1. Strains, Media and Compounds
C. albicans SC5314 was kindly provided by Prof. Cao (School of Pharmacy, Naval Medical University, Shanghai, China.). For all vitro experiments, 6.4 mg / mL SK (purity > 99%, Beijing Suolaibao Technology Co. Ltd, Beijing, China, dissolved in DMSO (Sigma)) was used as the stock solution stored at -20°C and added to the culture medium to obtain the required concentration. Media used in this study included Sabouraud dextrose agar plate (SDA, 1% peptone, 4% glucose, and 1.8% agar), yeast extract peptone dextrose (YPD, 1% w/v yeast extract, 2% w/v peptone, 2% w/v dextrose), phosphate buffered saline (PBS) [10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride (pH 7.4)] and RPMI 1640 (Gibco, Bethesda, MD, U.S.A.) supplemented with L-glutamine and buffered with morpholinepropanesulfonic acid (MOPS). C. albicans strains were grown in SDA plates medium, and cultivated in a liquid complete medium YPD medium at 30°C with constant shaking (200 rpm).
2. Methods
2.1 Protoplast preparation
To study the effect of test SK on apoptotic markers, protoplasts were prepared as described previously [10], with modifications. Briefly, mid-exponential phase cells were harvested and exposed to various concentrations of SK shaken at 30°C for 2 h at 200 rpm. The cells were then washed twice with PBS. Next, the cells was resuspended at a concentration of 3 × 107 CFU / mL and digested with lyticase (100 U / mL ) at 30°C for 30 min. The digested cells were centrifuged at 1500 rpm for 5 min again and the supernatant was discarded. Finally, the obtained protoplasts were washed twice with PBS and resuspended for further use.
2.2 Annexin V-FITC/PI-staining
This assay was used to identify necrosis (cellular integrity) and apoptosis (externalization of phosphatidylserine). Annexin-V-FLUOS kit (Roche Applied Science, Germany) was used following the manufacturer’s instructions [11]. Briefly, protoplasts of C. albicans cells were treated with different concentrations of SK, as mentioned in above section, and 10 mM H2O2 was used as a positive control. Then these preparation protoplasts were resuspended and incubated at 25°C for 15 minutes by 5 uL propidium iodide (PI) and FITC-labeled annexin-V. After incubation, 100 µL binding buffer was added to resuspend each sample, then which was analyzed using the flow cytometry (FACSCalibur, USA). The detection conditions are as follows: Annexin V-FITC: excitation wavelength 488 nm, emission wavelength 518 nm; PI: excitation wavelength 488-540nm, emission wavelength 617 nm.
2.3 Transmission Electron Microscope (TEM) [12]
The C. albicans SC5314 (3 × 107 CFU / mL) were firstly administrated by 4 µg / mL SK for 2 h at 37°C, and then harvested by centrifugation (3000 g, 5 min). The collected cells were promptly placed in 3% glutaraldehyde at 4°C overnight, fixed with 1% (w/v) osmium acid for 1 h. After gradient dehydration with ethanol and acetone, it was immersed, embedded, polymerized, and sliced. Finally, thin sections were prepared and stained by uranyl acetate for 2 h, being observed under Hitachi H-800 transmission electron microscope.
2.4 Cytochrome c oxidase (COX) activity
Firstly, C. albicans mitochondria of the cells were separated by Cell Mitochondria Isolation Kit (Beyotime). Then, COX detection kit (GENMED) was used to detect mitochondrial COX activity [13]. All experiments were performed according to the manufacturer’s instructions. Briefly, protoplasts of C. albicans cells were treated with 1, 2, 4 µg / mL SK as mentioned in above section, and 10 mM H2O2 was used as a positive control. Then these preparation protoplasts were homogenized in order to release the mitochondria. After centrifugation at a speed of 10 000 × g for 10 min at 4°C, the supernatant was discarded, and the precipitation particles (mitochondrial precipitate) were resuspended in 200 uL of storage solution. Protein concentration was determined by the TCA Lowry method [14]. After that, the mitochondrial COX activity need be determined. Firstly, these prepared mitochondrial samples were melted in ice. Secondly, set the spectrophotometer parameters: temperature 25°C, wavelength 550 nm, reading at 0s and 60 s each time. Thirdly, 850 µL buffer solution and 50 µL reaction working solution were added into the colorimetric cup, then which was detected for the background reading (0 s reading – 60 s reading) by spectrophotometer. Fourthly, added another 100µL sample (complete mitochondria containing 2 µg mitochondrial protein) into the foregoing colorimetric cup for detecting the sample reading (0 s reading – 60 s reading). Finally, calculation of sample activity: (sample reading - background reading) / [0.1 (sample volume, mL) × 21.84 (molar absorptivity)] = Unit / mL or µmol·min-1·mL-1.
2.5 Real time RT-PCR
RNA isolation and real-time RT-PCR were performed as described previously [9]. Total RNA was extracted according to the instructions of C. albicans RNA extraction kit (TIANZ, Beijing, China). Isolated RNA was resuspended in diethyl pyrocarbonate-treated water. The OD260 and OD280 were measured. First-strand cDNA was obtained using the cDNA synthesis kit (TaKaRa Biotechnology, Dalian, China) for RT-PCR according to the manufacturer’s instructions. Real-time PCR was performed using the 7500 Applied Biosystems. SYBR green I (TaKaRa Biotechnology, Dalian, China) is used for real-time monitoring of amplification products. CaMCA1 was amplified with the forward primer 5-TATAATAGACCTTCTGGAC-3 and the reverse primer 5-TTGGTGGACGAGAATAATG-3.
The PCR protocol consisted of denaturation program (95°C for 10 s); 40 cycles of amplification and quantification program: 95°C for 10 s (denaturation), 60°C for 20 s (annealing), 72°C for 30 s (extension); melting curve program: 60–95°C with a heating rate of 0.1°C per second; finally, a cooling step to 40°C. The changes in SYBR Green I fluorescence in every cycle were monitored by the LightCycler system software, and the threshold cycle (CT) above background for each reaction was calculated. The CT value of 18S ribosomal RNA (amplified with the forward primer 5-TCTTTCTTGATTTTGTGGGTGG-3 and the reverse primer 5-TCGATAGTCCCTCTAAGAAGTG-3) was subtracted from that of the tested gene to obtain a ΔCT value. The ΔCT value of an arbitrary calibrator (e.g., an untreated group) was subtracted from the ΔCT value for each sample to obtain a ΔΔCT value. The gene expression level relative to the calibrator was expressed as 2−ΔΔCT. Triplicate experiments were conducted to generate a mean value.
2.6 Assessment of Caspase Activity
Caspase activity was detected by the CaspSCREEN Flow Cytometric Apoptosis Detection Kit (BioVision, USA) [15]. The kit used dye D2R (rhodamine 110 containing two aspartic acid residues). The experimental procedures are as follows: protoplasts of C. albicans cells were treated with 1, 2, 4 µg / mL SK as mentioned in above section, then were resuspended in D2R staining solution (296 µL incubation buffer, 3µL 1M DTT and 1µL D2R reagent) at 30°C for 45 min before viewing and counting under a fluorescence microscope.
2.7 Statistical analysis
Data were analyzed using SPSS 19.0 (IBM Corp.) and presented as the mean ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. P < 0.05 was considered to indicate a statistically significant difference.