Bhi broth

S. pneumoniae strain DSM20566 (serotype 1) was obtained from the German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany). Bacteria were grown on Columbia blood agar plates (BD Biosciences, Heidelberg, Germany) at 37°C in 5% CO₂ overnight or cultured in brain heart infusion (BHI) broth (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) at 37°C as described elsewhere (Walther et al., 2015 (link)).
Precipitation of pneumococcal total proteins was described previously (Richter et al., 2015 (link)). Briefly, 200 μL of a pneumococci culture (grown overnight in BHI broth) were precipitated by 1.8 mL of ice-cold absolute ethanol (−20°C for 16 h). Aliquots were centrifuged at 3000 × g for 20 min at 4°C. The precipitate was washed with 1 mL of ice-cold 70% ethanol. After centrifugation at 3000 × g for 5 min at 4°C, the supernatant was removed and the pellet dried. The ethanol-precipitated proteins were dissolved in 50 μL of NA assay buffer and stored at −20°C until use.

Synchronised L4 to young adult C. elegans nematodes were infected by feeding on lawns of E. dermatitidis or C. neoformans cells for 24 h or 48 h, as described above. After the inoculation, the nematodes were washed from the culture plates with M9 buffer and were centrifuged at 500 × g for 5 min. After this first washing step, they were moved to an agar medium containing drinking water and 2% agar (% = w/v). The infected nematodes were allowed to crawl over the agar for at least 30 min for removal of fungal cells from the outer cuticula. Afterward, the infected worms were moved to a 96-microwell plate, containing a mix of M9 buffer and brain heart infusion (BHI) broth (modified from Breger et al.)^{32 (link)}: M9 buffer, 79%; BHI broth, 20%; cholesterol, 10 µL (concentration, 5 mg/L in ethanol; Carl Roth, Karlsruhe, Germany), ampicillin, 100 µL (concentration, 0.0025 mg/mL; ampicillin sodium salt; Sigma-Aldrich). The plates were implemented with 2 C. elegans worms per microwell, using 12 microwells per E. dermatitidis strain infection, and were observed daily for 72 h. Morphologic development of E. dermatitidis inside the infected C. elegans worm was observed by imaging (EVOS all-in-one digital inverted microscope; AMG, Bothell, WA, USA) and was listed together with information about death or health. Every test was repeated three times.