Trypsin

A complex synthetic wastewater of a controlled composition mimicking real wastewater was prepared according to a previous work (Layer et al. 2019 (link)) that has also been applied in conjugative experiments (Pallares-Vega et al. 2021 (link)). The detailed influent composition is shown in Tables S1 and S2 in the Supplementary Material. Briefly, this medium was composed of 1/3 volatile fatty acids (1/6 acetate + 1/6 propionate), 1/3 soluble and fermentable substrates (1/6 glucose, 1/6 amino acids), and 2 particulate substrates (1/6 peptone, 1/6 starch) in equal equivalents of chemical oxygen demand (COD). Amino acids were composed of L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, L-leucine, L-proline, and glycine in COD equivalents. Particulate substrates were peptone from casein, digested with trypsin (Carl Roth, Germany), and starch made from wheat (Merck Sigma, Germany). Nitrogen was supplied as a combination of soluble ammonium chloride and nitrogen from the aforementioned amino acids and peptone. Phosphorus was composed of soluble orthophosphate.

N52.E6 cells for cultivation of adenovirus, A549 cells for TCID-test and HEK 293 cells for western blotting were stored at -80°C. For cultivation cells were transferred either in D-MEM (HEK 293 and A549) or in alpha MEM (for N52.E6 cells) concentrated with 10% fetal calf serum, 1% glutamine, 0,1mg/ml penicillin and 0,1mg/ml streptomycin (Invitrogen Life technologies Corp., Carlsbad, USA). Cell medium was seeded and incubated at 37°C with 95% humidity and 5% CO2. At regular intervals cell culture were checked until a confluent cell lawn was formatted. Then cell medium was removed and the cells were washed with 5ml PBS and incubated with 3ml trypsin (0,25%, Carl Roth GmbH, Karlsruhe, Germany) for 3min. Generated cell titer was count in a Neubauer chamber (Neubauer improved counting chamber, Carl Roth GmbH, Karlsruhe Germany).

Genetically modified T. gondii RH-GFP tachyzoites (type I strain, kindly provided by Professor Dominique Soldati-Favre, University of Geneva Medical School, Switzerland) were harvested from infected human foreskin fibroblast (HFF) cultures. E. tenella Houghton-YFP strain (kindly provided by Professor Xun Suo, China Agricultural University, China) sporozoites were gained following an established protocol (Thabet et al. 2017 (link)) with slight improvement. Briefly, sporocysts were collected by mechanical destruction of the oocyst wall with 0.5-mm glass beads (BioSpec Products, Bartlesville, OK, USA). Sporocysts were incubated in 0.25% trypsin (w/v) (Carl Roth, Karlsruhe, Germany) and 4% sodium taurocholic acid (w/v) (Sigma-Aldrich, Taufkirchen, Germany) at 41 °C for 90 min for excystation. Then, sterile pluriStrainer® 5 μm (pluriSelect Life science, Leipzig, Germany) was used to purify excysted sporozoites with 1% glucose in PBS at pH 7.4 (follow buffer).