Figure 1.
Chemical structure of TET.
Figure 2.
TET inhibits C. albicans SC5314 biofilm formation in vitro.
(A) Effects of different concentrations of TET on biofilm formation. (B) Effects of different concentrations of Amphotericin B on biofilm formation. AmB: amphotericin B. (C) Effects of different concentrations of TET on the maintenance of mature biofilms. Biofilm formation was evaluated by XTT reduction assay, and the results were presented as the percentage compared to the control biofilms formed without TET treatment. Biofilm formation results represent the mean ± standard deviation for five independent experiments. * P<0.05 compared to the control biofilms, ** P<0.01 compared to the control biofilms. (D) Effects of different concentrations of TET on biofilms formed on silicone pads. Standard deviations are depicted and based on 6 silicone pad measurements. ** P<0.01. (E) Screen for TET-treated biofilms formed on silicone pads. The wells are shown for a: normal biofilm. b: cells were treated with 4 mg/L TET. c: 8 mg/L TET. d: 16 mg/L TET. e: 32 mg/L TET. f: uninoculated control. (F) Effects of different concentrations of TET on biofilm formation presented by using CLSM. a: Control. b: 8 mg/L of TET. c: 16 mg/L TET. d: 32 mg/L TET. (G) Effects of different concentrations of TET on biofilm formation presented by using SEM. The inset in the 500 ×, 2000× panels show the area that was magnificated.
Figure 3.
Effects of TET on fungal and bacterial biofilm formation in vitro.
(A) TET against C. neoformans H99; (B) Amphotericin B against C. neoformans H99, AmB: amphotericin B; (C) TET against A. fumigatus T308073458; (D) Amphotericin B against A. fumigatus T308073458, AmB: amphotericin B; (E) TET against S. aureus; (F) Penicillin against S. aureus; (G) TET against P. aeruginosa; (H) Ciprofloxacin against P. aeruginosa, CIP: ciprofloxacin. * P<0.05 compared to the treatment-free control biofilm, ** P<0.01 compared to the treatment-free control biofilm, *** P<0.001 compared to the treatment-free control biofilm.
Figure 4.
Effects of different concentrations of TET on CSH of C. albicans SC5314.
CSH was estimated by using the water-hydrocarbon two-phase assay. Standard deviations are depicted and based on three independent experiments. ** P<0.01, *** P<0.001.
Figure 5.
Time-growth curves of different concentrations of TET on C. albicans strain SC5314.
Table 1.
The MIC50 of TET and fluconazole against C. albicans strains.
Figure 6.
Effects of different concentrations of TET on hyphal formation in Spider medium.
(A) Log phase cells were incubated in liquid Spider medium at 37°C. Cells were photographed after 4 h of incubation in Spider medium. Observed with a inverted phase contrast microscope (AMG® EVOS xl) with a×40 objective. (B) Approximately 10 cells were plated on Spider solid medium. Incubation time and temperature were 5 d at 37°C.
Figure 7.
Gene expression changes of some important biofilm formation related genes.
The C. albicans strain tested was SC5314. The concentration of TET was 32 mg/L. All genes were examined by real-time RT-PCR with gene-specific primers. Gene expression was indicated as a fold change relative to that of the control group treated with DMSO. (A) in RPMI 1640 medium. (B) in Spider medium. Data are shown as mean ± SD from three experiments.
Figure 8.
Determination of intracellular cAMP level.
Exponentially growing C. albicans SC5314 cells incubated at 37°C in Spider medium in the presence of 32 mg/L TET and harvested at the 60 min time point. The cAMP content was measured using the cAMP Enzyme Immunoassay Kit according to the manufacturer’s instructions. ** P<0.01.
Figure 9.
Addition of exogenous cAMP reverts the morphological transition defect of C. albicans SC5314 caused by TET.
Exponentially growing C. albicans SC5314 cells were transferred to Spider medium. (A) Cells were incubated in liquid Spider medium supplemented without or with cAMP (final concentration 5 mM). The cells were incubated at 37°C for 4 h. Magnification 40 ×. (B) Hyphal formation on solid Spider medium plate with the same concentrations of cAMP and TET as in liquid Spider medium.
Figure 10.
TET prolongs the survival of C. elegans glp-4; sek-1 nematodes infected by C. albicans SC5314.
(A) Nematodes were infected with C. albicans for 4 h and then moved to pathogen-free liquid medium in the presence of TET (4 mg/L, 8 mg/L, 16 mg/L, 32 mg/L, P<0.0001), FLC (32 mg/L, P<0.0001) or DMSO. Dead worms were counted and removed daily. (B) After exposure to strain C. albicans SC5314, C. elegans nematodes were piped into 12-well plates that contain TET or DMSO. TET exhibited antifungal activity. a: Treatment free control group (DMSO added); b: TET 4 mg/L; c: TET 8 mg/L; d: TET 16 mg/L; e: TET 32 mg/L; f: FLC 32 mg/L.
Figure 11.
TET shows no toxicity on uninfected C. elegans glp-4; sek-1 nematodes.
C. elegans glp-4; sek-1 nematodes were pipetted into 12-well plates that contained different concentrations of TET, incubated at 25°C, and observed daily. On day 2, the worms were photographed. (A) 32 mg/L TET; (B) 64 mg/L TET; (C) 128 mg/L TET; (D) Treatment free group with the solvent DMSO added.