SWL-1 Reverses Fluconazole Resistance in Candida albicans by Regulating the Glycolytic Pathway

Abstract

Candida albicans is a ubiquitous clinical fungal pathogen. Prolonged use of the first-line antifungal agent fluconazole (FLC) has intensified fungal resistance and limited its effectiveness for the treatment of fungal infections. The combined administration of drugs has been extensively studied and applied. SWL-1 is a lignin compound derived from the Traditional Chinese Medicine Schisandra chinensis. In this study, we show that SWL-1 reverses resistance to fluconazole in C. albicans when delivered in combination, with a sharp decrease in the IC₅₀ of fluconazole from >200 to 3.74 ± 0.25 μg/ml, and also reverses the fluconazole resistance of C. albicans in vitro, with IC₅₀ from >200 to 5.3 ± 0.3 μg/ml. Moreover, killing kinetics curves confirmed the synergistic effects of fluconazole and SWL-1. Intriguingly, when SWL-1 was administered in combination with fluconazole in a mouse model of systemic infection, the mortality of mice was markedly decreased and fungal colonization of the kidney and lung was reduced. Further mechanistic studies showed that SWL-1 significantly decreased intracellular adenosine 5'-triphosphate (ATP) levels and inhibited the function of the efflux pump responsible for fluconazole resistance of C. albicans. Proteomic analysis of the effects of SWL-1 on C. albicans showed that several enzymes were downregulated in the glycolytic pathway. We speculate that SWL-1 significantly decreased intracellular ATP levels by hindering the glycolysis, and the function of the efflux pump responsible for fluconazole resistance of C. albicans was inhibited, resulting in restoration of fluconazole sensitivity in FLC-resistant C. albicans. This study clarified the effects and mechanism of SWL-1 on C. albicans in vitro and in vivo, providing a novel approach to overcoming fungal resistance.

Keywords: Candida albicans; SWL-1; combination; glycolysis; natural compounds; resistant.

Figure 1

Time-course of killing of SWL-1 and FLC against FLC-resistant C. albicans 23^#. (A) Chemical structure of SWL-1, which is derived from the Traditional Chinese Medicine Schisandra chinensis. (B) C. albicans 23^# was treated with SWL-1 or SWL-1 plus FLC at different concentrations. Each group was compared with the control group. ^*** p < 0.001

Figure 2

Effects of SWL-1 on fungal infection. Mice were infected with FLC-resistant C. albicans 23^# by intravenous injection (tail vein), and then treated with CMC-Na (control), FLC, SWL-1 combined with FLC, BBR combined with FLC in the model group. The condition of mice was monitored and the survival rate was calculated daily. (A) The weight of infected mice was recorded daily to reflect the antifungal activity of SWL-1. (B) Survival curves of mice infected with C. albicans. (C) Representative PAS- and H&E-stained sections of the kidney and lung from mice in the various groups 12 days after treatment. Results showed that the weight of mice in the combination therapy groups increased compared with that of mice in the model group. Each group was compared with the model group. SWL-1(H) represents the SWL-1 dose of 30 mg/ml, SWL-1(L) represents the SWL-1 dose of 15 mg/ml. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 3

The effect of SWL-1 on resistance-related gene expression in C. albicans. Expression of four genes (CDR1, CDR2, ERG11, and MDR1) was detected by RT-PCR. ns, not significant.

Figure 4

SWL-1 affected the pump activity of C. albicans. (A) Glucose-induced R6G efflux in FLC-resistant C. albicans 23^# and SWL-1-treated 23^#. (B) The intracellular ATP levels in FLC-resistant C. albicans 23^# and SWL-1-treated 23^#. ^*p < 0.05, ^***p < 0.001.

Figure 5

The effects of SWL-1 on the function of mitochondria. (A) Intracellular reactive oxygen species (ROS) production of FLC-resistant C. albicans 23^# strains and SWL-1 treatment 23^#. (B) Mitochondrial membrane potential of C. albicans 23^#. Data represent the mean ± SD. ^**p < 0.01, ^****p < 0.0001.

Figure 6

Proteins related to C. albicans metabolic routes identified by proteomics analysis. (A) Volcano map of C. albicans and SWL-1treated 23^#. (B) Hierarchical clustering analyses of proteins that were up- or downregulated in C. albicans and SWL-1 treated 23^#. (C) Proteins related to metabolism identified in GO analysis.

Figure 7

Impacts of SWL-1 treatment on glycolysis. SWL-1 inhibits some key enzymes in the glycolytic pathway, resulting in downregulation of the pathway. In contrast, some enzymes in the gluconeogenic pathway are upregulated.

Figure 8

The proposed mechanisms by which SWL-1 reverses drug resistance in C. albicans. The glycometabolism of C. albicans is changed by decreasing glycolysis but increasing gluconeogenesis, resulting in a decrease in ATP levels. The efflux pump-mediated removal of FLC from cell is blocked. The resistance to FLC is reversed and the antifungal effects of FLC are restored.