Slsv025ls

Monolayers containing freshly excysted parasites were scraped, syringe lysed, and filtered through a 5 µM syringe-driven filter (Fisher Scientific, SLSV025LS) after 24 hr of growth. Filtered parasites were pelleted and passed at MOIs of 0.5 (H. hammondi) and 0.1 for (T. gondii) onto coverslips containing a monolayer of HFFs. Three coverslips were infected per parasite per treatment group. After 2 days, media was changed to pH 8.2 bradyzoite switch media (DMEM with 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine 10 mM HEPES, 2 g/L NaHCO₃, and 1% FBS [Gay et al., 2016 (link)]) or cDMEM. Coverslips with pH 8.2 media were grown at 37°C in the absence of CO₂. Media was changed again at 3 DPI. After 4 DPI, cells and parasites were fixed as described above and stored in blocking buffer at 4°C until needed. DBA and counter-staining was conducted using Fluorescein or Rhodamine labeled DBA and rabbit Toxoplasma gondii Polyclonal Antibody as described above. The number of DBA-positive vacuoles was quantified in 15 FOVs in three coverslips for each strain grown at either pH 7.2 or pH 8.2 and the percentage of DBA positive vacuoles was determined. Images were obtained and analyzed as described above.

Sporozoites were obtained using the excystation protocol described above. After 24 hr, HFF monolayers infected with excysted sporozoites were scraped, syringe lysed 5X with a 25 gauge needle, and pelleted. The pellet was resuspended in cDMEM, filtered through a 5 µM syringe-driven filter (Fisher Scientific, SLSV025LS) and passed onto confluent monolayers of HFFs grown in T-25s. Infected host cells were incubated at 37°C 5% CO₂ from 6, 8, 10, 13, and 15 days. The media was replenished after 5–7 days. At each time point, infected host cells were scraped, syringe lysed 3X with a 25 gauge needle and 3X with a 27 gauge needle, and pelleted. Parasites were counted and diluted in PBS, and a dose of 50,000 parasites was injected intraperitoneally in 2 BALB/C mice for each time point. Serum samples were obtained from infected mice daily for 9 days post-infection and the mass of the mice was also monitored daily. After 9 days of infection, the mice were sacrificed and dissected. Tissue samples were preserved in 10% neutral buffered formalin (Sigma HT501128) until immunohistochemistry analysis was performed.

Sporozoites were obtained using the excystation protocol described above with the exception of syringe lysis and previously described host cell incubation. Six confluent monolayers of HFFs grown in T-25’s were infected with H. hammondi American (HhCatAmer) sporozoites at an MOI of ~2 (2.8 million sporozoites). After a three-hour incubation at 37°C, the Day 0 Transfer flask was scraped, syringed lysed 5X with 25 gauge needle, filtered through a 5 µM syringe-driven filter (Fisher Scientific, SLSV025LS), and passed to a new confluent monolayer of HFFs grown in a T-25. This process was repeated after 2, 5, 8, 11, and 15 days post-excystation. After 3 days of growth following passage, 4.17 cm² of the T-25 was monitored daily. Images of vacuoles were obtained with an Axiovert 100 inverted fluorescent microscope with Zen lite 2012 software with a 40X objective, and vacuole sizes were quantified using ImageJ software (NIH). Similar methods were used to generate parasite samples for RNAseq analysis, except cells were processed for RNA harvest using methods described below.