RNA quality. RT-PCR experiment: C = Compound 1 pretreatment (3 h/3 μM) only; E = HFF cells pretreated with both Compound 1 and siRNA(s) against either CDA1 or Lamin A/C. Protein blot: L = HFF cells pretreated with LipofectAMINE, but without siRNAs; si = HFF cells pretreated with siRNAs against either CDA1 or Lamin A/C, but without transfection reagent (LipofectAMINE); E = same as above.</p><p>(C) Co-transfection of HFF cells with siRNAs 1 to 4 separately and then treatment with Compound 1 prior to parasite infection demonstrate that siRNAs 2 and 3 were responsible for antagonizing the Compound 1–induced inhibition of parasite growth as measured by average parasites per vacuole at 48 h postinfection. Compare light bars for siRNAs 2 and 3 with untreated parasites (labeled no siRNA), and also with Compound 1–treated parasites (labeled Compound 1). siRNAs 1 and 4 do not significantly antagonize the Compound 1–induced inhibition of parasite replication.</p></div
Compound 1–Induced Parasite Growth Inhibition and BAG1 Expression
<div><p>(A) Parasites allowed to invade an HFF monolayer, and then treated 1, 2, 3, or 6 h with 3 μM Compound 1, express maximal levels of BAG1 after 3-h treatment (light bars). Similar BAG1 expression was observed with host monolayers that were pretreated with Compound 1 (dark bars).</p><p>(B) Phase image of pH-induced vacuole is shown at 72 h postinfection. Following immunostaining with dolichos lectin, note the phenotypic similarities between cyst wall formation in alkaline media and that induced by 3 μM Compound 1.</p><p>(C) Treatment of extracellular (out) or intracellular parasites (in) with 3 μM Compound 1 for 3 h was unable to induce BAG1 expression following infection of untreated HFF monolayers (72 h postinfection). Compare percent BAG1 induction with VEG parasites that remained in a pre- or post-treated culture for 72 h.</p><p>(D) Type I RH and GT-1 tachyzoites inoculated into Compound 1 (3 μM for 3 h)–pretreated HFF cells displayed little or no BAG1 expression (light bars), while in type III VEG or type II ME49B7 cultures, 68% to 75% of parasites express BAG1. Note that parasite growth is dramatically affected in VEG and ME49B7 cultures but not in RH or GT-1 parasites (black bars). The <i>x</i>-axis in this graph is used to plot both percent BAG1<sup>+</sup> and average vacuole size.</p></div
Compound 1–Induced Slowing of Parasite Growth and BAG1 Expression Is Mediated by New Host Cell Gene Transcription
<div><p>(A) HFF cells were treated or co-treated for 3 h with 3 μM Compound 1 and 30 or 60 μM RNA polymerase II inhibitor DRB. Following removal of the compounds, VEG parasites were inoculated and BAG1 expression was evaluated 72 h postinfection. Antagonism of Compound 1 induction of parasite BAG1 with DRB shows a dose effect when comparing co-treatment with 30 or 60 μM DRB and 3 μM Compound 1 (DRB 30/C1 and DRB 60/C1, respectively). DRB alone had no effect on parasite growth or BAG1 expression, while induction by 3 μM Compound 1 (CMPND1) is included here for reference.</p><p>(B) In untreated cultures, VEG parasites reach greater than 70 per vacuole by 72 h postinfection (black circle), but vacuoles with an average of only four parasites were observed in Compound 1–treated cells (grey box). Co-treatment with 60 μM DRB restored parasite replication to approximately 60 parasites per vacuole (black box) by 72 h postinfection, while reducing the level of BAG1+ to 12% (dark bars) from greater than 70% in cultures treated with Compound 1 alone (light bars).</p></div
Overexpression of CDA1 in Infected Cells Slows Parasite Growth and Induces Bradyzoite Development
<div><p>(A) HeLa cells expressing the tetracycline repressor protein transfected with the pcDNA4 TO-CDA1 plasmid inhibit parasite growth and induce cyst wall protein when grown in media containing tetracycline to induce expression of the CDA1 transgene. Compare parasite growth in transfected HeLa cells cultured without tetracycline (growth measured as average parasites per vacuole, PPV, open box with lines) with that of parasite growth in media containing tetracycline to induce CDA1 expression (shaded box with lines). Concurrent with the observed inhibition of parasite replication, cyst wall protein was also expressed. Compare background levels of cyst wall protein (light bars) with levels induced by overexpression of CDA1 (dark bars) at 36 and 48 h. Treatment of HeLa cells with tetracycline alone, transfection of HeLa cells with the pcDNA4 TO expression vector alone, or transfection of HeLa cells with the pcDNA4 TO vector and induced to expressed the coding sequence for yellow fluorescent protein (YFP) were all unable to slow parasite growth or induce the expression of cyst wall protein (unpublished data).</p><p>(B) Early passage type I GT-1 parasites (fewer than 12 passages from the oocyst) do not slow growth or initiate the expression of bradyzoite-specific cyst wall protein in a cell expressing CDA1 under control of the tetracycline-inducible promoter. Compare parasite growth and cyst wall protein measured by immunofluorescence in uninduced cells (open boxes, light bars) with growth and cyst wall protein in cells ectopically expressing the CDA1 protein (shaded boxes, dark bars).</p></div