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.
Introduction
Candida albicans is an opportunistic fungal pathogen that causes mucosal and systemic infections, particularly in immunocompromised individuals [Citation1]. Oral candidiasis is one of the most common fungal infections caused by C. albicans. Other oral diseases, such as oral cancer, periodontitis, early childhood caries, root caries, and refractory root canal infections, have also been associated with C. albicans [Citation2–6]. However, with the limited availability of antifungals, the emergence of drug resistance has posed a serious public health concern over the last two decades. Therefore, comprehensively investigating the antifungal mechanism against C. albicans is essential to provide a basis for developing new antifungal drugs.
Chlorhexidine has been extensively used in dental practice as an antiseptic agent since 1970. It exhibits broad-spectrum antimicrobial activity against oral bacteria, such as Porphyromonas gingivalis, Fusobacterium nucleatum, Enterobacter, Enterococcus faecalis, and Streptococcus mutans, and fungi, including C. albicans and other common non-albicans yeast species [Citation7]. Mouthwash containing 0.12–0.2% chlorhexidine digluconate (CHG), a sub-lethal dose of CHG, is often used as an endodontic irrigant and plaque control agent. Although the effects of sub-lethal concentrations of CHG on C. albicans cell structure have been reported, the precise mechanism remains poorly understood [Citation8,Citation9].
Ion homeostasis is considered crucial for all living cells. In C. albicans, it participates in membrane potential maintenance, cell wall integrity, oxidative stress responses, morphological transition, host tissue invasion, drug resistance, proliferation, and apoptosis [Citation10–14]. Copper and iron are the most abundant metal ions that serve as essential nutrients for the growth and metabolism of C. albicans and play important roles in regulating various cellular processes, such as oxygen transport, DNA synthesis, and the tricarboxylic acid cycle [Citation15]. However, excess metal ions can be potentially toxic, leading to cell death. This toxic effect is reportedly associated with the increased generation of reactive oxygen species (ROS), which can lead to mitochondrial dysfunction-mediated apoptosis [Citation16,Citation17].
In this study, we determined the effect of sub-lethal concentrations of CHG against C. albicans and investigated the potential mechanism from the perspective of intracellular ROS accumulation and metal ion homeostasis to better understand the antifungal mechanisms against fungal cells and help identify novel therapeutic antifungal agent.
Materials and methods
Strains, chemicals, and culture conditions
The C. albicans strains used in this study are listed in . Wild-type strain SN250 was used to investigate the antifungal activity of CHG. The library containing 165 transcription factor gene deletion mutants for screening associated genes was the same as previously described [Citation19,Citation20].
Table 1. Candida albicans strains used in this study.
The gene reconstituted strains were constructed using pNIM1 (for MAC1 and SFU1) and pSFS2A (for SEF2) plasmids. The primers used in this study are listed in Table S1. To generate the MAC1-reconstituted plasmid, fragments of the 3′-untranslated region (UTR) and that containing the open reading frame (ORF) plus 5′-UTR of MAC1 were amplified by PCR from the genomic DNA of SN250. The PCR products were digested with XhoI/BglII and SalI/BamHI respectively, and subcloned into the plasmid pNIM1 [Citation21]. The MAC1 reconstituted strain (mac1/mac1+MAC1) was constructed by transforming the mac1/mac1 mutant with the SalI-digested pNIM1-MAC1p-MAC1 plasmid. The SFU1 reconstituted strain (sfu1/sfu1+ SFU1) was constructed using the same method. To construct the SEF2-reconstituted plasmid, a fragment of the 3′-UTR of SEF2 and a fragment containing the SEF2 ORF plus 5′-UTR were amplified by PCR from the genomic DNA of SN250 and sequentially inserted into the SacII/SacI and ApaI/XhoI sites of the plasmid pSFS2A [Citation22]. The SEF2 reconstituted strain (sef2/sef2+SEF2) was constructed by transforming the sef2/sef2 mutant with ApaI/SacI-digested pSFS2A-SEF2p-SEF2 plasmid.
CHG was purchased from Sigma-Aldrich (St. Louis, MO, USA), diluted in sterilized ddH2O at a concentration of 1 mM (stock solution), and stored at 4°C. Yeast extract, peptone, and agar were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). Glucose was purchased from Sigma-Aldrich (St. Louis, MO, USA). C. albicans strains were routinely cultured in YPD medium (20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract). For C. albicans, the OD value (0.9 to 1) obtained at 600 nm would give an approximate measure for 107 CFU/mL. Solid medium was supplemented with 2% agar. YPD +25 μM CHG medium plates were used for spot dilution assays. All experiments were performed under normoxic conditions.
Determination of minimum inhibitory concentration (MIC)
Broth microdilution assay was used to determine the MIC of CHG in accordance with the guidelines provided by the Clinical and Laboratory Standards Institute for yeasts (M27-A3) [Citation23]. The mixture of fungal suspension and CHG solution was dispensed into the first 10 columns of a sterile U-bottomed 96-well microtiter plate. The final concentrations of CHG ranged from 0.78125 to 400 μM with two-fold serial dilutions, and the fungal suspensions were adjusted to around 150 cells per well. The eleventh and twelfth columns were used as blank and negative control wells without inoculation or CHG, respectively. The lowest concentration of CHG that prevented visible growth of C. albicans was noted as MIC after 24 h of incubation at 37°C.
Fungal cell viability assay
C. albicans cells were initially grown in liquid YPD to the stationary phase at 30°C, harvested, and washed with sterilized ddH2O. Cells (1 × 107 CFU/mL) were treated with different concentrations of CHG (0, 25, and 50 μM for 0.5, 2, and 4 h, or 2.5, 5, 10 μM for 2 h) at 30°C with shaking (200 rpm). The cells were washed twice with phosphate-buffered saline (PBS, 0.01 M, pH 7.4) and suspended in 1 mL PBS. The fungal suspensions were serially diluted and seeded onto YPD plates. Colony-forming units (CFU) were counted after 24 h incubation at 37°C.
Determination of cell permeability
The permeability of the cell wall and membrane was observed using propidium iodide (PI) staining. C. albicans cells (1 × 107 CFU/mL) were treated with different concentrations of CHG (0, 25, and 50 μM) for 0.5, 2, and 4 h at 30°C with shaking. After incubation, the cells were stained with 50 µg/mL PI (Sigma-Aldrich, USA) for 15 min in the dark and observed under a confocal laser scanning microscope (CLSM) (LSM880, Zeiss, Germany).
Cell wall structure changes of fungal cells
C. albicans cells (1 × 107 CFU/mL) were treated with different concentrations of CHG (0, 25, and 50 μM) for 30 min at 30°C with shaking. Following incubation, the cells were harvested by centrifugation and fixed with 2.5% (v/v) glutaraldehyde for 2 h at 4°C. The fixed samples were washed twice with PBS, dehydrated in a series of ethanol solutions (50, 75, and 90% for 10 min and then absolute alcohol for 10 min, twice), and subsequently treated with a series of tert-Butanol solutions (50, 75, and 90% for 10 min and then absolute tert-Butanol for 10 min, twice). The samples were freeze-dried, coated with a thin layer of gold-palladium, and observed under a scanning electron microscope (TM-3000, Hitachi, Japan).
Determination of metabolic activity
The metabolic activity of C. albicans cells was determined using a colorimetric 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2 H-tetrazolium-5-carboxanilide (XTT) assay [Citation24]. XTT sodium salt and phenazine methosulfate (PMS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Solutions containing 0.5 mg/mL XTT and 0.32 mg/mL PMS were freshly prepared by dissolving XTT powder and PMS powder in PBS and water, respectively. The solutions were filter-sterilized (0.22 μm pore size filter), mixed at a 9:1 XTT:PMS ratio, and protected from light. C. albicans cells (1 × 107 CFU/mL) were treated with different concentrations of CHG (0, 25, and 50 μM) for 2 h at 30°C with shaking. The cells were then washed twice with PBS, suspended in XTT:PMS solution, adjusted to 1 × 107 CFU/mL, and transferred to a 96-well microtiter plate, 100 μL for each well. The plate was incubated in the dark for 30 min at 37°C, and the optical density was measured at 492 nm using a microplate reader (Cytation 3, BioTek, USA).
Determination of ROS generation
ROS levels were measured using a ROS assay kit (Beyotime Biotech, China). C. albicans cells (1 × 107 CFU/mL) were treated with different concentrations of CHG (0, 2.5, 5, 10, and 25 μM) for 2 h at 30°C with shaking. After incubation, cells were stained with 10 µM of 2′,7′-dichlorofluorescein diacetate (DCFH-DA) for 30 min at 37°C in the dark. Intracellular fluorescence intensity of the treated cells was measured with the microplate reader at excitation and emission wavelengths of 488 and 525 nm, respectively.
The intracellular ROS levels of the mac1/mac1, sef2/sef2, and sfu1/sfu1 mutants were also determined using this method. After 2 h of incubation with or without treatment of 25 μM CHG, fungal cells were stained with DCFH-DA, and the fluorescence intensity was measured using the microplate reader.
Spot dilution assay
The effect of CHG on the growth of the gene deletion and complementary strains was evaluated using a spot dilution assay. The C. albicans suspensions were tenfold serially diluted in distilled water at final concentrations ranging from 1 × 103 to 1 × 108 CFU/mL [Citation25]. Subsequently, 2 μL of each dilution was spotted onto YPD +25 μM CHG plates. The growth conditions of the colonies were observed after 3 d of incubation at 37°C.
Determination of intracellular levels of Cu and Fe
C. albicans cells (1 × 107 CFU/mL) were incubated with different concentrations of CHG (0, 2.5, 5, and 10 μM) for 2 h at 30°C with shaking. After incubation, fungal cells were harvested by centrifugation, washed twice with Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and twice with deionized water, and adjusted to 1 × 106 CFU/mL. Cells were digested with 200 µL of 10% nitric acid for 1 h at 100°C and diluted to a final concentration of 2% nitric acid in deionized water. The Cu and Fe concentrations were determined using inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 8900, Agilent, USA) [Citation26,Citation27].
Gene expression analysis using quantitative real-time PCR (qRT-PCR)
C. albicans cells (1 × 107 CFU/mL) were incubated with different concentrations of CHG for 2 h at 30°C with shaking. Cells were harvested by centrifugation, and total RNA was extracted using a Fermentus GeneJET RNA Kit (Thermo Scientific, USA). Then 0.6 μg of total RNA per sample was used to synthesize cDNA with RevertAid Reverse Transcriptase (Thermo Scientific, USA) and diluted in DEPC-treated water. Quantitative reverse real-time PCR was performed in a Bio-Rad CFX96 real-time PCR detection system using SYBR Green qPCR mix (TOYOBO, Japan). The reactions were incubated at 94°C for 4 min and cycled 35 times at 94°C, 20 s, 54°C, 20 s, and 72°C, 20 s. The expression levels of each gene were normalized to those of ACT1. Primers used in this analysis are listed in Table S1.
Statistical analysis
Three independent experiments were performed for all assays, and the quantitative results are presented as mean ± standard deviation. Significant differences were assessed using the IBM SPSS Statistics software (version 22.0, IBM Corporation, USA). One-way ANOVA and Dunnett’s t test were performed to determine the cell viability, metabolic activity, intracellular ROS and metal ion levels, and gene expression of C. albicans cells treated with different concentrations of CHG. Student’s t-test was used to compare the intracellular ROS levels and gene expression of the gene deletion mutants with the wild-type strain.
Results
CHG exhibits antifungal activity against Candida albicans
The antifungal activity of CHG against C. albicans was evaluated using the cell viability assay. As shown in , compared with the controls, which were not treated with CHG, the cells exhibited significant growth inhibition or cell death after different exposure times at a concentration of 25 µM. When the concentration increased to 50 µM, most fungal cells died after 30 min of treatment, and the cells were completely killed after 2 and 4 h. Therefore, CHG inhibited the growth of C. albicans in a dose- and time-dependent manner, as reflected by a progressive decrease in the viable cells or activity with increasing concentration and exposure time.
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).
CHG-induced permeability impairment of C. albicans cells was investigated using PI staining. As shown in , after exposure to CHG (25 and 50 μM) for 30 min, most fungal cells were stained with PI, whereas no PI incorporation was observed in the negative control. Furthermore, after treatment for 2 and 4 h, even a relatively low concentration (25 μM) could lead to PI incorporation. The cellular morphology of C. albicans cells was observed using scanning electron microscopy (SEM). As shown in , the untreated cells were round with smooth surfaces, whereas many CHG-treated cells were abnormally shaped, with deep wrinkles and deformities. Some cells were either cracked on the surface or ruptured with leaking intracellular content. Collectively, CHG can also affect the C. albicans cell wall/membrane and may penetrate fungal cells, having toxic effects. Furthermore, the metabolic activity of C. albicans cells treated with CHG was quantified using an XTT assay. As shown in , CHG at concentrations of 25 and 50 μM exhibited similar inhibitory effects on the metabolic activity of C. albicans.
Treatment with CHG increases intracellular ROS
The effect of CHG on intracellular ROS production was measured using the fluorescence dye 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA). Considering that apoptotic cells often produce high levels of ROS, we investigated the effect of CHG on ROS production under concentrations that do not kill cells (). The results showed that even under a relatively low concentration (5 μM), CHG caused a significant increase of intracellular ROS. When the concentration increased to 10 and 25 μM, the intracellular ROS was much higher (). These results indicate that CHG leads to intracellular ROS accumulation, which may result in intracellular oxidative damage, cell membrane damage, and induction of apoptosis in C. albicans cells.
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).
Mac1, Sef2, and Sfu1 are involved in the response to CHG treatment
We screened a library containing 165 transcription factor gene deletion mutants by performing an MIC assay. As shown in , compared with the wild-type (WT) strain SN250, the MAC1, SEF2, and SFU1 deletion mutants were more sensitive to CHG. Among them, the MIC of CHG for the mac1/mac1 mutant exhibited the most significant difference, decreasing from 25 to 3.125 μM. The MIC for sef2/sef2 and sfu1/sfu1 mutants was reduced to 12.5 μM. A copy was re-integrated into the original locus of the corresponding mutant to verify that this effect was due to the inactivation of these three genes. The MIC of the re-constituted strains recovered to 25 μM.
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.
A spot dilution assay was used to confirm the results of the MIC assay. Consistent with the results of the MIC assay, MAC1, SEF2, and SFU1 deletion mutants showed increased susceptibility to CHG (). Growth of the mac1/mac1 mutant was almost completely inhibited in the presence of 25 μM CHG, whereas the reconstitution of MAC1 reversed this effect. Deletion of SEF2 and SFU1 also led to different degrees of growth defects.
CHG inhibits cell growth by raising the intracellular metal ion concentration
Mac1 plays crucial roles in copper import and ROS homeostasis, whereas Sef2 and Sfu1 regulate iron uptake. Therefore, we speculated that the antifungal activity of CHG was associated with dysregulation of intracellular metal ion homeostasis, consequently resulting in ROS accumulation in C. albicans cells. To verify this hypothesis, SN250 (WT) cells incubated with different concentrations of CHG were digested, and ICP-MS was used to measure the concentrations of metal ions. As shown in , even under relatively low concentrations (2.5 μM), CHG caused a significant increase of intracellular Cu and Fe. A progressive increase in levels of Cu and Fe was observed with increasing concentration of CHG (2.5–10 μM). Therefore, CHG promotes the accumulation of Cu and Fe inside C. albicans cells in a dose-dependent manner.
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).
The expression levels of MAC1, SEF2, SFU1, and the copper transporter-encoding gene CTR1 in the WT and mac1/mac1 mutants were detected using qRT-PCR. Considering that the MIC of CHG for the mac1/mac1 mutant is 3.125 μM, we performed qRT-PCR analysis under concentrations 1 and 2.5 μM. As shown in , after CHG treatment, the expression of MAC1, SEF2, and SFU1 was upregulated, which was consistent with the results of MIC assay, indicating that the metal-ion sensing regulators Mac1, Sef2, and Sfu1 play crucial roles in resisting CHG-mediated ion accumulation and ROS generation. In addition, Ctr1 was suppressed to reduce the uptake of Cu from the extracellular environment, whereas copper-dependent iron transport was activated by upregulating SEF1 and downregulating HAP43 to consume excess Cu. Compared with the WT, mac1/mac1 mutant exhibited significantly lower expression of SEF2, SFU1, and CTR1, whereas the expression of SEF1 and HAP43 were significantly higher.
As shown in , the intracellular ROS levels in the WT and mac1/mac1, sef2/sef2, and sfu1/sfu1 mutants were significantly elevated after treatment with 25 μM CHG. The three deletion mutants generated more intracellular ROS than that in the WT, both before and after CHG treatment. These results indicate that CHG promotes the generation of intracellular ROS by regulating metal ion homeostasis, and finally leads to cell death.
Discussion
CHG is the most effective ingredient in mouthwashes and is widely used as an antimicrobial agent in dental practice. It has a broad spectrum of activity against various bacteria, yeasts, dermatophytes, and lipophilic viruses, with few undesirable side effects [Citation28]. In this study, we verified the antifungal activity of CHG against the most common fungal pathogen, C. albicans, at a concentration of 25 μM, far below the concentration (0.12–0.2%, approximately 1.3–2.2 mM) used in mouthwashes. This provides evidence that CHG can be considered an adjunctive medication for oral fungal infections.
CHG is a cationic bisbiguanide comprising two symmetric 4-chlorophenyl rings and two biguanide groups connected by a central hexamethylene chain [Citation29]. According to previous studies, its efficacy can be attributed to the increased permeability of the microbial membrane and the alteration of osmotic equilibrium by binding its positively charged end to the negatively charged end of the microbial membrane or cell wall [Citation30,Citation31]. Therefore, CHG can be applied to some medical devices such as dental implants, needleless connectors, antimicrobial dressings, and vascular catheters. When absorbed onto the surface of medical devices, CHG can kill microorganisms, prevent microbial colonization, and subsequently block biofilm development [Citation32]. CHG can also be applied to some uncleaned surfaces like dental plaque, mouth tissue, and skin by directly binding its positively charged end to the microbial cell surface, and exerts antimicrobial effects for prolonged periods of time [Citation33]. Unlike other antimicrobials, antimicrobial activity of CHG can be retained in the presence of body fluids such as blood and some organic matters [Citation34,Citation35]. An antimicrobial effect sustained over time is referred to as substantive antimicrobial activity or substantivity [Citation36–38]. According to previous studies, the substantivity of 2% CHG has been reported to extend at least 24 h after 1 min of application, even prolonged up to 12 weeks after 10 min of application [Citation39,Citation40]. In this study, we verified the increased permeability of the membrane and the defects in the cell wall of C. albicans. Consequently, metal ions, such as copper and iron, may enter from extracellular to intracellular space through active or passive transport mechanisms. Previous studies have shown that excess intracellular metal ions can be toxic as they catalyze ROS production, which can severely damage biological molecules, including nucleic acids, proteins, and lipids, and is vital in triggering apoptosis in C. albicans [Citation11,Citation41–43].
Various transcriptional factors and transporter proteins regulate metal ion homeostasis. Mac1 is a crucial copper-sensing transcription factor involved in Cu/Fe utilization and stress response and is required for invasion and adhesion to host cells and antifungal tolerance [Citation44,Citation45]. Mac1 also controls ROS homeostasis by repressing Cu-containing superoxide dismutase and inducing Mn-containing superoxide dismutase [Citation46,Citation47]. Consistently, our results showed that the deletion of MAC1 resulted in increased intracellular ROS production and cell susceptibility to CHG.
In addition to Cu, Fe is an essential nutrient and the most abundant trace metal in fungal cells. Cu and Fe uptake in C. albicans are linked through a high-affinity ferric/cupric-reductive uptake system, which is the primary strategy used by C. albicans to acquire iron from the environment [Citation48]. The high-affinity iron permeases Ftr1 is the most critical component of this process. It can transport iron into the cell when it binds to the permease-coupled multicopper ferroxidase Fet3 [Citation49]. Since copper is crucial for Fet3, it is essential for iron uptake from the environment by C. albicans. This is consistent with our results which indicate that, in addition to copper, iron also accumulated after CHG treatment.
Among the iron homeostasis-associated genes, SEF2 and SEF1 encode Zn(2)Cys(6) DNA-binding proteins, which are involved in iron acquisition and are required for normal resistance to copper. Hap43 represses genes that encode iron-dependent proteins involved in mitochondrial respiration and iron-sulfur cluster assembly [Citation50]. SFU1 encodes a transcriptional repressor of siderophore uptake/biosynthesis that controls various iron-responsive genes [Citation10]. These transcription factors form an elaborate and interconnected transcriptional network that coordinately regulates iron acquisition, utilization, and other iron-responsive metabolic activities. In this study, the expression of these metal ion-associated genes was detected. The activation of MAC1, SEF2, and SFU1 after CHG treatment indicates their importance in regulating the homeostasis of metal ions and ROS generation in response to CHG. The deletion strains of these three genes showed higher sensitivity to CHG, consistent with the results of the MIC and spot dilution assays. Ctr1 is the most important copper transporter on the plasma membrane of fungal cells. It is responsible for high-affinity copper uptake and is transcriptionally regulated by Mac1 [Citation51]. To resist excess copper, high-affinity copper uptake was repressed by downregulating CTR1. In contrast, copper-dependent iron transport was activated by upregulating SEF1 and SEF2, leading to the upregulation of SFU1 and the downregulation of HAP43.
In conclusion, we revealed a novel and precise mechanism of CHG against C. albicans (): CHG induced apoptosis of fungal cells through ROS generation and metabolic alteration, and the disruption of metal ion homeostasis plays a crucial role in this process. This study provides a better understanding of the antifungal mechanism of CHG against C. albicans and a basis for identifying novel targets for the treatment of fungal infections [Citation52].
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.
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