Control organisms C. albicans SC5314 and additional 2 clinical isolates of C. albicans CA-R15 and CA-R21 were investigated in this study. C. albicans SC5314 was obtained from American Type Culture Collection. The 2 clinical isolates were obtained from the intensive care unit of Qingpu Branch of Zhongshan Hospital, Shanghai, China. All clinical isolates were recovered from oral, thigh and each represented a unique isolate from a patient. Yeasts were identified at the species level by CHROmagar (KANTO CHEMICAL) and Rapid Tm Yeast plug system (REMEL Inc) and stored at -70 °C in 10% glycerol. Before the initiation of the study, yeast isolates were sub-cultured on antimicrobial agent-free medium to ensure viability and purity.
4.2. Antifungal Agents
Fluconazole was purchased from Sigma-Aldrich (St. Louis, MO). 10 nm poly-vinylpyrrolidone (PVP) coated biopure silver nanoparticles were purchased from Nano Composix Inc. (San Diego, CA). The mass concentration of the AgNPs used in this study was 1 mg/ml. The size distribution is 8–12 nm.
4.3. In vitro antifungal assays
Minimum inhibitory concentrations of AgNPs and fluconazole were tested by the microdilution method as outlined in Clinical and Laboratory Standards Institute document M27-A3 and as previously described using 96-well microtiter plates . The concentrations for fluconazole were 0.125 µg/ml to 128 µg/ml. For AgNPs, the concentrations were set from 0.25-64 µg/ml. 100 µl of the inoculum suspension with a concentration of 2 × 103 cells/ml in RPMI 1640 medium without phenol red and sodium bicarbonate (Sigma-Aldrich. St. Louis, MO), was incubated with these various concentrations of antifungals in 96-well plates and cultured at 35◦C for 48 h. After incubation, the cell growth was detected by XTT reduction assay. Absorption at 492 nm was detected by the microplate reader. The MICs were defined as the lowest concentration of drugs that caused > 80% inhibition of C. albicans. When the MICs were higher than the top concentration, then the top concentration was regarded as the MICs. The types of interaction between AgNPs and fluconazole were analyzed with the checkerboard method. Briefly, various concentrations of AgNPs and fluconazole were combined to provide 80 combinations and incubated with C. albicans cells. The interaction of AgNPs and fluconazole was interpreted using fractional inhibitory concentration index (FICI) model: FICI = FIC AgNPs + FICFLC = (MIC of AgNPs in combination/MIC of AgNPs alone) + (MIC of fluconazole in combination/MIC of fluconazole alone). FICI values ≤ 0.5 synergy, 0.5 < FICI ≤ 1 additivity, 1 < FICI ≤ 4 indifference, > 4 antagonism. Each assay was performed in triplicate.
4.4. Biofilm studies
50 µl of C. albicans cells diluted in RPMI-1640 with a concentration of 1.0 × 106 cells/ml were added to 96-well plates to a final volume of 200 µl and cultured under static conditions at 35◦C for 6, 12 or 24 hours to form biofilm. RPMI-1640 without cells was used as a negative control. After that, the wells were carefully aspirated and gently washed three times with 200µL of sterile phosphate-buffered saline (PBS) to remove any remaining suspended cells. Then, an equal volume of fresh RPMI-1640 with various concentrations of antifungal fluconazole (2–256 µg/ml) and AgNPs (8–512 µg/ml) were added. After incubation for another 48 h at 35◦C, XTT reduction assay was used to measure the viability of biofilms. And absorption at 492 nm was detected by the microplate reader. The MICs were defined as the lowest concentration of drugs that reduces the absorption value by 50% relative to control. Each assay was performed in triplicate. The interaction of AgNPs and fluconazole was also interpreted using fractional inhibitory concentration index (FICI) model: FICI = FIC AgNPs + FICFLC = (MIC of AgNPs in combination/MIC of AgNPs alone) + (MIC of fluconazole in combination/MIC of fluconazole alone). FICI values ≤ 0.5 synergy, 0.5 < FICI ≤ 1 additivity, 1 < FICI ≤ 4 indifference, > 4 antagonism.
4.5. Ergosterol measurement
Sterols were extracted and analyzed as described in . Briefly, cells, after grown overnight in YPD medium in the presence of AgNPs (0, 15, 30 µg/ml), were harvested and boiled in 25% alcoholic KOH for 1 h. Then sterols were extracted from the boiled cells with petroleum ether. Ergosterol content was determined spectrophotometrically by analyzing the specific pattern of absorbance between 200 and 320 nm.
4.6. Quantification Analysis by Real-Time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
The RNA isolation and cDNA synthesis were performed as previously described. 18S ribosomal RNA (18S rRNA) was used as internal control.
4.7. Total protein extractions, Plasma Membranes isolation and western blotting
Cells were cultured in YPD liquid medium overnight in the presence of AgNPs (0, 15, 30 µg/ml) and harvested. Then cells were washed twice in PBS and resuspended in homogenization medium containing 50 mM Tris (pH 7.5), 2 mM EDTA, and protease inhibitor cocktail (Roche Diagnostics, Germany ). After incubation at 37℃ for 30 min, cells were disrupted by Braun cell homogenizer. Cell lysate was centrifuged at 2,000 × g for 10 min. The supernatant were collected to obtain total proteins. For plasma membrane isolation, the supernatant was ultra-centrifuged at 30,000 × g for 45 min to isolate crude membrane fraction. A micro-BCA protein assay kit was used to determine protein concentrations (Pierce, Rockford, IL). A total of 30 µg protein was separated on 6% or 8% SDS-PAGE gel. After that, the gel was transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA) with Trans Blot SD Semi-Dry transfer cell (Bio-Rad). Ponceau S reagent (0.1% Ponceau S in 5% acetic acid) staining was performed to visualize the transferred proteins on membranes. Subsequently, the membranes were incubated with anti-Cdr1p (1:1000) or anti-Cdr2p (1:1000) polyclonal antibodies, then with secondary anti-rabbit IgG peroxidase (1:5000, Santa Cruz Biotechnology, TX). The anti-Cdr1p and anti-Cdr2p polyclonal antibodies were generated in rabbits as described in .
4.8. Membrane Fluidity Assessment
The fluorescence polarization measurements were carried out to assess the physical state (fluidity) of the membrane. After corresponding treatment, cells were harvested and incubated with lyticase (Sigma-Aldrich. St. Louis, MO) according to the manufacturer’s introduction to get spheroplasts. Then the prepared spheroplasts were labeled with 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) (Aladdin Industrial Corporation, Los Angeles, CA). Fluorescence polarization (p value) was measured with spectrofluorometer Hitachi F-7000 (Hitachi, Tokyo, Japan) at excitation and emission wavelengths of 360 nm and 450 nm respectively.
4.9. Rhodamine 6G (R6G) efflux assay
Rhodamine 6G (R6G) purchased from Shanghai Macklin Biochemical Co. was used as a surrogate to investigate the uptake and efflux of azoles as described previously . The C. albicans cells were inoculated in SDA medium and cultured at 35◦C by at 200 rpm for 16 hours. After reaching exponential phase, cells were harvested by centrifugation at 3000 rpm and 4◦C for 5 min. Then wash the cells three times with precooling PBS. After that, cells were re-suspended in glucose-free PBS (pH = 7.0) and adjusted to 1 × 108 cells /ml.
R6G efflux assay was performed as follows: After adjusting to 1 × 108 cells /ml in glucose-free PBS and de-energized for 2 h, cells were treated with R6G (10 µM) at 35◦C for 60 minutes to preload the cells with R6G. Cells were then treated with precooling PBS to stop the uptake of R6G, and were centrifuged at 3000 rpm and 4◦C for 10minutes. After washed twice in PBS to remove the remaining extracellular R6G, cells were resuspended in PBS with 0.1 M glucose and AgNPs (0 µg/ml, 15 µg/ml, and 30 µg/ml). Cells were collected at specific time intervals. Mean fluorescence intensity of the intracellular R6G was detected by CytoFLEX (Beckman Coulter, USA) at excitation and emission wavelengths of 488 and 530 nm respectively. Each assay was performed in triplicate.
4.10. Ergosterol supplementation experiments
Ergosterol was dissolved in petroleum ether and added directly and concomitantly with AgNPs into the medium during inoculation to a final concentration of 10 µg/ml.
4.11. ROS production assay
CA-R21 cells were grown until mid-exponential phase. After that, cells were harvested and washed twice with PBS. Then, cells (1 × 108 cells/mL) were treated with fluconazole 0.5 µg/ml for another 4 hours, together with AgNPs 0 µg/ml, 0.5 µg/ml, and 1 µg/mL. After incubation, cells were centrifuged at 5,000 rpm for 10 minutes at 4 °C to harvested, followed by washing twice with PBS buffer to remove the media. Then cells were resuspended in PBS buffer and the fluorescent probe DCFH-DA was added to a final concentration of 10 µM. After incubation in the dark for 30 minutes, cells were washed in PBS for three times to remove the residual dye. Mean fluorescence intensity was detected by CytoFLEX (Beckman Coulter, USA) at excitation and emission wavelengths of 488 and 540 nm respectively.
4.12. In vivo experiment
Female, 5-week-old, BALB/c mice (CLEA Japan.Inc.) ,15 to 18 g, were used in the experiments (n = 18/group),and maintained in micro-isolator boxes with a standard rodent diet according to National Institutes of Health guidelines for animal care and in fulfillment of American Association for Accreditation of Laboratory Animal Care criteria. Immune-compromised mice were obtained by administrating 200 mg/kg cyclophosphamide (Sigma-Aldrich, St. Louis, MO) and 250 mg/kg cortisone acetate (Sigma-Aldrich, St. Louis, MO) intraperitoneally 2 days prior to yeast challenge and continued until the day of challenge. Clinically isolated C. albicans were sub-cultured overnight in YPD broth (Becton, Dickison and company) at 30 °C 1 day prior to challenge. On the day of challenge, the subculture was pelleted, and exponential-phase cells were harvested, washed, and resuspended in sterile saline. The concentration of blastospores was counted with an absorbance detector, and the blastospore suspension was adjusted to give 2 × 108 blastospores/ml for the in vivo experiments. Actual CFU in the inocula were determined by culturing serial dilutions of each preparation onto YPD plates. Murine models of systemic candidiasis were established by infecting immune-compromised mice intravenously with 0.2 ml of Candida cell suspension of these strains through the lateral vein. After that, mice were further divided into two sub-groups with one sub-group for tissue burden analysis (8 mice) and the other one for survival analysis (10 mice). Therapy began four hours after challenge and continued for 12 days. A dose of 10 mg/kg fluconazole was given intravenously once daily. AgNPs were administrated intravenously at doses of 1 mg/kg and 3 mg/kg separately. After 7 days of consecutive therapy, mice for tissue burden analysis were euthanized. Kidneys and livers were aseptically removed, and the entire organs were homogenized (Shake master, NEO bio medical science) in 1 ml of sterile saline. Serial 10-fold dilutions of the homogenates were plated on YPD agar (Becton, Dickison and company), incubated at 35 °C, and examined the number of CFU per organ of tissue. The mice for survival analysis were kept and monitored daily for mortalities in the next two weeks.
4.13. Statistical analysis
A one-way analysis of variance (ANOVA) was applied for analysis and where differences occurred, a two-tailed Tukey’s multiple-comparison test was done between paired groups. A p value of < 0.05 was considered significant. Kaplan-Meier survival plots were analyzed by a log-rank (Mantel-Cox) test. All statistical analysis was performed using GraphPad Prism, version 7.03 (GraphPad Software, Inc., San Diego, CA).