Fangchinoline inhibits growth and biofilm of Candida albicans by inducing ROS overproduction

Abstract

Infections caused by Candida species, especially Candida albicans, threaten the public health and create economic burden. Shortage of antifungals and emergence of drug resistance call for new antifungal therapies while natural products were attractive sources for developing new drugs. In our study, fangchinoline, a bis-benzylisoquinoline alkaloid from Chinese herb Stephania tetrandra S. Moore, exerted antifungal effects on planktonic growth of several Candida species including C. albicans, with MIC no more than 50 μg/mL. In addition, results from microscopic, MTT and XTT reduction assays showed that fangchinoline had inhibitory activities against the multiple virulence factors of C. albicans, such as adhesion, hyphal growth and biofilm formation. Furthermore, this compound could also suppress the metabolic activity of preformed C. albicans biofilms. PI staining, followed by confocal laser scanning microscope (CLSM) analysis showed that fangchinoline can elevate permeability of cell membrane. DCFH-DA staining suggested its anti-Candida mechanism also involved overproduction of intracellular ROS, which was further confirmed by N-acetyl-cysteine rescue tests. Moreover, fangchinoline showed synergy with three antifungal drugs (amphotericin B, fluconazole and caspofungin), further indicating its potential use in treating C. albicans infections. Therefore, these results indicated that fangchinoline could be a potential candidate for developing anti-Candida therapies.

Keywords: Candida albicans; ROS; antifungal; biofilm; fangchinoline; virulence factor.

FIGURE 1

The chemical structure of FN and the suppression of FN on the adhesion of Candida albicans cells to polystyrene surfaces. (A) The chemical structure of FN. (B) FN inhibited the adhesion of C. albicans SC5314 to polystyrene surfaces. Adhesion was considered as the relative viability of adherent fungal cells determined by MTT assay. (C) Representative graphs of adherent C. albicans cells left by FN treatment and PBS washing. (D) The total viable cells in each group after 1.5 h treatment with different concentrations of FN at 37°C (before PBS washing). *, p < 0.05 and **, p < 0.01.

FIGURE 2

The time‐kill curves of FN in Candida albicans SC5314 cells. Candida cells with different initial inoculum densities (10⁴ cells/mL (A), 10⁵ cells/mL (B) and 10⁶ cells/mL (C)) were treated with 0, 12.5, 25, 50, 100 and 200 μg/mL FN for 24 h. At indicated time points, 100 μL aliquot was taken from each group, diluted and spread on SD agar to count the viable cell number of each group.

FIGURE 3

FN inhibited the hyphal growth of Candida albicans. C. albicans hyphal growth induced by RPMI‐1640 medium at 37°C for 4 h was impeded by FN.

FIGURE 4

Escalating concentrations of FN inhibited both the formation and development of Candida albicans SC5314 biofilm. (A) The viability of C. albicans SC5314 biofilms formed in the presence of escalating concentrations of FN was determined through XTT reduction assay. (B) Twenty‐four hour biofilms formed in the absence of FN were exposed to FN for another 24 h. XTT assay was used to evaluate the influence of FN on the biofilm development. (C) Biofilms formed in the presence of 0, 25, 50 and 100 μg/mL FN were stained with Syto 9 and recorded by CLSM in 3D mode, followed by reconstruction using Imaris 7.2.3 software. *, p < 0.05, and **, p < 0.01.

FIGURE 5

The presence of FN in preformed biofilms attenuated the EPS production. Twenty‐four hour mature biofilms treated with 0, 25, 50 and 100 μg/mL FN for another 24 h were subjected to EPS determination. **, p < 0.01.

FIGURE 6

FN induced hyper‐permeability of Candida albicans SC5314 cell membrane. After a 4‐h co‐incubation with 0, 25, 50 and 100 μg/mL FN at 37°C, C. albicans cells were stained with PI for detecting cell membrane damages.

FIGURE 7

FN induced ROS overproduction in Candida albicans cells. DCFH‐DA was used to stain the ROS produced in C. albicans cells challenged with 0, 25, 50 and 100 μg/mL FN for 4 h at 37°C.

FIGURE 8

NAC rescued biofilm inhibition caused by FN treatment. (A) Addition of 150 μg/mL NAC could save part of viability of Candida albicans biofilms treated with 100 μg/mL FN. **, p < 0.01. (B) The presence of 150 μg/mL NAC could increase the MIC of FN. (C) Representative graphs of C. albicans biofilms formed in the absence and presence of NAC and exposed to 0 and 100 μg/mL FN.