SUPPLEMENTARY INFORMATION

Control of Candida albicans metabolism and biofilm formation by Pseudomonas aeruginosa phenazines by Morales, D.K. et al.

Supplementary Materials and Methods

Co-culture growth conditions

C. albicans-P. aeruginosa co-cultures were performed on YPD by inoculating P. aeruginosa strains on YPD agar, incubation for 37oC for 5 hours, followed by inoculation of C. albicans from overnights, then incubation for 24 hours.

Preparation of phenazines and methylene blue.

When indicated, media were supplemented by adding phenazines from stock solutions of 25 mM PMS (Acros-organics) dissolved in water and 30 mM PYO (Cayman Chemicals) in ethanol to sterile liquid medium or molten agar. Phenazine-1-carboxylic acid (PCA) was added from a 20 mM stock solution, and methylene blue was added from a 3 mM stock solution made in water. All assays were carried out in the dark to prevent photoinactivation of phenazines.

Beta-galactosidase assay in colonies. To measure the expression of HWP1 or ACT1, C. albicans CAI-4 lacZ fusion strains (1), were spot inoculated onto Glu+AA agar with 200 µg/ml of X-Gal in the presence or absence of 5 µM PMS, and incubated for 72 hours prior to visualization.

Anaerobic growth conditions

For colonies grown under anaerobic conditions, 10 µl of a YPD-overnight were inoculated on Glu+AA medium with either 10 or 100 mM of glucose and supplemented with 20 µM of oleic acid (in 100% methanol) and 80 µM nicotinic acid, two nutrients that were previously reported to be necessary for supporting C. albicans anaerobic growth (2). Plates were incubated for 48 hours in an anaerobic jar (Torbal) using a gas pack system with indicator for anaerobiosis (Becton Dickinson).

Growth-curve analysis and enumeration of cells within colonies.

To measure C. albicans growth in the presence and absence of phenazines, C. albicans overnight cultures were diluted down to 0.01 (OD600) in 5 ml modified Glu+AA medium with 100 mM glucose and grown to an absorbance of 0.03. Cells were washed twice with sterile water, then resuspended in water and diluted 1:1 in 2X liquid media with or without phenazines. Growth was evaluated every hour by measuring the absorbance (OD600) using a SpectraMax M2 instrument.

To determine the total number of cells expressed as colony-forming units (CFU) within each phenazine-treated or untreated colony, C. albicans was grown on 0.2 µm membranes (Millipore) placed on Glu+AA agar medium with or without PYO or PMS (20 and 5 µM, respectively) under non-inducing conditions. The membranes were removed and the cells were suspended in saline prior to serial dilution and plating on YPD agar. After 3 days of incubation at room temperature, the final number of colonies was counted.

C. albicans colony biofilm assays and analysis of extracellular pH

C. albicans strains from stationary phase cultures with an adjusted absorbance (OD600) of 8.0 in 5 ml of YPD were spot inoculated (10 µl) onto agar media. Extracellular alkalinization was qualitatively evaluated by adding 0.01 % of the pH indicator bromocresol purple in ethanol (Sigma) to the medium. To measure fermentation products from colony biofilms, C. albicans colonies grown in the liquid assay were cultured onto a 0.2 µm membrane (Millipore) that was floating on 600 µl of Glu+AA liquid medium with or without 5 µM PMS and 20 µM PYO in 24-well plates. 5 µl of a C. albicans was inoculated, and colonies were grown for 24 hours at 37oC. Acidification of the extracellular milieu was assessed by adding 0.01 % of the pH indicator bromocresol green (Sigma) in ethanol after the cells were cultured. Fermentation products were measured as described below.

Quantification of ammonia, glucose and fermentation-related products

To quantify the production of volatile ammonia by phenazine treated and untreated colonies, a previously described and slightly modified acid trap technique was used (3, 4). Briefly, individual colonies were grown under inducing conditions in 96-well plates with 150 µl of YNB Glu+AA agar alone or with 5 µM PMS or 20 PYO µM. Acid traps, with 100 µl of 10 % (w/v) citric acid per well of a 96-well plate, were situated directly on each well of inverted 96-well plates with growing colonies. After 24 hours at 37˚C, the ammonia concentrations within the citric acid solution were determined. The solution was diluted 10-fold, and 20 µl was added to 80 µl of Nessler’s reagent (Fisher). Samples were incubated at room temperature for 30 min prior to measurement of the absorbance at OD400. Ammonia concentrations (ppm) were determined using an NH4Cl standard curve (3-50 ppm).

To determine production of fermentation products, 500 µl of culture media were collected and centrifuged (10 min at 13,200 rpm) to pellet cells. Supernatants (450 µl) were filtered by using a HPLC Spin-X centrifuge tube filter (Costar) and then stored at -80oC until analysis. Filtered samples were acidified with 10% (w/w) H2SO4 and subsequently analyzed by HPLC with an Aminex-87H column (Bio-Rad Laboratories, Hercules, CA) operated at 60oC (5) with 0.01 % (v/v) H2SO4 mobile phase using dual detection of refractive index and ultra violet (λ =210nm). Peak heights were measured and identified by comparison of retention times with authentic standards and their concentrations determined by the external standard method.

C. albicans respiratory activity analyses and oxygen measurement analysis

To analyze the respiratory activity of colonies, we employed the previously described TTC-overlay technique (6). This method that was originally described by Lenhard in 1956 (7) has been broadly used to determine the ETC activity of microbial populations. Colorless 2,3,5 triphenyltetrazolium chloride (TTC) is able to pass through the plasma membrane and reach the mitochondria, where it is converted into reddish 1,3,5-triphenylformazan (TTF) by reducing equivalents and where the colored products then remain immobilized (6, 8). To assess metabolic activity in colonies, 2 ml of the TTC-agar solution (55˚C) was overlaid onto 55 mm plates containing spot-inoculated colonies grown as described in previous sections. Red color development was observed 24 hours after the addition of TTC at room temperature. The TTC-overlay agar solution consisted of 0.067 M potassium phosphate buffer at pH 7.0 and 0.1% TTC.

Supplementary References

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