Figure 1.
Optimization of target protein knockdown and Toxoplasma invasion assay for high-throughput RNAi screen.
A. HeLa-GFP cells were transfected with non-targeting (NT) siRNA or siRNA targeting GFP by reverse transfection and examined by immunofluorescence 48 h and 72 h post-transfection. B. HeLa-GFP cells were mock-transfected or transfected with non-targeting siRNA or siRNA against GFP and analyzed by immunoblotting 72 h post-tranfection using anti-GFP or anti-actin antibodies. C. HeLa cells were reverse-transfected with either non-targeting siRNA (lysed cells, NTsiRNA or tachyplegin) or PLK1 in 384-wells. 48 h post-transfection, a set of HeLa cells transfected with NT siRNA were treated with passive lysis buffer to lyse cells. 72 h post-transfection, freshly harvested U-Luc parasites untreated or treated with tachyplegin (chemical inhibitor of invasion) were added onto host cells and incubated at 37°C for 3 h. Steadylite Plus™ was added to the plate and luciferase activity was measured using PHERstar system. n = 3 independent experiments each with triplicate samples. Error bars, SEM. D. Host cell viability was assessed by adding CellTiter-Fluor™ and the values obtained with cells transfected with NT siRNA only were set at 100%. n = 3 independent experiments each with triplicate samples. Error bars, SEM.
Figure 2.
Confirmation of gene silencing in a screening format and overview of the screening procedure.
A. Eighteen target genes represented in the library were silenced by siRNA transfection of HeLa cells in 96 well plates. The percent decrease in mRNA levels of target genes was determined using qRT-PCR 72 h post-transfection. n = 2 independent experiments, each with triplicate samples. Error bars, SEM. B. Schematic flowchart showing the siRNA screening strategy for identification of host factors playing a role in Toxoplasma invasion. The strategy consisted of three main steps of transfection, invasion and measurement. Host cell viability was assessed using CellTiter-Fluor™, which measures protease conversion of a quenched membrane permeable peptide substrate (glycine-phenylalanine-7-Amino-4-trifluormethylcoumarin). Parasite invasion was assessed using the luciferin-based reagent Steadylite Plus™, which measures the inducible luciferase activity of U-luc parasites.
Figure 3.
Results of primary screen of an siRNA library targeting human druggable proteins to identify host factors involved in Toxoplasma invasion.
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.
Figure 4.
Validation of the primary screen hits with secondary luciferase-based invasion and red-green invasion assays.
A. The top 100 hits of primary screen with at least 90% host cell viability were subjected to rescreening with the luciferase invasion assay. The values obtained with non-targeting siRNA controls were set at 100% invasion. A total of 81 genes (red asterisks) showed significant decrease (p<0.05, one sample t-test) in parasite invasion compared to NTsiRNA controls. n = 3 independent experiments each with duplicate samples. Error bars, SEM. B. 81 genes that were validated by secondary luciferase assay were subjected to secondary screen using high-content microscopy based red-green assay. A total of 24 genes (arrows) showed significant reduction (p<0.05, one sample t-test) in the percentage of invaded parasites in comparison to NT siRNA control. n = 4 independent experiments each with triplicate samples. Error bars, SEM.
Table 1.
Categorization of the validated hits.
Figure 5.
Validated host factors that affect actin dynamics.
A. Host cells transfected with NT siRNA or test siRNAs were fixed 72 h post-transfection and stained for F-actin using rhodamine-phalloidin. The images for NT siRNA and test siRNAs were captured with same exposure time. B. Quantification of F-actin at the periphery of the host cell 72 h post-transfection with control (NT siRNA) or test siRNAs. The results are based on three independent experiments each measuring the intensity and thickness of cortical F-actin in 10 cells at four uniformly spaced sites using line-scan analysis with standardized parameters (see also Fig. S2). Error bars, SEM.
Figure 6.
Validation of target gene knockdown at mRNA and protein levels.
A. The percent decrease in mRNA levels of target genes transfected with gene-specific siRNA were determined using q-RT PCR 72 h post-transfection. n = 2 independent experiments, each in triplicates. Error bars, SEM. B. Immunoblot analysis of host cells transfected with gene-specific siRNA (PTK9L or PHPT1) 72 h post-transfection using anti-PTK9L, anti-PHPT1 and anti-actin (loading control) antibodies. A loading series of the NT siRNA transfected sample (100%, 50%, etc) was included to estimate the percentage knockdown.
Figure 7.
A schematic showing the location of 24 validated host-factors that appear to play roles in host cell entry by Toxoplasma.
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.