Fig 1.
Copy number analysis of AAH2 in T. gondii strains.
(A) Copy number variation (CNV) of Tg_ME49_212740 (AAH2) in five representative strains. (B) CNV at each base across the AAH2 gene in ME49. Red lines indicate the start and stop codons of AAH2. (C) PCR using primers (Z28+ Z65) (S3 Table) common to both AAH1 and AAH2 were used against the ME49 genome to amplify both genes for Sanger sequencing and single nucleotide polymorphism (SNP) determination. (D) Sanger sequencing of PCR fragments against the AAH genes of the wild type ME49 Δhxg::Luc strain shows a 2:1 ratio of AAH2 to AAH1 SNPs, indicating a duplication of the AAH2 gene. Each SNP in the chromatograph is marked by a red dot. In the Δaah2::HXG knockout, all AAH2 SNPs are no longer visible, indicating loss of both copies of the AAH2 gene.
Fig 2.
Disruption of the AAH1 and AAH2 genes.
(A) Schematic of the AAH2 knockout strategy in the wild-type ME49 Δhxg::Luc strain (referred to as WT). A CRISPR-Cas9 construct with guide RNAs targeted to the 5’ and 3’ UTRs of AAH2 was co-transfected with the pΔaah2::HXG plasmid (S2 Table) and selected for with MPA +Xanthine to delete AAH2 to produce the clone Δaah2::HXG (Δh2-HXG). Subsequently, the HXG gene was replaced with either a clean fusion of the AAH2 5’ and 3’ UTRs (pΔaah2) or an AAH2 cDNA construct (pAAH2). (S2 Table) using 6-thioxanthine selection against the HXG locus to create the clean knockout clone Δaah2 (Δh2) (upper panel) and the complement clone Δaah2::AAH2 (Δh2-H2) (lower panel). Yellow Bars: CRISPR targeting sites. Black bars: PCR screening primer target regions (S3 Table). (B) Schematic of the knockout strategy for AAH1. A CRISPR-Cas9 construct with guide RNAs targeted to the 5’ and 3’ UTRs of AAH1 was co-transfected with the pΔaah1::DHFR-Ts repair construct (S2 Table) into WT or Δaah2 parasites to create the clones Δaah1 (Δh1) and Δaah1Δaah2 (Δh1Δh2). Transfectants were selected for via pyrimethamine resistance. Subsequently, using pΔuprt::AAH1::HXG, a cDNA copy of AAH1 driven by its native 5’ and 3’ UTRs was complemented into the UPRT locus by means of the HXGPRT drug resistance marker selected for with MPA +Xanthine, negative selection against UPRT with FUDR, and a single-cutting CRISPR-Cas9 construct targeted to the UPRT gene (S2 Table), creating the complement clones Δaah1-AAH1 (Δh1-H1) and Δaah1Δaah2-AAH1 (Δh1Δh2-H1). Brown & Yellow Bars: CRISPR targeting sites. Black bars: PCR screening primer target regions (S3 Table). (C) PCR verification of successful ablation and complementation of knockouts. Expected product sizes: Tubulin (Tub): 0.378kb. AAH1 (H1): 0.745kb (Native), 0.278kb (cDNA). AAH2 (H2): 0.745kb (Native), 0.278kb (cDNA). (D) Growth assays of parasites seeded into 96-well plates and allowed to proliferate for 24 h, then quantified using a luciferase assay. The WT, Δh1, Δh2, Δh1Δh2, Δh1-H1, Δh2-H2, and Δh1Δh2-H1 parasites showed no significant difference in total growth (Kruskal-Wallis test, P = 0.0672, N = 3 per strain).
Fig 3.
Development of bradyzoites in vitro and in vivo.
(A) There was no significant difference in bradyzoite differentiation in vitro across the parasite lines in either tachyzoite conditions (left) (P> 0.99) or bradyzoite conditions (right) (P> 0.99) (Two-way ANOVA).(B) Representative pictures of tachyzoites, partial cysts, and complete cysts produced in vitro as assessed by DBL staining. (C) Brain cyst yields in mice 1–2 months post-infection. All parasite lines produced similar numbers of tissue cysts in vivo (Kruskal-Wallis, ns).
Fig 4.
Development of oocysts following infection of cats.
(A) Yields of oocysts shed from infected cats. The yield of knockout mutants as a whole were significantly reduced relative to the wild-type (Kruskal-Wallis, P ≤0.05), however due to low sample size, pairwise comparisons between each mutant and the WT approached, but did not reach significance (Δh1 P = 0.116, Δh2 P = 0.821, Δh1Δh2 P = 0.116). (B) The sporulation success rate of shed oocysts shows a significant defect in mutant lines (Dunn’s multiple comparisons test, for wild type vs. Δh2 P = 0.0008; and for the wild type vs. Δh1-H1 P = 0.0178 and Δh1Δh2-H1 P< 0.0001). The oocyst yields of Δh1 and Δh1Δh2 parasites were not sufficient to allow quantification (not done = N.D.). Each result is displayed as the Mean ±SD of three replicate counts of oocysts (n ≥ 50 per count) from one cat.
Fig 5.
Representative fluorescence microscope images of dityrosine autofluorescence in sporulated and unsporulated oocysts of WT, Δh1, Δh2, and Δh1Δh2 oocysts.
All images were taken at 1000-1600ms exposure using a DAPI UV filter, but due to rapid photobleaching and differing levels of background signal in different oocyst fecal suspensions, direct comparison and quantification of fluorescence is not feasible. Scale bar = 10μm.
Fig 6.
Representative images of cat intestinal ileum infected with WT (A), Δh1 (B), and Δh2 (C) parasites stained with a polyclonal anti- T. gondii antibody and Streptavidin-HRP (brown). Parasites are located throughout the intestinal villi (dark deposits). Scale bar = 50μm. (D) Parasite density per villus in intestinal sections infected with WT, Δh1 and Δh2 parasites. Δh1-infected intestines showed significant reduction of parasite density (P< 0.0001, Dunn’s Multiple Comparisons test, N = 50 villi counted per section, 4 (WT, Δh2) or 6 (Δh1) independent sections counted per strain).
Fig 7.
Representative images of hematoxylin and eosin-stained tissue sections of cat intestinal ileum infected with WT (A, B), Δh1 (C, D), and Δh2 (E, F) parasites.
Multiple stages of the parasite’s sexual cycle can be seen. m: merozoite, is: immature schizont, s: schizont, fg: female gamont, mg: male gamont. Scale bar = 5μm.
Fig 8.
Distribution of parasite sexual developmental stages in cat intestinal ileums infected with WT, Δh1 or Δh2 mutant parasites.
(A) Pooled counts from four (WT, Δh2) or six (Δh1) independent intestinal sections from an infected cat are summarized. (B) The relative proportion of each stage observed across the four or six samples are shown +/- SD. No significant differences in the distribution of parasite stages were seen (P> 0.9999, Two-way ANOVA).