Antifungal activity and mechanism of novel peptide Glycine max antimicrobial peptide (GmAMP) against fluconazole-resistant Candida tropicalis

Abstract

Background: There is a pressing need to create innovative alternative treatment approaches considering the overuse of antifungal drugs causes the number of clinically isolated fluconazole-resistant Candida species to increase. Glycine max antimicrobial peptide (GmAMP) is a novel peptide screened by us using artificial intelligence modeling techniques, and pre-tests showed its strong antimicrobial activity against clinically fluconazole-resistant Candida tropicalis.

Methods: The study aimed to comprehensively investigate the antimicrobial activity and mechanisms of GmAMP against fluconazole-resistant C. tropicalis. The antifungal activity of GmAMP against fluconazole-resistant C. tropicalis was assessed by using broth microdilution method, growth and fungicidal kinetics, hypha transformation, and antibiofilm assay. To further uncover the potential mechanisms of action of GmAMP, we performed scanning electron microscopy, flow cytometry, cell membrane potential probe 3, 3'-Dipropylthiadicarbocyanine Iodide (DiSC₃(5)), and reactive oxygen species (ROS) probe 2', 7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) detection to assess the cellular morphology and structure, membrane permeability, membrane depolarization, and ROS accumulation, respectively. At the same time, we used cytotoxicity and degree of erythrocyte hemolysis assays to assess GmAMP's toxicity in vitro. Cytotoxicity and treatment efficacy were evaluated in vivo by utilizing the Galleria mellonella larvae infection model.

Results: GmAMP exhibited significant antifungal activity against fluconazole-resistant C. tropicalis with a minimum inhibitory concentration (MIC) of 25 µM and demonstrated fungicidal effects at 100 µM within 2 h. GmAMP prevented the transition from yeast to hypha morphology, inhibited the biofilm formation rate of 88.32%, and eradicated the mature biofilm rate of 58.28%. Additionally, GmAMP treatment at 100 µM caused cell structure damage in fluconazole-resistant C. tropicalis, whereas GmAMP treatment at concentrations ranging from 25 to 100 µM caused membrane permeability, depolarization of cell membrane potential, and intracellular ROS accumulation. Moreover, GmAMP enhanced the survival rate of 75% for G. mellonella with fluconazole-resistant C. tropicalis infection as well as reduced fungal burden in vivo by approximately 1.0 × 10² colony forming units per larva (CFU per larva).

Conclusion: GmAMP can disrupt the cell membrane of fluconazole-resistant C. tropicalis and also shows favorable safety and therapeutic efficacy in vivo. Accordingly, GmAMP has the potential to be an agent against drug-resistant fungi.

Keywords: Antifungal activity; Antimicrobial peptide; Candida tropicalis; Drug-resistance; GmAMP.

Figure 1. Physicochemical properties of GmAMP.

(A) Helical wheel analysis of GmAMP. Positively charged amino acids are indicated in blue, the red ‘N’ represents the starting position and the arrow represents the hydrophobic moment. (B) Predicted three-dimensonal spatial structures of GmAMP. (C) The physical and chemical properties of GmAMP.

Figure 2. Effect of GmAMP on the growth of fluconazole-resistant C. tropicalis.

(A) Growth kinetics of fluconazole-resistant C. tropicalis. (B) Time-killing kinetics of fluconazole-resistant C. tropicalis. (C) The transformation from yeast phase to mycelial phase of fluconazole-resistant C. tropicalis. Scale bar, 25 µm.

Figure 3. Effect of GmAMP on the biofilm of fluconazole-resistant C. tropicalis.

Inhibition (A) and eradication (B) effects of fluconazole-resistant C. tropicalis biofilms treated with GmAMP at different concentrations observed by confocal laser scanning microscopy. Images obtained by live/dead staining (SYTO 9, green; PI, red). Scale bar, 20 µm. The activity level of biofilm under different concentrations of GmAMP was determined by the XTT reduction method (C and D), and the colorimetric absorbance was measured at OD_490nm. The error bar represents the standard deviation of the three independent experiments. ***P < 0.001 compared with the control group.

Figure 4. Effects of GmAMP cell morphology and cell membranes of fluconazole-resistant C. tropicalis.

The control group (A) and GmAMP group treated with 100 µM (B) of fluconazole-resistant C. tropicalis morphological images by scanning electron microscopy. (C) Cell membrane permeability of GmAMP on the fluconazole-resistant C. tropicalis was determined by flow cytometry, and with SYTO 9 and PI as pore formation mechanism marker. (D) The bar chart showed the percentage of PI positive cells. (E) DiSC₃(5) was used to detect the cell membrane depolarization of fluconazole-resistant C. tropicalis. (F) The ROS-induced accumulation of DCFH-DA is a pore formation mechanism marker. The error bar represents the standard deviation of the three independent experiments. *** P < 0.001 compared with the control group.

Figure 5. The hemolytsis and cytotoxicity effects of GmAMP.

(A) The cytotoxicity of GmAMP against RAW 264.7 cells. (B) The hemolysis rate of 2% human red blood cells.

Figure 6. In vivo toxicity and therapeutic activity of GmAMP in the G. mellonella model.

(A) Schematic diagram of the GmAMP treatment. (B) The toxicity of GmAMP in G. mellonella larvae model. (C) Survival of larvae after treatment with GmAMP. (D) Fungal burden of larvae after treatment with GmAMP. *P < 0.05; ***P < 0.001 compared with the group of fluconazole-resistant C. tropicalis + PBS.