0.5 mm glass bead

Total RNA was extracted from the cell pellets via mechanical rupture with 0.5 mm glass beads (BioSpec) by vortexing for 10 min, followed by the addition of Tri-Reagent (Ambion). The lysate was incubated for 10 min at room temperature, and subsequently 200 μl of chloroform per ml of Tri-Reagent was added, mixed, and centrifuged for 5 min at 4,000 x g. The aqueous phase was recovered, and two consecutive extractions with acidic phenol: chloroform (1: 1) were performed. The RNA was precipitated with two volumes of isopropanol for 10 min at room temperature, and the RNA was washed with 75% ethanol and suspended in RNase-free water. RNA samples at a 260/280 ratio >1.9, measured using a V-630 UV–vis Spectrophotometer, were used for next-generation sequencing.

Cells released from α-factor–induced G1 synchrony were harvested every 10 min and frozen in liquid nitrogen. Cells were lysed using 0.5 mm Glass Beads (BioSpec) in a FastPrep-24 bead beater (MP Biomedical), and RNA was extracted using RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. 1 μg of the harvested RNA was treated with 1U DNase I (Thermo Fisher Scientific), and cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen). Gene expression was analysed in technical duplicates by quantitative real-time PCR using 12.5 ng of cDNA in a 20 μl reaction containing 0.4 μM forward and reverse primers (Table 2) and 10 μl 2x SyGreen Mix (#PB20.14-05; qPCRBIO) in a LightCycler instrument (Roche). The 180 s of preincubation at 95°C was followed by 40 cycles of amplification consisting of 95°C for 10 s, 56°C for 10 s, and 72°C for 1 s with single signal acquisition, and melting curve of 95°C for 5 s, 65°C for 60 s, and 97°C at 0.2°C/sec with continuous signal acquisition. Cq values determined by LightCycler 96 software (Roche) were used to calculate the expression of the cyclin genes relative to actin using the Pfaffl method (57 (link)). Statistical comparisons between selected timepoints of individual strains were performed by a standard two-tailed t test in GraphPad Prism (GraphPad Software, Inc.).

Proteins were extracted from cells fixed with 5% trichloroacetic acid by lysing cells in protein breakage buffer (50 mM Tris at pH 7.5, 1 mM EDTA, 27.5 mM DTT) with 0.5 mm glass beads (BioSpec) on the Mini-Beadbeater-96 (BioSpec). Samples were denatured in SDS loading buffer (62.5 mM Tris (pH 6.8), 2% β-mercaptoethanol, 10% glycerol, 3% SDS and 0.017% Bromophenol Blue) and separated by SDS-PAGE in Tris-glycine buffer (25 mM Tris base, 192 mM glycine, 0.1% SDS). V5-epitope tagged proteins were detected using anti-V5 primary antibodies (Invitrogen, 1:2000, mouse) and IRDye 800CW (LI-COR, 1:15000) or HRP-conjugated (GE Healthcare, 1:8000) secondary antibodies. For equal loading Hxk1 protein levels were determined using anti-hexokinase primary antibodies (Stratech Scientific, 1:2000, rabbit), and IRDye 680RD (LI-COR, 1:15000) or HRP-conjugated (GE Healthcare, 1:8000) secondary antibodies. Images were acquired on the Odyssey CLx imaging system (LI-COR) or by ECL Prime (GE Healthcare) using Amersham Imager 600 (GE Healthcare). Uncropped versions of the western blots presented in Fig. 1e, g, Supplementary Fig. 4a are provided in the Source Data file.

Total RNA from S. microadriaticum was extracted by grinding the cell pellets with 200–300 μl of 0.5 mm glass beads (BioSpec Products, Bartlesville, OK) in liquid nitrogen-chilled mortar and pestle prior to using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The quality of extracted total RNA was assessed using a Bioanalyzer 2100 (Agilent, Santa Clara, CA). Strand-specific RNA-seq libraries were generated from oligo-dT selected total RNA using the NEBNext Ultra Directional RNA Prep Kit (New England Biolabs, Ipswich, MA) according to manufacturer's instructions. A total of 397 million paired-end read pairs (read length: 101 bp, insert size: 180 bp) were retrieved from 4 lanes on the HiSeq 2000 platform (Illumina, San Diego, CA).

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).

The placental samples (15 mg) were ground in a BeadBug homogenizer (D1030-E, Benchmark Scientific, Sayreville, NJ, USA) with two 0.5-mm glass beads (#11079105, BioSpec, Bartlesville, OK, USA) and 1 mL lysis buffer [5% (w/v) sodium dodecyl sulfate (SDS, #L3771-100) in 100 mmol/L Tris-HCl buffer containing 1% (v/v) phenylmethylsulfonyl fluoride (PMSF, #P7626), 1% phosphatase inhibitor (#P5726), and 1% protease inhibitor (#P8340), all from Sigma-Aldrich, St. Louis, MO, USA]. After centrifugation at 20,000 × g for 15 min at 4 °C, the soluble fractions were collected and the protein concentrations were measured using a bicinchoninic acid (BCA) standard assay (9470BCAstand, Cyanogen, Bologna, Italy), then snap-frozen and stored at −80 °C.

Liver samples (~100 mg) were homogenized in modified RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors (Halt™, Thermo Fisher Scientific, Waltham, MA, USA) in tubes filled with 0.5 mm glass beads (BioSpec, Bartlesville, OK, USA). Following centrifugation at 16,000× g, supernatants were collected, and protein concentrations were determined by the BCA method. A total of 600 µg of liver protein was analyzed on the V-PLEX immunoassay platform for TNFα. The data were collected on the MESO Sector S 600 instrument and then analyzed using Discovery Workbench v. 4.0 software (all from MesoScale Discovery, Rockville, MD, USA).