Inhibition of Respiration of Candida albicans by Small Molecules Increases Phagocytosis Efficacy by Macrophages

Media and reagents.Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Lonza (Verviers, Belgium). YPD medium (yeast extract [10 g/liter], peptone [20 g/liter], and glucose [20 g/liter]), MOPS-buffered YPD medium (YPD medium plus MOPS 165 mM), yeast nitrogen base (YNB) medium, 100× penicillin, and streptomycin were purchased from Sigma (Saint Louis, MO, USA). Dimethyl sulfoxide (DMSO) was from Biomol GmbH (Hamburg, Germany). The inhibitors antimycin A (AA), rotenone, and salicylhydroxamic acid (SHA) were from Sigma, thenoyltrifluoroacetone (TTFA) was from Fluka (Saint Louis, MO, USA), and potassium cyanide (KCN) and H₂O₂ were from Roth (Karlsruhe, Germany) and Merck (Darmstadt, Germany), respectively. Anti-β-1,3-glucan antibodies were from Biosupplies (Parkville, Australia). MOPS, fluorescein isothiocyanate (FITC), FITC-labeled concanavalin A (FITC-ConA), propidium iodide (PI), and the formalin solution were purchased from Sigma, and stock solutions were prepared: 1 mg/ml in 0.9% NaCl for FITC-ConA, 100 mg/ml in dimethyl sulfoxide (DMSO) for FITC, and 20 mM for propidium iodide also in DMSO. Trypan blue was from Fluka.

Cell culture.The murine macrophage cell line RAW 264.7 was purchased from the American Type Culture Collection (Manassas, VA). The cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a 5% CO₂-in-air atmosphere.

Microorganisms and culture conditions.If not mentioned otherwise, growth was followed via determination of optical density at 620 nm (OD₆₂₀) in a 180-μl sample volume using the μQuant microplate reader (BioTek Instruments, Bad Friedrichshall, Germany).

C. albicans strains SC5314 (ATCC MYA-2876) and CAF2-1 (29) were cultivated overnight in 100-ml flasks in 20 ml of YPD medium at 30°C with orbital shaking at 160 rpm. From this overnight culture, a preculture was prepared by dilution to an OD₆₂₀ of 0.2 in 25 ml YPD medium.

The cells were allowed to grow for 3 h so that they reached the exponential growth phase. The working culture was prepared by diluting the preculture to an OD₆₂₀ of 0.1 in 25 ml YPD medium supplemented with or without electron transfer chain inhibitor. The cultures were allowed to grow at 30°C with orbital shaking at 160 rpm overnight. The final concentrations of electron transfer chain (ETC) inhibitors were 40 mg/liter of rotenone, 5 mg/liter of TTFA, 50 mg/liter of SHA, 0.5 mg/liter of AA, and 5 mg/liter of KCN. The concentrations were chosen according to previous studies (15) and were based on inhibitory effects on oxygen consumption and reactive oxygen species (ROS) induction.

For fluorescence labeling, 1 × 10⁸ yeast cells were harvested by centrifugation (13,000 rpm, 5 min, 24°C), washed twice with 1 ml phosphate-buffered saline (PBS), and stained with 1 ml of 500 mg/liter FITC at 4°C overnight. Finally, yeast cells were carefully washed three times in PBS to remove excessive dye before use.

Growth and viability of C. albicans.C. albicans was cultivated overnight in YPD medium with and without AA and methanol as solvent control as described above. Growth of the cultures was estimated from measurements of optical densities at 620 nm. The investigations were independently performed three times with three samples per test.

The viability of the yeast cells was determined by PI staining. One milliliter of a yeast suspension was centrifuged at 10,000 × g for 3 min to pellet the cells. The supernatants were removed, and the cells were washed with PBS once and resuspended in 1 ml PBS. The samples were diluted to 10⁶ cells/ml, and 10 μl of the PI stock solution (2 mM) was added. Each sample was gently vortexed. The samples were incubated at room temperature or 37°C in the dark for 15 to 30 min. Cells were analyzed by flow cytometry without additional washing. Flow cytometric analysis was performed with a FACSCanto cytometer (Becton Dickinson, USA) (see Fig. S1 in the supplemental material).

Phagocytosis assay.Phagocytosis of C. albicans by macrophages was quantified as described previously (4). Briefly, 100 μl of 2 × 10⁶ macrophages/ml was seeded in each well of 96-well microplates (Nunc, Germany) followed by incubation for 2 h to let the cells adhere to the plates. The supernatant was replaced by 100 μl of a suspension of 4 × 10⁶ FITC-labeled yeasts in DMEM supplemented with 10% FBS, so that the ratio of macrophages to yeast was 1:2. Phagocytosis was allowed to proceed at 37°C in 5% CO₂. At indicated time points, the yeast suspension was removed and 100 μl trypan blue (250 mg/liter in PBS) was added to quench the fluorescence of yeasts which were not internalized. After incubation at room temperature for 1 min, the trypan blue solution was removed. The number of internalized yeasts was estimated from fluorescence measurements (λ_Ex = 480 nm and λ_Em = 520 nm) through the bottom of the plates by a fluorescence microplate reader (Synergy 4; BioTek, Bad Friedrichshall, Germany).

Data analysis was based on the mean of the fluorescence values of eight wells per experiment, and each experiment was independently repeated at least twice. Background fluorescence was determined from the fluorescence of wells containing only FITC-labeled yeasts but no macrophages. Data are presented as the difference between the total fluorescence and the background.

As a control for the microplate assay, we investigated the ingestion of yeasts by fluorescence microscopy. Seven hundred fifty microliters of 5 × 10⁵ macrophages/ml was seeded in each of the 4 compartments of a Cellview glass-bottom dish (Greiner Bio-one, Frickenhausen, Germany) followed by incubation for 2 h to let the cells adhere to the plates. A C. albicans suspension was added at a 1:2 ratio of macrophages to yeast. Phagocytosis was allowed to proceed at 37°C in 5% CO₂ for 1 h. Wells were washed with PBS and fixed with a 2% formalin solution. The numbers of macrophages and yeasts were recorded in arbitrarily chosen pictures, and at least 200 macrophages were counted. The phagocytosis index was defined as the ratio of the number of macrophages with intracellular yeasts to the total number of macrophages per picture.

Detection of β-(1,3)-glucans and mannans on yeast cells.β-(1,3)-Glucans were stained with specific Alexa Fluor 488-conjugated monoclonal antibodies (30). One milliliter of 10⁶ cells/ml was washed with PBS containing 2% FBS and then incubated with 200 μl of the anti-β-(1,3)-glucan antibody (1:150 dilution) for 1 h. Cells were washed in PBS containing 2% FBS 3 times and incubated with the secondary antibody (Alexa Fluor 488-labeled goat anti-mouse; 1:250 dilution) for 45 min. As a negative control, yeast cells were incubated only with the secondary antibody. After washing, cells were fixed in 0.4% formalin and analyzed by flow cytometry.

The detection of mannans was based on binding of concanavalin A (31). Suspensions from AA- and H₂O₂-treated C. albicans cultures were adjusted to an OD₆₂₀ of 0.1 using sample volumes of 180 μl and the μQuant microplate reader. Cells were washed twice with 0.9% NaCl, collected by centrifugation, and resuspended in 500 μl of an FITC-concanavalin A solution (1:50 dilution with 0.9% NaCl of the stock solution of 1 mg/ml). After 30 min, cells were collected by centrifugation and washed twice with 1 ml 0.9% NaCl. After washing, cells were fixed in 0.4% formalin and analyzed by flow cytometry. All incubation and washing steps were done at room temperature.

Gene expression analysis by real-time PCR (RT-PCR).C. albicans was cultivated overnight in 250-ml flasks with 50 ml YPD medium at 30°C. A preculture was prepared by diluting the overnight culture in 25 ml YPD (OD₆₂₀ of 0.2) and was cultivated for 3 h to reach the exponential growth phase. For the working culture, the preculture was diluted to an OD₆₂₀ of 0.1 in YPD supplemented with or without 1.5 mg/liter AA. After cultivation at 30°C for 20 min, the cells were harvested by centrifugation, and the cell pellets were shock-frozen in liquid nitrogen. Frozen pellets were suspended in 0.6 ml RLT buffer (Qiagen) and mechanically disrupted using glass beads (425 to 600 μm; Sigma). RNA was isolated on RNeasy minicolumns with added DNase (Qiagen) as recommended by the manufacturer. The quality and integrity of total RNA of a subset of the samples were controlled with the Agilent Technologies 2100 Bioanalyzer (Agilent Technologies).

Three micrograms of total RNA was employed in reverse transcription, with Superscript II reverse transcriptase (RT) and random and oligo(dT)_12–18 primers, according to the manufacturer’s recommendations (Invitrogen). Quantitative real-time PCR was carried out on a 96-well LightCycler 480 system using the LightCycler 480 SYBR green I master (Roche), as recommended by the manufacturer (95°C, 60°C, and 72°C for 10 s each for 45 cycles). Gene sequences were obtained from the C. albicans Genome Database (32), and gene-specific oligonucleotides (Table S1) were designed by Roche’s Probe Library Assay Design Center and synthesized by Eurofins MWG Operon. Specificity was controlled against the C. albicans genome sequence by using BLAST. Real-time analysis data (crossing points) were normalized with respect to the actin gene ACT1, and relative gene expression levels were calculated. The average and standard deviations of the gene expression levels relative to solvent controls in three independent experiments were calculated, and the significance of the changes in gene expression was tested by Student’s t test of normalized data (P < 0.05) (Fig. S2).

Ultrastructural analysis of the cell walls of C. albicans after AA treatment using TEM.Cells were treated according to the literature (33). Briefly, a suspension of C. albicans cells (1.5 × 10⁸ cells/ml) was prepared from yeast cultures grown for 24 h at 37°C in YPD with or without different concentrations of AA. The samples were centrifuged. The resultant pellets were each resuspended in 5 ml PBS. After washing two times, the fungal cells were fixed by resuspending each pellet in 1 ml of 4% formaldehyde and 1% glutaraldehyde in PBS. After 2 h of incubation at room temperature, the samples were stored at 4°C until they were stained with 1% osmium tetroxide (OsO₄) at room temperature. After washing with PBS to remove excess OsO₄, a dehydration series was performed with 25, 50, 75, 95, and 100% ethanol diluted in distilled water (dH₂O). The samples were additionally dehydrated with propylene oxide, embedded in a resin, and left to harden for 48 h at 60°C. The resin capsules were cut using an ultramicrotome (Leica Ultracut UCT) and a 45° diamond knife. Ultrathin sections of approximately 95 nm were obtained and visualized using a CM100 TEM (Philips, Netherlands).

Ethanol and acetate determination.A C. albicans preculture was prepared as described above. Twenty-milliliter working cultures were prepared by diluting the preculture to an OD₆₂₀ of 0.1 with YPD medium supplemented with 1.5 mg/liter AA or the respective amount of solvent (methanol). The sample volume was 200 μl of the cell suspension. Samples were taken every hour and immediately centrifuged at 14,000 rpm and 4°C for 10 min. Ethanol concentrations were determined by enzymatic assays based on ethanol dehydrogenase (21). Quantification was based on the photometric determination of NADH at 340 nm, and the assays were performed in volume-reduced 96-well transparent microplates (Corning). For acetate determination, C. albicans was grown with 0.5 and 5 mg/liter AA or the respective amount of solvent (methanol). Samples were taken after 1 h and 24 h. Acetate concentration was detected by high-performance liquid chromatography (HPLC) (Agilent, USA).

pH measurement.C. albicans was grown in YPD medium supplemented with or without various concentrations of AA at 30°C overnight. The suspension was collected by centrifugation at 5,000 rpm for 5 min, and the pH of the cultivation medium was determined with a pH meter.

Extraction of intracellular metabolites.C. albicans preculture was prepared as described above. Twenty-milliliter working cultures were prepared by diluting the preculture to an OD₆₂₀ of 0.1 with YNB medium supplemented with 1.5 mg/liter AA or the respective amount of solvent (methanol) for 4 h. Six biological replicates were used for the treated group and the control group. The extraction of intracellular metabolites was done as described in literature (34, 35). Briefly: the cell pellet was obtained by 5 min of centrifugation (10,000 × g, 4°C), fixed with 60% cold MeOH in NaCl buffer, and washed with 5 ml cold phosphate-buffered saline (PBS) 3 times. The cell pellets were stored at −80°C and then lyophilized in a freeze drier.

The metabolic extract was prepared according to literature (36). Briefly, 100 μl of cell pellets was spiked with internal standard (15 μl heptadecanoic acid in water, 1 mg/ml) and vortexed for 2 min. The mixed solution was extracted with 400 μl methanol and centrifuged for 10 min at a rotation speed of 13,000 rpm at 4°C. An aliquot of 450 μl of the supernatant was transferred to a clean glass sampling vial and dried under a gentle stream of nitrogen at room temperature. For derivatization, 80 μl methoxamine hydrochloride (20 mg/ml in pyridine) was added to the vial and kept at 60°C for 4 h followed by the addition of 60 μl of 1% N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA; 1% TMCS) for 1 h at 70°C. An aliquot of 2 μl of the derivatized sample was injected into an Agilent 7890B gas chromatograph coupled with an Agilent 5977A mass spectrometer. Separation was performed on an HP-5MS capillary column (30 m by 0.25-mm inside diameter [i.d.], 0.25-μm film thickness; Agilent, USA) with He as the carrier gas at a constant flow rate of 1 ml/min. The solvent delay time was set to 5 min. The temperatures of injection, transfer interface, and ion source were 250°C, 290°C, and 230°C, respectively. The GC temperature programming was set to 2 min of isothermal heating at 80°C, followed by 5°C/min oven temperature ramps to 240°C and 8°C/min to 300°C, and a final 6-min maintenance at 300°C. Ionization mode was electron ionization (EI) mode with the electron energy of 70 eV. A full scan mode with a scannning range of m/z 50 to 600 was used.

Data analysis.The GC-MS raw data were converted to an mzXMLfile and then imported into R software (http://cran.r-project.org/) for noise filtering, baseline correction, and ion peak alignment. The data were then probability quotient normalized and analyzed by orthogonal signal correction partial least squares discriminant analysis (OSC-PLS-DA) to differentiate each group. The OSC-PLS-DA model was cross-validated using a 7-fold method by default; the validity of the models against overfitting was assessed by the parameters R²Y, and the predictive ability was described by Q²Y (37). Negative or very low Q²Y values indicate that the differences between groups are not statistically significant. Metabolites were identified by comparing the mass fragmentations with NIST 05 standard mass spectral database (National Institute of Standards and Technology [NIST], Gaithersburg, MD) with a similarity of more than 70% and finally verified by available reference standards.

Statistics.Diagrams were created with Excel 2010 and GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA), respectively. For comparative statistical analysis, Student’s unpaired t test was performed using Microsoft Excel 2010. P values of <0.05 were considered significant.

Data availability.All data have been provided in the paper and in the supplemental material.