Fig. 2: Cell culture models of bradyzoite development fail to express SRS22A.

a (right images) SRS22A expression was not detected in any parasite vacuole irrespective of the pH condition used. The presence of SRS9+ vacuoles that were the result of tachyzoite-to-bradyzoite development in pH 8.2 media (48 h) confirm early bradyzoite differentiation in these cultures. The infected HFF cell cultures were fixed and co-stained for SRS9 (bradyzoites, red) and SAG1 (tachyzoites, green), or separately for SRS22A (red) and SAG1 (green), which was required because SRS9 and SRS22A antisera were raised in rabbits. DAPI was included to visualize nuclei. Scale bar = 10 μm. (left graphs) Quantification of vacuole size for SAG1+ only, both markers (D+ indicates vacuoles with both markers: SAG1 + /SRS9 + ), and SRS9+ only vacuoles demonstrated cultivation in pH 8.2 media caused overall slower parasite growth, which is known to be associated with the alkaline-stress bradyzoite differentiation model. Alkaline-shift experiments were repeated three times and all vacuoles in each coverslip were counted. b Immunofluorescence images showing SRS22A staining in ME49EW excysted, free bradyzoites and ex vivo bradyzoite-infected primary astrocyte cultures at days 1-, 3-, and 7- post-infection. Free bradyzoites and infected-astrocyte cultures were co-stained for SRS22A (red), SAG1 (tachyzoite-specific, green), and DBA (cyst wall-specific, magenta). Notably, SRS22A staining diminishes very quickly (within the 1st 24 h) in ex vivo bradyzoite-infected astrocytes, and thus, at day 3 (D3 EW) and day 7 (D7 EW) post-infection no parasites stained positive for SRS22 A. Note that SRS22A was also not expressed or re-expressed in bradyzoite vacuoles where cyst wall formation was active (DBA + ). Scale bar = 20 μm. Ex vivo experiments were repeated at least three times. Source data are provided as a Source Data file for this figure.