Department of Pharmacology & Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, USADepartment of Microbiology & Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
Characterization of AP2IX-4 protein expression. (A) Schematic showing the strategy used to generate parasites expressing AP2IX-4 tagged at the C terminus with 3xHA epitopes (AP2IX-4HA) in RHQ parasites. The AP2IX-4 genomic locus is aligned with the pLIC-3xHA construct used for endogenous tagging via single homologous recombination. The construct contains a 1,514-bp homology region and a DHFR*-TS drug selection cassette. UTR, untranscribed region. (B) Western blot (WB) of parental RHQ and AP2IX-4HA parasites probed with anti-HA antibody. Anti-β-tubulin was used to verify the presence of parasite protein. kD, kilodaltons. (C) IFAs performed on AP2IX-4HA tachyzoites using anti-HA (green). To monitor cell cycle phases, parasites were costained with anti-IMC3 (red) and DAPI (blue). G1, gap phase; S, synthesis phase; M, mitotic phase; C, cytokinesis. Scale bar, 3 µm. (D) Quantification of the proportion of parasite vacuoles (of 100) in the asynchronous population of AP2IX-4HA parasites exhibiting G1/S IMC3 staining with absence of HA signal (G1/S HA-), G1/S IMC3 staining with positive HA signal (G1/S HA+), or M/C staining with positive HA signal (M/C+). Error bars represent standard deviations of results from 3 independent experiments.
Generation of PruQΔap2IX-4 parasites. (A) Schematic illustrating the generation of the AP2IX-4 knockout by allelic replacement with a DHFR*-TS minigene. (B) Genomic PCRs were performed with the indicated primers (A to G) to verify replacement of the AP2IX-4 genomic locus with the DHFR*-TS minigene. RT-PCR performed with primers B and E confirmed the absence of AP2IX-4 transcripts in PruQΔap2IX-4 parasites. Primers for GAPDH were used as a positive control for the RNA preparation. (C) Representative doubling assay showing the number of parasites/vacuole at 24- and 36-h time points postinfection for parental PruQ versus PruQΔap2IX-4 parasites. Three independent assays were performed with similar results. P > 0.05 (unpaired two-tailed Student’s t test). (D) Plaque assays were performed to compare the in vitro viability of PruQ parasites to that of PruQΔap2IX-4 parasites. Three independent assays were performed with similar results. P = 0.98 (unpaired two-tailed Student’s t test).
Complementation of the AP2IX-4 knockout. (A) Schematic illustrating the generation of the complemented knockout (Δap2IX-4::AP2IX-4HA). A cDNA-derived version of AP2IX-4 tagged at the C terminus with 3xHA (complementation construct) was targeted to the ablated genomic locus in Δap2IX-4 parasites. (B) Genomic PCRs were performed with the indicated primer pairs to confirm proper integration of the complementation construct into Δap2IX-4 parasites. Primers corresponding to GAPDH were used as a positive control of the DNA preparation. (C) Western blot of Δap2IX-4 and Δap2IX-4::AP2IX-4HA parasites with anti-HA, using anti-β-tubulin as a control to verify the presence of parasite protein. (D) IFAs were performed on Δap2IX-4::AP2IX-4HA tachyzoites as described for Fig. 1C. (E) Quantification of the proportion of parasite vacuoles (of 100) in the asynchronous population of AP2IX-4HA parasites performed as described for Fig. 1D.
Expression of AP2IX-4 in developing bradyzoites in vitro. (A) IFAs were performed on Δap2IX-4::AP2IX-4HA parasites during the tachyzoite and developing bradyzoite stages for up to 8 days (8d) in alkaline pH. FITC-conjugated Dolichos lectin was used to visualize bradyzoite cyst walls (the LDH2-GFP reporter also appears in bradyzoites in this channel). AP2IX-4ha protein was visualized using anti-HA (red), and DAPI (blue) was used to stain DNA. The percentage of parasites expressing AP2IX-4HA within 50 random vacuoles (%HA+) was recorded; note that it was not possible to accurately discern the total number of parasites in tissue cysts beyond 4 days postinduction. ND, not determined. Values represent the averages and standard deviations of results from 3 independent experiments. (B) IFAs performed with anti-IMC3 antibody (yellow) on Δap2IX-4::AP2IX-4HA parasites following 4 days in alkaline pH. The LDH2-GFP reporter (green) identifies bradyzoites. AP2IX-4ha protein was visualized using anti-HA (red). Arrows point to budding daughter cells in dividing parasites expressing AP2IX-4HA. Scale bar, 3 µm.
Loss of AP2IX-4 results in lower frequency of bradyzoite cyst formation in vitro. Parental PruQ, Δap2IX-4, and Δap2IX-4::AP2IX-4HA parasites were cultured in alkaline medium for 4 days and then stained with Dolichos lectin to visualize tissue cyst walls. For each sample, 100 random vacuoles were surveyed for the presence or absence of lectin staining to determine the frequency of cyst formation. n = 4 for parental and Δap2IX-4; n = 3 for Δap2IX-4::AP2IX-4HA. *, P = 0.04; **, P = 0.006 (unpaired two-tailed Student’s t test). Error bars represent standard errors of the means.
Virulence and cyst burden in mice infected with Δap2IX-4 parasites. (A) Mice (n = 8 per group) were infected with 107, 106, or 105 parental, Δap2IX-4, or Δap2IX-4::AP2IX-4HA parasites and monitored over 14 days for survival. *, P = 0.01; **, P = 0.02 (log-rank test). (B) Mice were infected with 106 of the designated parasites (n = 4 per group) and allowed to progress to chronic infection for 35 days before brain cyst burden was assessed. *, P = 0.03 (unpaired one-tailed Student’s t test).
Differentially expressed genes in Δap2IX-4 and Δap2IX-4::AP2IX-4HA parasites under bradyzoite induction conditionsa
↵a Differentially expressed genes were defined as those corresponding to a fold change (FC) difference of ≥2. Genes that were previously found to be upregulated during bradyzoite induction are highlighted in gray (see Materials and Methods).