In Vitro Antibiofilm Activity of Eucarobustol E against Candida albicans

Abstract

Formyl-phloroglucinol meroterpenoids (FPMs) are important types of natural products with various bioactivities. Our antifungal susceptibility assay showed that one of the Eucalyptus robusta-derived FPMs, eucarobustol E (EE), exerted a strong inhibitory effect against Candida albicans biofilms at a concentration of 16 μg/ml. EE was found to block the yeast-to-hypha transition and reduce the cellular surface hydrophobicity of the biofilm cells. RNA sequencing and real-time reverse transcription-PCR analysis showed that exposure to 16 μg/ml of EE resulted in marked reductions in the levels of expressions of genes involved in hyphal growth (EFG1, CPH1, TEC1, EED1, UME6, and HGC1) and cell surface protein genes (ALS3, HWP1, and SAP5). Interestingly, in response to EE, genes involved in ergosterol biosynthesis were downregulated, while the farnesol-encoding gene (DPP3) was upregulated, and these findings were in agreement with those from the quantification of ergosterol and farnesol. Combined with the obvious elevation of negative regulator genes (TUP1, NRG1), we speculated that EE's inhibition of carbon flow to ergosterol triggered the mechanisms of the negative regulation of hyphal growth and eventually led to biofilm inhibition.

Keywords: Candida albicans; antibiofilm; eucarobustol E; formyl-phloroglucinol meroterpenoids; natural antimicrobial products; negative regulation.

FIG 1

Chemical structure of EE.

FIG 2

Time-kill curves of EE against C. albicans strain SC5314. Different working concentrations of EE were added to 1 × 10⁶ cells/ml of C. albicans suspensions in RPMI 1640 medium. The cell suspension with 0.5% DMSO was used as a control. Cell numbers are means ± SDs calculated from three independent experiments.

FIG 3

EE inhibits C. albicans biofilms in vitro. (a and b) An XTT reduction assay was performed to determine EE's effect on mature biofilms (a) and biofilm formation (b). The mature biofilms were left to grow for 24 h, and a biofilm formation assay was conducted after cell adhesion for 1.5 h. (c) The crystal violet method was conducted to quantify biofilm formation. Bars represent means ± SDs from three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

Effects of EE on C. albicans biofilm formation shown in CLSM images. (a) Control group; (b) group treated with 8 μg/ml of EE; (c) group treated with 16 μg/ml of EE; (d) group treated with 32 μg/ml of EE. FDA and PI were used to distinguish viable and dead cells.

FIG 5

SEM images show C. albicans biofilm inhibition under treatment with different concentrations of EE. Each treatment group was visualized under magnifications of 500×, 2,000×, and 5,000×. The boxes with dashed lines in the first and second columns are the areas enlarged in the second and third columns, respectively.

FIG 6

Effects of different concentrations of EE on CSH of C. albicans SC5314. CSH was evaluated using a water-hydrocarbon two-phase assay. Standard deviations based on three independent experiments are depicted. ***, P < 0.001.

FIG 7

EE inhibits the yeast-to-hypha transition in a dose-dependent manner. (a) Effects of different concentrations of EE on hyphal formation in liquid media. Bars, 50 μm. (b) Effects of different concentrations of EE on colony morphology on solid Spider medium.

FIG 8

Gene expression levels after EE exposure. RT-PCR was us ed to validate the differential expression of genes found by RNA sequencing (EFG1, CPH1, TEC1, NRG1, UME6, HGC1, ECE1, EED1, HWP1, ALS3, SAP5, ERG6, and ERG11). Other important genes (RAS1, CYR1, PDE2, TUP1, DPP3, and CSH1) related to C. albicans biofilm formation were also quantified by RT-PCR. GSP1 was used to normalize the levels of expression. Data are means ± SDs from three experiments. **, P < 0.01; ***, P < 0.001.

FIG 9

Quantification of ergosterol and farnesol content under EE treatment. (a) EE decreased the level of ergosterol biosynthesis, as measured by spectrophotometric assay; (b) EE treatment elevated the farnesol content, as measured by HPLC. Each experiment was performed in triplicate. ***, P < 0.001.