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Antifungal activity of hypocrellin compounds and their synergistic effects with antimicrobial agents against Candida albicans

Summary Candida albicans is a common human fungal pathogen. The previous study revealed that quinone compounds showed antimicrobial activity against C. albicans by inhibiting cell growth. However, it was unclear whether quinones have other antifungal effects against C. albicans in addition to fungicidal effects. In this study, we assessed the inhibitory activity of a total of 25 quinone compounds against C. albicans morphological transition, which is essential for the pathogenicity of C. albicans. Several quinones exhibited strong inhibition of mycelium formation by C. albicans SC5314. Three leading compounds, namely hypocrellins A, B and C, also exhibited marked attenuation of C. albicans SC5314 virulence in both human cell lines and mouse infection models. These three compounds significantly suppressed the proliferation of C. albicans SC5314 cells in a mouse mucosal infection model. Intriguingly, hypocrellins not only attenuated the cytotoxicity of a nystatin‐resistant C. albicans strain but also showed excellent synergistic effects with antifungal agents against both wild‐type C. albicans SC5314 and the drug‐resistant mutant strains. In addition, hypocrellins A, B and C interfered with the biological functions and virulence of various clinical Candida species, suggesting the promising potential of these compounds for development as new therapeutic agents against infections caused by Candida pathogens.


SI Methods
Candida cell growth analysis.
Candida cells cultured overnight were diluted in YPD medium and inoculated into fresh medium to an OD600 of 0.05 with or without compounds at the indicated final concentrations. Then, 200 µl of inoculated culture was grown in each well at 30°C under low-intensity shaking using the Bioscreen-C Automated Growth Curves Analysis System (OY Growth Curves AB, Finland).

Viability assay of Candida cells
The viability of cells was tested using 1% Evan's Blue dye, which is excluded by viable cells (1). Briefly, candida cells cultured overnight were diluted in YPD medium and inoculated into fresh medium to an OD600 of 0.5 with or without compounds at the indicated final concentrations of 10 μM. The inoculated cultures were grown in each well at 30°C as indicated with shaking at 220 rpm for 4 h. Then the same volume of 0.5% Evan's blue solution was added, and the cells were dyed at room temperature for 10 min. Finally, the samples were measured for the viability by an optical microscope.

cells (lung cancer cells) and human intestinal epithelial cells (HIEC) in DMEM
supplemented with 10% FBS were seeded in 96-well tissue culture plates at 1.5×10 4 cells/well one night in advance and incubated at 37°C with 5% CO2. To test the cytotoxicity of the compounds, the medium in the seeded plate was replaced by 100 μL DMEM supplemented with 1% FBS and containing different concentrations of the tested compounds as indicated, the same volume of 0.2% DMSO was used as the control. To test the effects of the compounds against C. albicans cytotoxicity, the medium was replaced by C. albicans cells (OD600=0.1) treated with different compounds at different concentrations in DMEM supplemented with 1% FBS, the same volume of 0.2% DMSO was used as the control. 10 µL of 5 mg/mL MTT stock solution was added to each well and mixed gently and incubate for 4 hours. Then the culture medium was aspirated from each well carefully to prevent disruption of the cell monolayer. And 100 µL of formazan solubilization solution was added into each well and then the plates were placed on a shaker to mix gently for 10 minutes to dissolve the formazan crystals. The cytotoxicity was determined by measuring the absorbance at 570 nm using a microplate reader.

Analysis of C. albicans cell numbers in mouse tongue
The mouse tongue was collected and weighed, then ground in sterilized water. The mouse tongue suspension was serially diluted and spread on SD plates. The plates were grown at 30°C for two days. Then the numbers of C. albicans colony-forming unit (CFU) on the plates were counted.

Histopathological diagnosis
In order to study the oral cavity of mice infected with C. albicans, the tongues of mice were observed by pathological section. The histological sections were stained with Periodic Acid-Schiff stain for histopathological diagnosis (3). The mouse tongues were fixed with 4% formaldehyde, then dehydrated and paraffin embedded. The sections were dewaxed to water and stained with Periodic Acid-Schiff stain for histopathological diagnosis. Finally, they were observed and imaged with a microscope.

Analysis of susceptibility of Candida strains to compounds or antifungal agents
MIC values of antifungal agents and hypocrellins against Candida spp. used in this study were determined based on the broth microdilution protocol of Clinical and Laboratory Standards Institute (CLSI) document M23-A7 with small modifications (4,5). Briefly, overnight cultured cells were inoculated at an OD600 of 0.05 in YPD medium supplemented with compounds or antifungal agents. One hundred microliters of inoculated cultures were grown in each well at 30°C as indicated with shaking at 250 rpm for 24 hours. MIC was defined as the lowest concentration of antibiotic in which Candida spp. growth in the well was not measureable by determination of the turbidity at 600 nm following the method from the Clinical and Laboratory Standards Institute (CLSI).

Western blotting
Expression levels of EFG1, HWP1 and TEC1 were determined by Western blotting (6).    The cells stained in blue color were dead. *P < 0.05; ***P < 0.001 vs DMSO (unpaired t-test). Compounds were dissolved in DMSO, and the same amount of DMSO (0.2%) used as the solvent for the compounds was used as a control. Data are means ± standard deviations from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 vs DMSO (unpaired t-test).