Figure 1

Characterization of Brazilian (TgBr EGS) T. gondii , originally isolated from amniotic fluid in 1994, establishes novel in vitro model. (a) In vitro EGS cultures in HFF cells form cysts. Note green dolichos immunstaining of the perimeter cyst wall, red bradyzoite antigen 1 (BAG1) immunostaining, and blue DAPI staining of DNA. These cultures produce oocysts when fed to cats (see Fig. 2). (b) Genetic characterization of EGS strain. Neighbor-net analysis based on 790,168 genome-wide SNPs common to EGS strain plus 62 parasite isolates representing all major population haplogroups that have been described for T. gondii demonstrated that EGS belongs to clade B, haplogroup 4. Groups A to F indicate major population clades of T. gondii. Haplogroup numbers are indicated within parentheses based on previous designation. Names within red rectangles denote the representative strains from each haplogroup. (c). Full genome sequence analysis of EGS compared with canonical and geographically closely related parasite genomic sequences reveal a non-synonymous mutation and disordered c terminal sequence in Apetela 2(AP2) IV-iv, a bradyzoite repressor. Because AP2 IV-iv is a bradyzoite repressor, a mutation could create a parasite like EGS that remains as an encysted bradyzoite. Biological process GO representation among EGS genes carrying non-synonymous SNPs not present in other canonical and phylogenetically closely related parasite genomic sequences. This analysis revealed unique non-synonymous SNPs in Apetela 2 transcription factor genes and other genes shown in Supplement A: Figure S1 and Supplement B: Excel Table S1 which shows the Go Slim Analysis. The effect on transcription may reflect the disordered region and nonsynomous mutation in AP2 IV-iv. (d) Cysts of EGS enlarge with time in culture in HFF. This enlargement is visible over the initial 96 hours in culture. (e) DGE analysis of EGS cultures in HFF reveals changes over time in both host and parasite transcriptomes. Left and middle panels depict heat maps of the 50 most upregulated HFF and EGS genes respectively. Right panel: MDS plot of HFF gene expression profiles from uninfected human fibroblast or infected with EGS parasites for 2, 18 or 48 hours. The full data set is in Supplement B: Excel Table S2.