A Forward Genetic Screen Reveals that Calcium-dependent Protein Kinase 3 Regulates Egress in Toxoplasma
Figure 8
Establishment and analysis of a Tgcdpk3 knockout strain.
A. Schematic diagram of the TgCDK3 genomic locus and the region disrupted in the knockout strain. The top black line represents the genomic DNA region with each tick being 1,000 bases. The gray box represents the region of the TgCDPK3 locus that is replaced by the hpt selectable marker (dhfrHXGPRTdhfr), which is transcribed in the opposite direction from TgCDPK3 (as indicated by arrow pointing left). The relative positions of exons (white boxes) and introns (dashed lines) are shown below. Black arrowhead indicates position of start codon while the white arrowhead points at the stop codon. Lines labeled a, b, c and d represent the regions amplified by PCR to test for the disruption of TgCDPK3. B. PCR products of the parental strain, RHΔhptΔku80, and the TgCDPK3 KO strain, RHΔhptΔku80Δcdpk3+HPT are shown. Labels above lanes indicate the DNA fragment amplified. Fragments a, b and c cannot be amplified if the TgCDPK3 locus has been disrupted by the hpt marker. Fragment d is expanded from both the parental and knockout strain and it serves as a control. C. Intracellular parasites of either the parental or knockout strain were treated with 1 µM A23187 for the time indicated. Percentage egress represents the number of lysed vacuoles divided by the total number of vacuoles (lysed+intact). Each data point represents the average of four independent experiments and the error bars represent the standard deviation. D. Extracellular parasites of either the parental or the knockout strain were exposed to 1 µM A23187 for 45 minutes before being added to cells. Percentage survival was calculated by dividing the number of plaques formed by parasites treated with A23187 divided by the number of plaques formed by untreated parasites. Each data point represents the average of three independent experiments and the error bars represent the standard deviation. E. Parasites were allowed to settle on glass in intracellular buffer, which was changed to extracellular buffer to initiate motility. After 15 minutes parasites were fixed and the percentage of parasites that had moved was determined by counting parasites that were associated with trails of surface proteins left behind during gliding. Data is from 3 independent studies and the error bars are the standard deviation.