A Cecropin-4 Derived Peptide C18 Inhibits Candida albicans by Disturbing Mitochondrial Function

Abstract

Global burden of fungal infections and related health risk has accelerated at an incredible pace, and multidrug resistance emergency aggravates the need for the development of new effective strategies. Candida albicans is clinically the most ubiquitous pathogenic fungus that leads to high incidence and mortality in immunocompromised patients. Antimicrobial peptides (AMPs), in this context, represent promising alternatives having potential to be exploited for improving human health. In our previous studies, a Cecropin-4-derived peptide named C18 was found to possess a broader antibacterial spectrum after modification and exhibit significant antifungal activity against C. albicans. In this study, C18 shows antifungal activity against C. albicans or non-albicans Candida species with a minimum inhibitory concentration (MIC) at 4∼32 μg/ml, and clinical isolates of fluconazole (FLZ)-resistance C. tropicalis were highly susceptible to C18 with MIC value of 8 or 16 μg/ml. Additionally, C18 is superior to FLZ for killing planktonic C. albicans from inhibitory and killing kinetic curves. Moreover, C18 could attenuate the virulence of C. albicans, which includes damaging the cell structure, retarding hyphae transition, and inhibiting biofilm formation. Intriguingly, in the Galleria mellonella model with C. albicans infection, C18 could improve the survival rate of G. mellonella larvae to 70% and reduce C. albicans load from 5.01 × 10⁷ to 5.62 × 10⁴ CFU. For mechanistic action of C18, the level of reactive oxygen species (ROS) generation and cytosolic Ca^{2 +} increased in the presence of C18, which is closely associated with mitochondrial dysfunction. Meanwhile, mitochondrial membrane potential (△Ψm) loss and ATP depletion of C. albicans occurred with the treatment of C18. We hypothesized that C18 might inhibit C. albicans via triggering mitochondrial dysfunction driven by ROS generation and Ca^{2 +} accumulation. Our observation provides a basis for future research to explore the antifungal strategies and presents C18 as an attractive therapeutic candidate to be developed to treat candidiasis.

Keywords: Candida albicans; G. mellonella; ROS; antifungal activity; cecropin-4 derived peptide; mitochondrial dysfunction.

FIGURE 1

Physicochemical properties of C18. (A) The helical wheels of C18. The helical wheels were constructed with NetWheels (https://heliquest.ipmc.cnrs.fr/cgi-bin/ComputParams.py). (B) The predicted 3D conformation of the synthetic peptide C18 (https://zhanggroup.org/I-TASSER/).

FIGURE 2

Inhibition and killing kinetics of C18 against Candida albicans. (A) Growth curve of C. albicans within incubation for 48 h. Optical density at 630 nm (OD_630nm) of C. albicans SC5314 in the presence or absence of C18 at the concentration of 32∼128μg/ml was measured. (B) Time-killing kinetics of C18 against C. albicans SC5314. Fungal cells were incubated with C18 at concentrations of 32∼128 μg/ml for 8 h, and the colonies of C. albicans suspension were counted at every 2 h. The drug-free strain-containing medium was set as the control and fluconazole (FLZ) was used as the positive drug. Each data point represents mean ± SD of three independent experiments.

FIGURE 3

Effect of C18 on morphogenesis and biofilm in Candida albicans. (A) The hyphal formation of C. albicans exposed to C18 was detected by invert microscope. To induce hyphae formation, RPMI-1640 with 15% FBS was added and incubated at 35°C for 2 h, 4 h, and 6 h. C18 was treated at 32 μg/ml and 64 μg/ml, the drug-free strain-containing medium was set as the control, and 128 μg/ml FLZ was set as the positive control. (B) The morphogenesis inhibitory effect of C18 was evaluated and recorded as a percentage. Candida albicans cells was incubated with C18 (32 μg/ml and 64 μg/ml) or FLZ (128 μg/ml) in RPMI-1640 medium containing 15% FBS at 35°C for 6 h, and at least 300 cells was counted using a microscope. C. albicans cells without any treatment were set as control. (C,D) Effect of C18 on biofilm formation (C) and preformed biofilm (D) in C. albicans by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl) 2H-tetrazolium-5-carboxanilide sodium salt (XTT) reduction assay. For biofilm formation, C. albicans cells was incubated with C18 (32 μg/ml, 64 μg/ml, and 128 μg/ml) or FLZ (128 μg/ml) in RPMI-1640 medium at 37°C for 24 h. For preformed biofilm, C. albicans cells was incubated in RPMI-1640 medium at 37°C for 24 h to form mature biofilm. Next, supernatant was discarded and C18 (128 μg/ml, 256 μg/ml, and 512 μg/ml) or FLZ (512 μg/ml) was added and co-incubated for another 24 h. XTT reduction was performed to determine the level of biofilm viability at various treatment and colorimetric absorbance was measured at 490 nm. Error bars represent standard deviation of three independent experiments. ***p < 0.001 compared to the control group.

FIGURE 4

In vivo toxicity and therapeutic activity of C18 in a Galleria mellonella model. (A) The toxicity of C18 in G. mellonella larvae model. A range of concentrations (4 mg/kg∼32 mg/kg) of C18 injected to the larvae (10 μl pre larva) was applied to evaluate toxicity of the peptide. Only 10% of the larvae died when treated with 32 mg/kg C18 for 5 days. (B) Survival assays for 3 replicate experiments combined (n = 30). Galleria mellonella larvae were infected with 10 μl inoculum of 5 × 10⁷ CFU/ml of Candida albicans and treated with various concentrations of C18 or 4 mg/kg FLZ. For each replicate, a group of 10 larvae in each treatment was monitored for survival over 5 days. **p < 0.01, compared to the group of C. albicans + phosphate buffer solution (PBS). (C) Fungal burden for three replicate experiments (n = 3). For each replicate, three larvae from each group were sacrificed at 24 h post treatment, homogenized, cultivated on yeast extract peptone dextrose (YPD) medium. Colonies are counted and converted to obtain to the C. albicans counts per larva. ***p < 0.001 compared to the group of C. albicans + PBS.

FIGURE 5

Scanning electron microscopic (SEM) images of Candida albicans in the absence and presence of C18 at 35°C for 3 h. (A1-A2) SEM images of C. albicans treated without C18. (B1-B2) SEM images of C. albicans treated with 32 μg/ml of C18. (C1-C2) SEM images of C. albicans treated with 64 μg/ml of C18. Red arrow indicates the cell wall and membrane was destroyed leading to intracellular component leaked away. Blue arrow indicated the surface was folded and depression occurred in the cells. Green arrow indicated the size of cells was different and many cells were swollen.

FIGURE 6

Effect of C18 on the cell membrane permeability of Candida albicans. (A) Fluorescence intensity of propidium iodide (PI) (10 μg/ml) after C18 treatment was detected by sectrofluoro-photometer. Error bars represent standard deviation of three independent experiments. ^***p < 0.001 compared to the control group. (B) Fluorescence images of C. albicans cells stained by SYTO9 (10 μM) and PI (10 μg/ml) after C18 treatment for 6 h using confocal laser scanning microscopy (CLSM). Green represents live microbes while red represents dead microbes, and the merge shown in both green and red are combined from the two channel images. Scale bar 20μm.

FIGURE 7

Effect of C18 on intracellular ROS generation and Ca^{2 +} accumulation of Candida albicans. (A1,B1) Bar diagram showed the level of intracellular ROS and Ca^{2 +} in C. albicans treated with C18 at concentrations of 32 μg/ml and 64 μg/ml. The fluorescence intensity of dichlorofluorescein (DCF; indicates ROS) or Fluo-3/AM (indicates cytosolic Ca^{2 +}) was detected by sectrofluoro-photometer, respectively. Error bars represent standard deviation of three independent experiments. ^**p < 0.01,* p < 0.05, ^***p < 0.001 compared to the control group. (A2–B2) Fluorescence images of C. albicans cells stained by 2′,7′-dichlorofluorescein diacetate (DCFH-DA) or Fluo-3/AM was captured by CLSM after C18 treatment for 6h respectively. Blue represents cell nucleus stained by 4′,6-diaminidino-2-phenylindole (DAPI), while green represents ROS accumulation (A2) and cytosolic Ca^{2 +} (B2), the merge shown in both green and blue are combined from the two channel images. Red arrow indicates dispersive or degradative DNA with DAPI staining in the high level of ROS expression cells. Scale bar 10 μm.

FIGURE 8

Mitochondrial Dysfunction of Candida albicans treated with or without C18. (A1) △Ψ_m of C. albicans was measured by 5,5′,6′,6′-terachloro-1, 1′3,3′-tetraethyl-benzimidazolyl carbocy-anine iodide (JC-1) staining in the presence or absence of 32 μg/ml or 64 μg/ml C18 for 3 h, 6 h, and 12 h. Bar diagram showed the ratio of red fluorescence intensity to green fluorescence intensity (△Ψm) detected by sectrofluoro-photometer. Carbonyl cyanide m-chlorophenyl hydrazine (CCCP), a mitochondrial uncoupler, can depolarize △Ψm of cells and was used as the positive control. The results are presented as mean ± SD of three independent experiments. ^***p<0.001, compared to the control group. (A2) Fluorescence images of C. albicans cells stained by DAPI and JC-1 after C18 treatment for 6h was captured by CLSM. Blue represents cell nucleus while red fluorescence represents JC-monomers, and green fluorescence represents JC-aggregates. The color of merged images reflects the degree of △Ψm loss. Scale bar 10 μm. (B) Intracellular ATP of cells were measured by sectrofluoro-photometer. The results are presented as mean ± SD of three independent experiments. ^***p<0.001, compared with the control group.

FIGURE 9

The mode of C18 against Candida albicans. C18 has an significant inhibitory efficacy on C. albicans, such as abnormal morphogenesis, cell wall damage, and alteration of membrane permeability, resulting in a series of destructive effects in cytoplasm. Causally, C18 plays an essential role in ROS generation and intracellular Ca^{2 +} increase, which contributes to dysfunctional mitochondria in form of △Ψ_m collapse and ATP depletion. In short, C18 can trigger mitochondrial dysfunction driven by excessive ROS production and Ca^{2 +} accumulation, leading to the cell death.