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The standard strain C. albicans SC5314 was cultured in YPD (1% yeast extract, 2% peptone, and 2% glucose) liquid medium and incubated overnight in an orbital shaker at 200 rpm and 35 ℃. Fungal cells were harvested through centrifugation and washed twice prior to use in experiments. The egg yolk agar plate used in the phospholipase activity assay consisted of 1% glucose, 1% peptone, 2% agar, 10% sterile yolk, 1 mol/L NaCl, and 0.005 mol/L CaCl2[29]. Itraconazole was purchased from Sigma, and BDSF was synthesized in accordance with a previous report[30]. BDSF stock solution (0.3 mol/L) was prepared in an ethanol/water mixture (1:1), and itraconazole was suspended in DMSO. BDSF and itraconazole stock solutions were diluted to the concentration of 250 μmol/L using the corresponding medium and suspended in 0.3% Noble agar. The vehicle was added as the control. Each dilution was administered separately through intravaginal instillation.
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TR146 oral epithelial cells and VK2/E6E7 vaginal epithelial cells were cultured in six-well plates until reaching 95% confluence and used to evaluate the adhesive ability of C. albicans cells. The cells were infected with C. albicans at the rate of 2 × 102 yeast cells per well in DMEM and treated with 0, 3, 30, or 300 μmol/L BDSF. The number of adherent cells was verified through colony counting. After 0.5 and 1 h of incubation, dissociative yeast cells were removed by thrice rinsing the wells gently with 3 mL of PBS. The wells were then covered with YPD agar, and the number of adherent cells was quantified through colony counting. Adhesion efficiency was expressed as the ratio of the number of adherent cells treated with BDSF to that of the cells cultured in DMEM only[31].
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Candida cells were suspended and diluted in fresh YPD after overnight culture to obtain a 103 CFU/mL suspension for use in the agar invasion assay. Aliquots (20 µL) of yeast suspension was spread on YPD plates supplemented with 0, 3, 30, 100, 200, or 300 μmol/L BDSF. After 72 h of incubation at 37 ℃, the plates were then rinsed under running water to discard noninvasive cells. The washed plates were then incubated for an additional 8 h at 37 ℃ for the observation of the invasive ability of Candida cells.
The epithelial cell invasion assay was performed as previously described[32]. Briefly, TR146 and VK2/E6E7 cells were cultured on circular coverslips with diameters of 12 mm. After 2 days of culture, the cell monolayer was infected with 1 × 105 CFU yeast cells suspended in DMEM medium containing 0, 3, 30, 100, 200, or 300 μmol/L BDSF and placed for 3 h in a humidified incubator. The supernatant was discarded, and cells were rinsed three times with PBS to remove dissociative fungal cells. The cells were fixed with 4% paraformaldehyde. Any fungal cells that remained attached to epithelial cells were incubated for 1 h with rabbit anti-C. albicans antibodies (ab53891), rinsed thrice with PBS, and stained again with Cy3-tagged anti-rabbit IgG antibodies (ab97075). Fungal cells that did not invade epithelial tissue were labeled with red (Cy3) fluorescence. Next, epithelial cells were washed with PBS, and 0.5% Triton X-100 in PBS was added to the wells to permeabilize epithelial cells. Fungal cells were then incubated with FITC-tagged anti-C. albicans antibodies (ab21164) and labeled with green (FITC) fluorescence. Finally, the prepared coverslips were rinsed with PBS and visualized under fluorescence microscopy (Olympus X71).
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The phospholipase activity of C. albicans was evaluated on egg yolk agar plates[29]. In brief, freshly cultured cells were diluted with PBS to an optical density of 0.6 at 600 nm. Next, 4 µL aliquots of fungal solution were spotted onto egg yolk plates supplemented with 0, 3, 30, 100, 200, or 300 μmol/L BDSF. After 5 days of incubation at 37 ℃, the diameters of colonies and the dense white precipitation zone on each plate were measured. Phospholipase activity (Pz value) was calculated as the colony diameter divided by the total diameter of the colony and precipitation zone.
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The ability of C. albicans to cause cell damage was determined on the basis of lactate dehydrogenase (LDH) release. LDH release was quantified by using a commercial LDH cytotoxicity detection kit (Roche Applied Science). TR146 cells were seeded in 96-well tissue plates and cultured to 95% confluence. Each well was then infected with 2 × 104 fungal cells suspended in DMEM medium containing 0, 3, 30, 100, 200, or 300 μmol/L BDSF and cultured overnight in a humidified incubator. TR146 cells incubated with DMEM medium were considered as the low control and DMEM containing 0.5% Trion X-100 were considered as the high control. C. albicans cells were also seeded in the absence of epithelial cells. The damage incurred by epithelial cells infected with C. albicans cells was calculated in accordance with the following formula: (test sample release − low control release of TR146 and Candida cells)/(maximum cell release − low control release). The final result was expressed as (damage observed in the BDSF treatment sample)/(cells infected only with C. albicans) × 100[33].
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A total of 110 female ICR mice (SLAR laboratory animal, Shanghai) aged 8-10 weeks were randomly assigned to four groups. The mouse model was constructed in accordance with a previous report[34, 35]. Mice were subcutaneously injected with 0.5 mg of estradiol valerate at 3 days before infection to induce transient pseudoestrus. On infection day, animals were inoculated intravaginally with 20 μL of yeast cell suspension containing 108 cells/mL. Animals in the negative control group were inoculated with sterile PBS. Mice in the positive control group were infected and received 0.3% Noble agar intravaginally, whereas those in the negative control group were reared without any treatment save for subcutaneous injections of estradiol valerate. The mice then received drug treatment at 24 h after infection. All animal experiments were performed in accordance with the guidelines of the National Institutes of Health on animal care. The study protocol was approved by the Animal Ethics Committees of Fudan University.
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BDSF and itraconazole (250 μmol/L) were dispersed in 0.3% Noble agar and given intravaginally twice a day at the volume of 20 μL on 1-6 days postinfection. On days 1, 2, 4, and 7, the vaginas of the mouse models were swabbed with 200 μL of PBS. Next, 100 μL aliquots of each dilution (10−1 to 10−5) were inoculated on SDA plates for the quantification of fungal load (CFU/mL). Mice were then sacrificed, and blood was taken from the ventral vein and centrifuged to collect serum, which was then stored at −25 ℃. Serum was used to test the activity of superoxide dismutase (SOD) with a SOD testing kit (Jiancheng bioengineering institute). Vaginal tissue was also used for RNA extraction and histopathological examination (Boyuan biotechnology Co.). Vaginal inflammation was scored as follows: 0- normal; 1- slight inflammatory infiltration and swelling; 2- grade 1 changes involving 50% of the specimen; 3- obvious inflammatory infiltration, swelling, and mild ulceration; 4- grade 3 changes involving 50% of the specimen; and 5- extensive ulceration and necrosis[36].
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The primers used in this study are shown in Table 1. RNA was obtained from murine vaginal tissue and C. albicans cells by using an RNA fast extracting kit (Tiangen Biotech Co.) and EASY spin yeast kit (Aidlab), respectively. DNA was degraded with DNase (Tiangen Biotech Co.). TevertAid first-strand cDNA synthesis kit (Fermentas) was used to synthesize cDNA, which was then diluted (1:16) with sterile Milli-Q water (Millipore) for real-time RT-PCR. Real-time RT-PCR was performed using SYBR Premix Ex Tag kit (TaKaRa) with a Stratagene Mx3000p System. A series (1:22 to 1:27) of cDNA dilutions were used to construct a standard curve for each primer pair. The reaction procedure consisted of 5 min of incubation at 95 ℃, 40 cycles of amplification (95 ℃ for 15 s, 55 ℃ for 20 s, and 72 ℃ for 30 s), and melting curve construction (95 ℃ for 15 s, 55 ℃ for 30 s, and 95 ℃ for 30 s). Then, the expression of target genes was normalized to that of the housekeeping gene ACT1 and calculated through comparative Ct method (ΔΔCt). Data were expressed as fold changes in the expression of target genes relative to that of the untreated control.
Gene Primer Direction Sequence (5′-3′) C. albicans ALS3 Forward TCCACTTCACAATCCCCATC Reverse CAGTAGTAGTAACAGTAGTAGTTTCATC ALS9 Forward AATGTTCCTGCTGGGTATCG Reverse GATCACATCCCCGCTAGAGT MNT2 Forward CCACCACAATCACCTTCATC Reverse TTGTTTCTCTTGTTGCTGTGG PLB4 Forward AAAGCTCATGAAGATGTGGCT Reverse CCAATCCCGCCATCTATAAC LIP1 Forward ACTGACAATTTGCGTCAAGG Reverse TCCATAACCTCCAGGGAAAG ECE1 Forward AAGAGAGATGTTGCTCCAGCTG Reverse AATGGCATATCAGCAATGATAC ACT1 Forward TTGACCAAACCACTTTCAACTC Reverse AGAAGATGGAGCCAAAGCAG Mouse IGFBP3-1 Forward GGAACTGTGGGAGAGGATATG Reverse ACCATTATTTGCGACATCTCTG MCP-1 Forward ATTCACATGGAAAGCCCCC Reverse TTGAACACAAAGAGTACCAGAG ACT1 Forward TGTTACTGAGCTGCGTTTTAC Reverse CACCGTTCCAGTTTTTAAATCC Table 1. Primers Used in This Study
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All experiments were performed in triplicate, and results were expressed as means ± SD. One-way ANOVA was applied for the comparison of all groups. Student's t-test was used to compare paired groups. Differences were considered significant when P ≤ 0.05 and nonsignificant when P ≥ 0.05.