Sub-lethal concentrations of chlorhexidine inhibit Candida albicans growth by disrupting ROS and metal ion homeostasis

Abstract

Candida albicans is a normal resident of the human oral cavity. It is also the most common fungal pathogen, causing various oral diseases, particularly in immunocompromised individuals. Chlorhexidine digluconate (CHG) is a broad-spectrum antimicrobial agent widely used in dental practice and has been recommended to treat oral candidiasis. However, its action mechanism against the fungal pathogen C. albicans remains poorly understood. The aim of the present study was to investigate the effect of CHG at sub-lethal concentrations against C. albicans. CHG inhibited the growth of C. albicans in a dose- and time-dependent manner. Cells treated with CHG exhibited altered membrane permeability, reduced metabolic activity, and enhanced metal ion and reactive oxygen species (ROS) accumulation. Copper-sensing transcription factor Mac1, iron-sensing transcription factors Sfu1 and Sef2, and copper transporter Ctr1 regulated intracellular metal ion and ROS homeostasis in response to CHG. Deletion of MAC1, SFU1, or SEF2 increased intracellular ROS production and cell susceptibility to CHG. This study revealed a novel mechanism by which CHG induced apoptosis of C. albicans cells through the disruption of metal ion and ROS homeostasis, which may help to identify new targets for fungal infections.

Keywords: Candida albicans; antifungal activity; chlorhexidine; metal ion homeostasis; reactive oxygen species.

Figure 1.

Antifungal activity of CHG against C. albicans. C. albicans cells were treated with CHG (0, 25 and 50 μM) for different time periods (0.5, 2, and 4 h for cell viability and propidium iodide (PI) influx assay, 2 h for XTT assay, and 0.5 h for morphology observation). a. cell viability determined by counting CFU after 24 h of incubation on YPD plates at 37°C. b. cell membrane permeability observed under CLSM using PI influx assay. Scale bar, 20 μm. c. cell wall structure changes observed under the scanning electron microscope (SEM). The wrinkles (red arrows) and cracks (green arrows) are presented. Scar bar, 5 μm. d. the metabolic activity detected by XTT assay. Three biological replicates were performed. For a and D, the statistical significance of the differences between various groups is indicated (***p < 0.001, one-way ANOVA and Dunnett’s t test, two-tailed). The strain used was SN250 (WT).

Figure 2.

Effect of CHG on intracellular ROS generation in C. albicans. C. albicans cells were treated with different concentrations (0, 2.5, 5, 10, and 25 μM) of CHG for 2 h. a. cell viability determined by counting CFU after 24 h of incubation on YPD plates at 37°C. b. intracellular ROS levels. Three biological replicates were performed, and statistical significance of the differences between various groups are indicated (***p < 0.001, one-way ANOVA and Dunnett’s t test, two-tailed). The strain used was SN250 (WT).

Figure 3.

Effect of CHG on MAC1, SEF2, and SFU1 deletion mutants in C. albicans. a. sensitivity of different strains to CHG by MIC assay. C. albicans cells were treated with twofold serial dilutions of CHG. The lowest concentration of CHG that prevents visible growth of C. albicans was noted after 24 h of incubation at 37°C. b. growth phenotype of different strains. C. albicans cells were tenfold serial diluted with concentrations ranging from 1 × 10³ to 1 × 10⁸ CFU/mL (left to right in each panel), and 2 μL of each dilution were spotted onto YPD +25 μM CHG plates. All growth was observed after three days of incubation at 37°C. The control strain (WT) was SN250.

Figure 4.

Effect of CHG on copper or iron homeostasis in C. albicans. a. intracellular levels of Cu and Fe. SN250 (WT) cells incubated with different concentrations (0, 2.5, 5, and 10 μM) of CHG for 2 h were digested, and concentrations of metal ions were detected using ICP-MS. Ppb, parts per billion. b. expression of metal ion-associated genes. Cells of SN250 (WT) and mac1/mac1 mutant were incubated with different concentrations (0, 1, 2.5 and 25 μM for WT; 0, 1, and 2.5 μM for mac1/mac1 mutant) of CHG for 2 h. The relative gene expression levels of MAC1, SEF2, SFU1, CTR1, SEF1, and HAP43 were detected by qRT-PCR. c. intracellular ROS levels of SN250 (WT), mac1/mac1, sef2/sef2, and sfu1/sfu1 mutants incubated with or without 25 μM CHG for 2 h. After DCFH-DA staining, the fluorescence intensity was detected with a microplate reader. Three biological replicates were performed, and the statistical significance of the differences between various groups is indicated. (*p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA and Dunnett’s t test for different concentrations of CHG, Student’s t-test for comparing gene deletion mutants with the WT, two-tailed).

Figure 5.

Potential antifungal mechanism of CHG against C. albicans. CHG binds to the membrane or cell wall of fungal cells and increases their permeabilities, leading to elevations of intracellular metal ions and ROS, which further induce mitochondrial dysfunction and apoptosis in C. albicans. Some Cu/Fe-responsive transcriptional factors are involved in this process to regulate the homeostasis of metal ions and ROS generation in response to CHG.