Glucose

Transfected islet cells were first incubated at 37 °C for 45 min in a Krebs-Ringer bicarbonate buffer (KRBH) containing 25 mM HEPES, pH 7.4, 0.1% BSA, and 2 mM glucose (Sigma). Thereafter, the cells were incubated at 37 °C for 45 min in KRBH-BSA solutions with 2 mM (basal) or 20 mM (stimulatory) glucose, or with 2 mM glucose and 30 mM (stimulatory) KCl (Sigma). After incubation, supernatants were collected. The cells kept at basal glucose were harvested using acid ethanol (75% ethanol, 0.55% HCl), and those incubated at stimulatory glucose were lysed using Triton X-100 lysis buffer to determine insulin and protein contents, respectively. Insulin levels were measured by ELISA (Mercodia) and cellular protein contents by Bradford assay (Thermo Fisher).

The yeast strain GRX1-GFP-HIS3MX6 was purchased from Thermo Ficher Scientific. All the primers used in this study are listed in Supplementary Table 1. To create the GRX1-GFP-tdTomato-kanR strain, a PCR fragment containing tdTomato-kanR and homologies to GFP-HIS3MX6 was amplified with primers C1 and C2 from pfa6a-tdTomato-kanR (constructed in our lab) and transformed to the GRX1-GFP-HIS3MX6 strain.
All the strains were grown in liquid YPD medium containing 20 g/L glucose (Sigma), 10 g/L peptone (Euromedex) and 10 g/L yeast extraction (Euromedex). When needed, 20 g/L agar (Euromedex) was added to make solid plates. YNB-URA⁻ plates [20 g/L glucose (Sigma), 20 g/L agar (Euromedex), 1.71 g/L yeast nitrogen base without amino acids and nitrogen (Euromedex), 5 g/L ammonium sulfate (Sigma), and 0.77 g/L CSM-URA⁻ (Euromedex)] or 5-FOA plate [20 g/L glucose (Sigma), 20 g/L agar (Euromedex), 1.71 g/L yeast nitrogen base without amino acids and nitrogen (Euromedex), 5 g/L ammonium sulfate (Sigma), 0.79 g/L CSM-URA⁻ (Euromedex), and 1 g/L 5-FOA (Euromedex)] were used to select transformants.

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.

One non-oleaginous S. cerevisiae strain and five oleaginous yeast strains were used in this study. S. cerevisiae CEN.PK113-7D was kindly provided by Peter Kötter (Entian and Kötter 2007 (link)). Trichosporon oleaginosus DSM11815, Rhodotorula graminis DSM 27356, L. starkeyi DSM 70296, and R. toruloides DSM 70398 were purchased from the culture collection of the DSMZ (Braunschweig, Germany). Y. lipolytica CBS 6124 was purchased from the CBS-KNAW Fungal Biodiversity Centre (Utrecht, The Netherlands). Rich media, YPD or YM, were used for cultivation of these six yeasts. The YPD medium was prepared with 10 g/l yeast extract (Merck Millipore), 20 g/l peptone (Merck Millipore), and 20 g/l glucose (Merck Millipore). The YM medium was constituted of 3 g/l yeast extract (Merck Millipore), 3 g/l malt extract (Oxoid), 5 g/l peptone from soybeans (Merck Millipore), and 10 g/l glucose (Merck Millipore). The nitrogen-limited medium (named NLM medium in the text) was prepared as described in the literature using 70 g/l glucose (Merck Millipore) as carbon source (Yang et al. 2014 (link)).

Bacterial suspension was added (10%) to four different media three of which are newly synthesized media and the other is MRS broth. The new media compositions included SGSL: 1% tryptone (Difco, Le Ponte de Claix, France), 1% peptone, (Difco, Le Ponte de Claix, France), 1% yeast extract (Difco, Le Ponte de Claix, France), 5% glucose (Merck, Darmstadt, Germany), 0.05% ascorbic acid (R & M Chemicals, Essex, UK), 0.2% sodium citrate (Peking Chemical Works, Peking, China), 0.005% manganese(II) sulphate (BDH Chemicals Ltd, Poole, England), 0.025% magnesium sulphate (Halewood Chemical Ltd, Middlesex, England), 0.02% sodium chloride (John Kollin Corporation, USA) and 0.1% Tween 80 (Sigma-Aldrich, Missouri, USA), TPYGMA: 1% tryptone, 1% peptone, 1% yeast extract, 5% glucose, 1% 2-morpholinoethanesulphonic acid, MESA (Merck, Darmstadt, Germany) and 0.05% ascorbic acid, TPYGCaT80: 1% tryptone, 1% peptone, 1% yeast extract, 5% glucose, 0.1% CaCO₃ and 0.1% Tween 80. Incubation at 37 °C was done after broth inoculation. OD was monitored over a period of 24 h. Fermencin SA715 production was determined by well diffusion assay using Micrococcus luteus as the bacterial target.

Following growth media were selected:

YPD (Yeast extract peptone dextrose: yeast extract 1% (Sigma-Aldrich Life Science, St. Louis, USA), glucose 2% (Merck KGaA, Darmstadt, Germany), peptone water 2% (Oxoid LTD, Hamshire, England)) and FBS (Fetal Bovine Serum: (Gibco, Life Technologies Carlsbad, California, USA)) were used as control growth media and to prepare the following mixtures:

BHI 30% (Sigma-Aldrich life science, St. Louis, USA) + YPD 70%

FBS 30% + YPD 70%

FBS 30% + RPMI 70% medium + 2% glucose (RPMI 1640: 20,8 g RPMI-1640 (Sigma-Aldrich life science, St. Louis, USA), 69,06 g MOPS (Sigma-Aldrich life science, St. Louis, USA) 36 g glucose (Merck KGaA, Darmstadt, Germany)

FBS 30% + YNB 70% (Yeast nitrogen base 0,67% with ammonium sulphate without dextrose or amino acids (Sigma-Aldrich life science, St. Louis, USA), glucose 2% (Merck KGaA, Darmstadt, Germany)

YPD 50% + RPMI 1640 50%

The set of C. glabrata strains used in this work are listed in Table 1. The different strains were batch-cultured at 30° in liquid minimal medium (MM) or in RPMI growth medium with orbital agitation (250 rpm). MM contains, per L, 20 g glucose (Merck), 1.7 g yeast nitrogen base with amino acids (Difco), and 2.65 g (NH₄)₂SO₄ (Merck). The RPMI growth medium contains, per L, 20 g of RPMI medium powder without glutamine (Sigma, St. Louis, MO), 20 g of glucose (Merck), and 0.3 g of glutamine (Sigma). Whenever needed, the MM and the RPMI growth medium were adjusted to pH 4 using HCl as the acidulant. Cell viability was assessed in Yeast Peptone Dextrose (YPD) growth medium which contains, per L, 20 g of glucose (Merck), 20 g bactopeptone (Difco), and 10 g yeast extract (Difco). Solid media was obtained supplementing the corresponding liquid growth medium with 2% agar (Iberagar).