Yeast extract peptone dextrose

C. glabrata CBS138, KUE100^{59 (link)}, and L5U1 (cgura3∆0 cgleu2∆0) strains, the later kindly provided by John Bennett, NIAID, NIH, Bethesda, were used in this study. Cells were batch-cultured at 30 °C with orbital agitation (250 r.p.m.) in the following growth media. Yeast extract Peptone Dextrose (YPD) growth media, containing per liter: 20 g glucose (Merck), 10 g yeast extract (Difco), and 20 g bacterial-peptone (LioChem). Basal minimal (BM) minimal growth medium contained per liter: 20 g glucose (Merck), 2.7 g (NH₄)₂SO₄ (Merck), and 1.7 g yeast nitrogen base without amino acids or (NH₄)₂SO₄ (Difco). SDB contained 40 g glucose (Merck) and 10 g peptone (LioChem) per liter.
The VK2/E6E7 human epithelium cell line (ATCC^® CRL-2616™) was used for adhesion assays. This cell line is derived from the vaginal mucosa of healthy premenopausal female submit to vaginal repair surgery and immortalized with human papillomavirus 16/E6E7. Cell maintenance was achieved with KSF medium, containing 0.1 ng/mL human recombinant epidermal growth factor, 0.05 mg/mL bovine pituitary extract, and additional 44.1 mg/L calcium chloride. Cells were maintained at 37 °C, with 95% air and 5% CO₂.

The activation of competent E. coli Top 10 cells was carried out in 100 mL of LB medium (Luria–Bertani, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20 mmol/L of MgCl₂ solution, incubated at 37 °C for 18 h. Komagaetella phaffii X-33 yeast was reactivated in 15 mL YPD liquid medium (Yeast Extract Peptone Dextrose, Sigma-Aldrich) for 24 h/30 °C with shaking at 150 RPM.

Candida albicans HE169 and C. parapsilosis P69 that were originally isolated from blood and ear, respectively, were obtained from the mycological collection of the Faculty of Medicine, Palacky University, Olomouc, Czech Republic. The yeasts were cultivated overnight in the YPD medium (yeast extract-peptone-dextrose; Sigma-Aldrich) and harvested by centrifugation at 4000 g for 10 min. Then, the DNA from the yeasts was isolated as described by Hoffman and Winston (1987 (link)). PHO15 sequences were amplified using the primers 5΄-gccgcgcggcagccaGTCAATTAAAATTACTTCTAAAGA-3΄ and 5΄- gtcatgctagccataCTAATTGTTAAGCTCATGGA-3΄ for C. albicans; gccgcgcggcagccatATGTCAGTAAAAATAACTG and gtcatgctagccataTCAATGGGTAAATTCGTA for C. parapsilosis. The sequences complementary to CaPHO15 and CpPHO15 are capitalised. PCR products were inserted into NdeI site of pET28b vector using the InFusion HD Cloning Kit (Clontech). The resulting vectors pET28-albi-Pho15 and pET28-para-Pho15 enabled expression of CaPho15p and CpPho15p with His-tags at the N-termini.

The yeast strain DBY746 (MAT leu2-3,112 his31 trp1-289a ura3-52 GAI+) culture was started with frozen stock plated onto a YPD medium (yeast extract peptone dextrose) (Sigma-Aldrich, Saint-Quentin Fallavier, France). Extract (CAJ-USE at a final concentration of 1 mg/mL) and resveratrol (RES, positive control, at a final concentration of 10 µM) were dissolved in cell culture-grade dimethyl sulfoxide (DMSO; Sigma-Aldrich, Saint-Quentin Fallavier, France) and applied at a final DMSO concentration of 0.1% (v/v). Control yeast was inoculated with the same DMSO concentration. Survival was determined as previously described [65 (link)].

The yeast strain DBY746 (MAT leu2-3,112 his31 trp1-289a ura3-52 GAI+) culture was started with frozen stock plated onto an YPD medium (yeast extract peptone dextrose) (Sigma-Aldrich, Saint-Quentin Fallavier, France). Extract (CAJ-USE at a final concentration of 1 mg/mL) and resveratrol (RES, positive control, at a final concentration of 10 µM) were dissolved in cell culture grade dimethyl sulfoxide (DMSO; Sigma-Aldrich, Saint-Quentin Fallavier, France) and applied at a final DMSO concentration was 0.1% (v/v). Control yeast was inoculated with the same DMSO concentration. Survival was determined as previously described [58 (link)].

The yeast strain DBY746 (MAT leu2-3,112 his31 trp1-289a ura3-52 GAI+; ATCC 204660) culture was started with frozen stock plated onto an YPD medium (yeast extract peptone dextrose) (Sigma-Aldrich, Saint-Quentin Fallavier, France). Extracts (at a final concentration of 1 mg/mL) were dissolved in cell culture grade dimethyl sulfoxide (DMSO; Sigma-Aldrich, Saint-Quentin Fallavier, France) and applied at a final DMSO concentration was 0.1% (v/v). Control yeast was inoculated with the same DMSO concentration. Resveratrol was used as positive control (at a final concentration of 10 µM). The impact on yeast survival was asserted as previously described [38 (link)].