Ypd broth

S. cerevisiae CMCC17452 was cultivated with YPD broth (Becton, Dickinson and Company, Sparks, MD, USA) at 28 °C for 48 h. P. notoginseng root (PNR) and pure water were were mixed to a ratio of 1 to 10 before sterilization for 15 min at 121 °C. S. cerevisiae CMCC17452 was inoculated into PNR solution at a concentration of 5% (V/V) and fermented at 28 °C for 24 h. The PNR solution without S. cerevisiae CMCC17452 was used as a blank control. Samples were centrifuged and the supernatant collected for analysis and named fermented P. notoginseng root liquid (FPNR) and water-extracted P. notoginseng root liquid (WPNR).

C. neoformans strains H99 (serotype A, mating type α) H99γ (serotype A, mating type α, an interferon-gamma producing strain derived from C. neoformans H99^{25 (link)}), and H99 calcineurin mutant (cna1Δ, a kind gift from Dr. Joseph Heitman, Duke University) were recovered from 15% glycerol stocks stored at −80 °C and maintained on yeast-extract-peptone-dextrose (YPD) agar (1% yeast extract, 2% peptone, 2% dextrose, and 2% Bacto agar). Candida albicans strain SC5314 was maintained on YPD media. Staphylococcus aureus strain UAMS-1 was maintained on Luria-Bertani (LB) agar. Yeast cells were grown for 15–17 h at 30 °C with shaking in YPD broth (Becton Dickinson and Company, Sparks, MD), collected by centrifugation (2500 × g), washed three times with sterile phosphate-buffered saline (PBS), and viable yeast quantified using trypan blue dye exclusion in a hemacytometer. S. aureus was grown for 12 h at 37 °C with shaking in LB broth (Fisher Scientific, Waltham, MA), collected by centrifugation (2500 × g) and washed three times with sterile PBS. Yeast cells were heat-killed at 65 °C for 30 min. S. aureus was heat-killed at 95 °C for 1 h. All cultures were confirmed dead by culturing for growth on LB or YPD agar.

C. gattii strain R265 (VGII molecular genotype) was recovered from 15% glycerol stocks stored at −80°C. The strain was maintained on yeast extract peptone dextrose (YPD) agar (1% yeast extract, 2% peptone, 2% dextrose, and 2% bacto agar). Yeast cells were grown in YPD broth (Becton Dickinson and Company, Sparks, MD) for about 16–18 hours at 30°C with constant shaking. Yeast cells were collected by centrifugation and washed with sterile phosphate buffered saline (PBS) for further protein extraction. Quantification of viable yeast was performed using trypan blue dye exclusion in a hemocytometer for pulmonary infection.

The isolates used in this study are part of a strain collection deposited at the Department of Translational Research, University of Pisa, and stored in YPD broth (yeast extract, peptone, dextrose) (Difco BD, Italy) supplemented with 40% glycerol at −20 and −80°C. Clinical isolates of Candida spp. were collected at the Mycology Unit, Azienda Ospedaliero-Universitaria Pisana (Pisa, Italy), identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Germany), and tested for antifungal susceptibility according to EUCAST guidelines (http://eucast.org/clinicalbreakpoints/). The strains used in this study were clinical isolates selected on the basis of caspofungin sensitivity and two reference strains (Table 4). For each experiment, cells were inoculated in YPD broth and incubated overnight at 30°C. Next, cells were washed twice in sodium phosphate buffer (NaPB) (0.01 M [pH 7]), and diluted at the desired concentration.

SAPs were isolated as previously described^{65 (link),66 (link)}. Briefly, C. albicans was grown in YPD broth (BD, Sparks, 21152) for 24 h at 26 °C. Cells were removed by centrifugation (8500 r.p.m./6800 × g, 5 min, 4 °C) and the supernatants containing SAPs were concentrated 25 times in a Pierce Protein Concentrator (10 kDA MWCO, #88535, Thermo Fisher Scientific, Waltham, MA). Concentrated SAPs were then purified by passage through a Pierce Strong Anion Exchange Spin Column (#90011, Thermo Fisher Scientific, Waltham, MA). 20 mM Tris/HCl (pH 6.0) was used for column binding and 2 M Tris/HCl (pH 6.0) was used for elution. SAPs were then concentrated again as described above. SAP concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific).