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

Materials

The peptide GmAMP (SPGKKKKKKKKKKKTKKKKKK) was synthesized by solid phase chemical synthesis method by Gil Biochemical Co., Ltd (Shanghai, China), purified by reversed-phase high-performance liquid chromatography (RP-HPLC) (Fig. S1A), and the purity was >95%. They were dissolved in deionized water at a stock concentration of 5 mg/mL before use. The molecular weight of GmAMP was determined using electrospray ionization (ESI) mass spectrometer (ESI MS) (Fig. S1B). We used the online website Heliquest software (https://heliquest.ipmc.cnrs.fr/index.html) to predict the helix diagram and hydrophilicity, and AlphFold2 to predict the 3D structure of peptides. The molecular weight of the peptide was predicted by Expasy ProtParam (https://web.expasy.org/protparam/).

Strains and cell culture conditions

The fluconazole-resistant C. tropicalis 4171, 4252, 6984, and 8402 were collected from infected patient’s blood in the affiliated hospital of Guizhou Medical University. C. tropicalis ATCC 20962 was purchased from the Shanghai Conservation Biotechnology Center. All strains were grown in yeast extract peptone dextrose medium (YPD, Solarbio, Beijing, China) at 35 °C with shaking at 200 rpm until the cultures reached the logarithmic phase. RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS) (Sigma-Aldrich) was used as the culture medium for hypha growth of C. tropicalis. The mouse monocyte-macrophage cell line RAW 264.7 was donated by Jiahong Wu from the Key and Characteristic Laboratory of Modern Pathogen Biology, Guizhou Medical University, and cells were cultured in DMEM medium (Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin (Gibco, Waltham, MA, USA), and 100 µg/mL streptomycin (Gibco, Waltham, MA, USA) and maintained at 37 °C in a humidified 5% CO₂ incubator.

Antifungal activity

The minimum inhibitory concentration (MIC) of GmAMP for four fluconazole-resistant C. tropicalis from clinical isolates, and a standard strain of C. tropicalis ATCC 20962 was determined by using broth microdilution method according to the Standards of Clinical and Laboratory Standards Institute (CLSI) (Clinical and Laboratory Standards Institute (CLSI), 2023). In brief, these yeasts of fungal strains were cultured in YPD broth medium at 35 °C to the logarithmic growth stage, and the cultures were washed by phosphate-buffered saline (PBS, 10 mM, pH 7.4) three times and resuspended to 0.5 × 10³~2.5 × 10³ CFU/mL, then 100 µL above fungal suspension was added to a 96-well plate with a series concentration of GmAMP (100, 50, 25, 12, 6, 3 µM) or fluconazole (3,343 µM to 3 µM) (Yuan Ye, Shanghai, China) and 10 mM PBS and medium were used as the negative and blank controls, respectively. After co-incubation at 35 °C for 24 h, The drug concentration corresponding to the well without visible fungal growth was regarded as MIC, corresponding to 90% inhibition of fungal growth. The experiment was performed in triplicate and repeated three times.

Growth and fungicidal kinetics

To analyze the antifungal or fungicidal process of GmAMP against fluconazole-resistant C. tropicalis, the growth kinetics and time-kill kinetics of GmAMP on fluconazole-resistant C. tropicalis were further investigated at different times after GmAMP treatment as to previously described (Ramesh et al., 2023). The concentration of the prepared fungal cells was 1.0 × 10⁶ CFU/mL according to the previously mentioned method and incubated with 25, 50, and 100 µM of GmAMP at 35 °C for 48 h. A microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) was used to record the OD_630nm every 2 h during co-cultivation. In the meantime, the cultivated yeasts were harvested at certain intervals (0, 2, 4, 6, 8, 10, and 12 h), and the agar solid plate experiment was conducted following gradient dilution. The negative and blank controls in the experiment were 10 mM PBS and medium. Fungal colonies were counted after incubation at 35 °C for 24 h. The results were presented as the average of triplicate measurements from three independent assays.

Effect of GmAMP on hypha formation

To analyze the effect of GmAMP on the transition of yeast-to-hyphal phase in fluconazole-resistant C. tropicalis as described previously (Jiang et al., 2016). The concentration of the prepared fungal cells was 1.0 × 10⁶ CFU/mL in RPMI 1640 medium (Gibco, Waltham, MA, USA) which contained 15% fetal bovine serum (Gibco, Waltham, MA, USA) according to the previously mentioned method. A total of 500 µL of fungal suspension was incubated with GmAMP at concentrations (25, 50, and 100 µM) in 24 well plates and 10 mM PBS as the negative control. Cells were co-incubated with peptides for 3, 6, 9, 12, and 24 h at 37 °C, then the hypha formation was observed and photographed under an inverted microscope.

Antibiofilm assay

Fungal cells grown to the logarithmic phase were adjusted to 1.0 × 10⁶ CFU/mL with RPMI-1640 liquid medium and added to 96-well polypropylene plates, which were then incubated at 37 °C for 90 min (biofilm formation assay) or 48 h (biofilm eradication assay) according to previous method (Zou et al., 2024). And 100 µL newly prepared 2, 3-bis(2-methoxy-4-nitro-5-sulfophenyl) 2H-tetrazolium5-carboxamide sodium salt (XTT) solution (Yuan Ye, Shanghai, China) was added to each well for 2 h at 37 °C after different concentrations of GmAMP were added and continued to co-incubate for 24 h. A microplate reader set to OD_490nm was used to measure absorbance.

Sterile polylysine cell crawls were then placed on the bottom of a 24-well plate and 500 µL of the fungal suspension at the above concentration was added. The biofilm formation and mature biofilm were prepared according to the above method, and 500 µL of SYTO 9 (Invitrogen, Waltham, MA, USA) and propidium iodide (PI; Sigma) solution with the final concentration of 10 µM were added and incubated for 20 min while 10 mM PBS was used as the negative control. Seal the cover glass with nail polish, laser confocal microscopy (Olympus SpinSR10; Olympus, Tokyo, Japan) was used to observe and obtain images.

Scanning electron microscope

The concentration of the prepared fungal cells was 1.0 × 10⁶ CFU/mL based on the previous description with minor modifications (Alfaro-Vargas et al., 2022), incubated in GmAMP with culture medium at 35 °C for 2 h, and 10 mM PBS was used as a negative control, then the suspension was centrifuged at 5,000 rpm for 10 min, fixed with 2.5% glutaraldehyde overnight at 4 °C, and dehydrated with 50%, 75%, 95% and 100% series of ethanol solutions for 10 min. It was then dried in a vacuum evaporator and coated with a thin layer of gold-palladium. The samples were observed by scanning electron microscopy and the image acquisition was performed using a Hitachi Regulus SU8100 (Tokyo, Japan).

Flow cytometry analysis

The concentration of the prepared fungal cells was 1.0 × 10⁶ CFU/mL according to the previous description with minor modifications (Torres et al., 2023), the GmAMP with different concentrations was incubated at 35 °C for 1 h, and 10 mM PBS was used as the negative control. After that, the fungal suspension was incubated with SYTO 9 and PI staining with a final concentration of 10 µM at 35 °C for 15 min, and the stained cells were analyzed by flow cytometry.

Membrane potential

The concentration of the prepared fungal cells was 1.0 × 10⁶ CFU/mL as described in the previous study (Decker et al., 2024), then added to 96-well plates while the membrane potential DiSC₃(5) probe was added into the fungal suspension. The fungal suspension was then treated with different concentrations of GmAMP, and 10 mM PBS was used as the negative control for measured fluorescence intensity. The change of fluorescence intensity in 1 h was continuously and dynamically monitored with by RF-5301PC sectrofluoro-photometer (Bio-Tek Synergy HTX, Winooski, Vermont, USA).

Reactive oxygen species level

The levels of ROS were determined by using 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Yuan Ye, Shanghai, China) according to previously described methods (Shaban, Patel & Ahmad, 2024). The 1.0 × 10⁶ CFU/mL of fungal suspension was added to 96-well plates while the Reactive Oxygen DCFH-DA probe (10 µM) was added into the fungal suspension. The fungal suspension was then treated with different concentrations of GmAMP. 10 mM PBS and 10 µM NAC (N-Acetylcysteine) were used as the negative and positive control. The change of fluorescence intensity in 1 h was continuously and dynamically monitored by RF-5301PC sectrofluoro-photometer (Bio-Tek Synergy HTX, Winooski, VT, USA).

Cytotoxicity Assays

Mouse RAW 264.7 cells were used to evaluate the cytotoxicity of GmAMP on mammalian cells as previously described (de Oliveira et al., 2023). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 1% penicillin-streptomycin (Gibco, Waltham, MA, USA), and maintained at 37 °C in a humidified 5% CO₂ incubator. Firstly, 100 µL of RAW 264.7 cells suspension (2 × 10⁴ cells/mL) was added to 96-well plates for cultivating overnight. A total of 100 µL with different concentrations of GmAMP solution was added and then incubated at 37 °C for 24 h. 10 mM PBS and complete medium were used as the negative and blank controls, respectively. When the incubation period was over, 10 µL of CCK8 solution was added to each assay well following the manufacturer’s protocols (MedChemExpress, Monmouth Junction, NJ, USA) and incubated for 1 h. Absorbance values were detected at OD_450nm, and the percentage of cell survival was counted.

$$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{\%})=\left(\frac{\mathrm{A}\mathrm{b}{\mathrm{s}}_{450\mathrm{n}\mathrm{m}}\mathrm{o}\mathrm{f}\mathrm{G}\mathrm{m}\mathrm{A}\mathrm{M}\mathrm{P}\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}-\mathrm{A}\mathrm{b}{\mathrm{s}}_{450\mathrm{n}\mathrm{m}}\mathrm{o}\mathrm{f}\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}{\mathrm{A}\mathrm{b}{\mathrm{s}}_{450\mathrm{n}\mathrm{m}}\mathrm{o}\mathrm{f}\mathrm{P}\mathrm{B}\mathrm{S}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{A}\mathrm{b}{\mathrm{s}}_{450\mathrm{n}\mathrm{m}}\mathrm{o}\mathrm{f}\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\right)\times 100\mathrm{\%}.$$

Hemolysis of human red blood cells

The human red blood cells (hRBCs) were used to evaluate the hemolytic activity of GmAMP based on previous descriptions with minor modifications (Larrán et al., 2022). The GmAMP with different concentrations and 2% hRBCs were added to the 96-well plate and incubated for 1 h in 37 °C. 1% Triton X-100 (Solarbio, Beijing, China) was the positive control, and 10 mM PBS was used as the negative control. When the incubation period was over, as the samples were gathered and centrifuged for 10 min at 1,000 rpm, the supernatant was moved to a fresh 96-well plate, and the OD_540nm absorbance value was used to determine the extent of hemolysis. The hemolysis test of the new peptide has been approved by Ethics Committee of GuiZhou Medical University, approval number: 2022 Ethical Approval (302).

$$\mathrm{H}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{i}\mathrm{s}(\mathrm{\%})=\left({\displaystyle \frac{\mathrm{A}\mathrm{b}{\mathrm{s}}_{540\mathrm{n}\mathrm{m}}\mathrm{o}\mathrm{f}\mathrm{G}\mathrm{m}\mathrm{A}\mathrm{M}\mathrm{P}\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}-\mathrm{A}\mathrm{b}{\mathrm{s}}_{540\mathrm{n}\mathrm{m}}\mathrm{o}\mathrm{f}\mathrm{P}\mathrm{B}\mathrm{S}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}{\mathrm{A}\mathrm{b}{\mathrm{s}}_{540\mathrm{n}\mathrm{m}}\mathrm{o}\mathrm{f}\left(\mathrm{T}\mathrm{r}\mathrm{i}\mathrm{t}\mathrm{o}\mathrm{n}\mathrm{X}-100\right)-\mathrm{A}\mathrm{b}{\mathrm{s}}_{540\mathrm{n}\mathrm{m}}\mathrm{o}\mathrm{f}\mathrm{P}\mathrm{B}\mathrm{S}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\right)\times 100\mathrm{\%}.$$

Galleria mellonella infection model

The larvae used in the experiment were purchased from Huiyude Biotechnology Co., Ltd., (Tianjin, China), each weighing 250~300 mg and about 2~3 cm in length. As previously described with slight modifications (Fernandes, Weeks & Carter, 2020). All larvae were placed in a dark incubator at 35 °C overnight before the experiment. Ten larvae were randomly divided into each group to inject 10 µL of GmAMP solution with a concentration of 8~32 mg/kg into the last left proleg of larvae to evaluate the toxicity of GmAMP. The negative control was given an equal volume of sterile PBS. In order to assess the effectiveness of GmAMP, 12 larvae were randomly assigned to each group, and 10 µL of a fungal suspension containing roughly 5.0 × 10⁸ CFU/mL was injected into the final left proleg of the larvae. After 1 h in the incubator, the same volume of GmAMP was injected into the last right proleg using the same method. Live and dead counts were taken every 24 h during the five days of incubation at 35 °C. Larvae were deemed dead when they went dark or mushy and had no discernible tactile reaction. Three larvae per group were chosen at random and placed into 1.5 mL of sterile PBS solution for high-speed homogenization and grinding after 24 h following GmAMP injection. The 10 µL gradient dilution was dropped onto a sterile solid YPD plate and incubated for 24 h. The number of fungal single colony was recorded, and the C. tropical burden of each larva was counted.

Data processing

Statistical mapping and data analysis were performed using GraphPad Prism 8.0 software (GraphPad Software). Data were expressed as mean ± SD and analyzed by one-way ANOVA. Long-rank test was used for the analysis of Mantel-Cox survival curves for the G. mellonella survival experiment. P < 0.05 was considered statistically significant.

GmAMP chemical characteristics

The antibacterial activity of antimicrobial peptides is significantly influenced by their secondary structure. The antimicrobial peptide’s interaction with the cell membrane is facilitated by the alpha-helical structure, which increases the antibacterial activity. (Personne et al., 2023). The antimicrobial peptide GmAMP, composed of 21 amino acids, is predicted to have an α-helical structure (Figs. 1A and 1B). The molecular weight (MW) of GmAMP was confirmed to be 2,539.35 Da by mass spectrometry. Furthermore, GmAMP has a net charge of +17 and a hydrophobicity value of −0.757 (Fig. 1C), these characteristics imply that GmAMP is a cationic hydrophilic peptide.

Antifungal activity

The results of the antifungal activity assay showed that GmAMP had strong antimicrobial effects against clinical fluconazole-resistant C. tropicalis, with MICs ranging from 25 to 50 µM (Table 1). In comparison to this, the MIC values of fluconazole against C. tropicalis ATCC 20962 was 13.06 µM (4 µg/mL), which was consistent with the Clinical and Laboratory Standards Institute standard (CLSI). The MIC values of fluconazole against clinical isolates exceeded 3,343 µM. Consequently, to gain a more comprehensive understanding of the impact of GmAMP on fluconazole-resistant Candida tropicalis clinical isolate, the 4252 isolate (MIC = 25 µM) was selected for further investigation.

Growth kinetics and fungicidal kinetics

To elucidate the effect of GmAMP on the growth process of fluconazole-resistant C. tropicalis, the growth curve of fluconazole-resistant C. tropicalis under GmAMP treatment was further plotted, as shown in Fig. 2A, the fungal cells in the control group entered the logarithmic phase within 6 h and reached the stationary phase within 18 h. In comparison, GmAMP treatments at concentrations of 25 and 50 µM were found to slow down the proliferation rate of fluconazole-resistant C. tropicalis and prolong the time to reach the logarithmic phase. When treated with GmAMP at a concentration of 100 µM, GmAMP showed an inhibitory effect on fluconazole-resistant C. tropicalis and prevented the natural growth and reproduction of fluconazole-resistant C. tropicalis. The time-fungicidal kinetic curve was further plotted to clarify the fungicidal effect of GmAMP (Fig. 2B). Compared with the control group, GmAMP exhibited a powerful inhibitory effect on fluconazole-resistant C. tropicalis when the concentrations of GmAMP were 25 and 50 µM. Notably, dealing with the concentration at 100 µM of GmAMP, the fluconazole-resistant C. tropicalis was killed within 2 h. These results indicate that GmAMP manifests effective antimicrobial activity and fungicidal effect against fluconazole-resistant C. tropicalis. Low-concentration GmAMP exerts an inhibitory effect on the growth of drug-resistant Candida tropicalis, whereas high-concentration high GmAMP directly kills Candida cells.

The transformation of yeast to the mycelial phase

The transformation of Candida mycelial morphology is closely related to its pathogenicity. Morphological changes during the transformation of the fluconazole-resistant C. tropicalis yeast phase to the mycelial phase were observed by using an inverted microscope which is shown in Fig. 2C. The length of the mycelium of the fungus in the control group increased with incubation time, after 9 h of incubation, the yeast cells were observed to grow and form bundles of hyphae while forming branches of various sizes and lengths and intertwining with each other to form a net structure, and a more tightly netted biofilm is formed over time. Interestingly, the development of fluconazole-resistant Candida tropicalis into its hyphal form was inhibited to varying degrees following treatment with GmAMP. Specifically, the morphological transformation from yeast to hyphae was partially inhibited at concentrations of 25 and 50 μM of GmAMP, whereas it was completely suppressed at a concentration of 100 μM. These results suggest that GmAMP inhibited the morphological transformation process of fluconazole-resistant C. tropicalis from the yeast phase to the mycelial phase.

Inhibition of biofilm formation and eradication of mature biofilm

In order to investigate whether GmAMP has an anti-Candida tropicalis biofilm effect, we visualized the results by using confocal laser scanning microscopy. In the control group of the biofilm formation assay, a compact and intact biofilm was observed to emit predominantly green fluorescence colored by SYTO9, a dye that penetrates cells. Nevertheless, after GmAMP treatment, intact biofilm could no longer be formed and only dispersed incomplete membranes of different sizes were observed, while only single yeast cells could be seen in the high-concentration group (Fig. 3A). In the control group of the biofilm eradication assay, it was observed that the tightly structured and complete biofilm mainly emitted green fluorescence colored by SYTO9. After GmAMP treatment, we found that the tightly structured biofilm became loose and the network structure was reduced and thinned, while a reduction in the number of yeasts and an increase in the number of fungal damages was observed, with a predominantly red fluorescence colored by PI, a dye that penetrates injured cells (Fig. 3B). In addition, the biofilm activity of C. tropicalis was determined by XTT quantitative method. Compared with the control group, GmAMP at concentrations of 50, 100, and 200 µM inhibited biofilm formation by 49.75%, 71.28%, and 88.32%, respectively (Fig. 3C), and eradicated mature biofilm by 17.53%, 42.07%, and 58.28%, respectively (Fig. 3D). These results indicated that GmAMP inhibited biofilm formation and eradicated a certain amount of mature biofilm in fluconazole-resistant C. tropicalis.

Antifungal mechanism

To investigate the impact of GmAMP on the cell morphology of fluconazole-resistant C. tropicalis, the morphological changes induced by GmAMP treatment of fungal cells for 2 h were directly observed by scanning electron microscopy. Untreated fungal cells were morphologically intact, with cell surfaces remaining round and smooth (Fig. 4A). When cells were exposed to 100 µM of GmAMP for 2 h, the cells were destroyed and the surface appeared rough and irregular (Fig. 4B). Thus, these outcomes suggested that the cellular structural integrity of fluconazole-resistant C. tropicalis has been impaired. To further investigate the interaction of GmAMP on the cell membrane of fluconazole-resistant C. tropicalis, PI and SYTO9 fluorescence staining were used to determine the effect of GmAMP on the integrity of the cell membrane. PI and STOY9 are DNA-binding dyes emitting red and green fluorescence, respectively, and the former only penetrated the membrane-damaged cells, while the latter stained both live and dead cells (Jin et al., 2005). As a result, when cells were exposed to 25, 50, and 100 µM of GmAMP, 45.02%, 76.88%, and 85.02% of the cells stained positively for PI, respectively. Moreover, PI staining positivity showed a dose-dependent correlation with GmAMP (Fig. 4D), which indicated that GmAMP disrupted the cell membrane integrity of fluconazole-resistant C. tropicalis. The membrane depolarization was detected using the membrane potential probe DiSC₃(5). As shown in Fig. 4E, compared with the control group, we found that GmAMP treatment caused depolarization of the cell membrane potential of fluconazole-resistant C. tropicalis and the level of membrane potential rose significantly with increasing concentration of GmAMP. Reactive oxygen species (ROS) generally maintain low levels within normal cells, but the accumulation of higher levels of ROS can damage cellular structures (Huang et al., 2020). Here, different concentrations of GmAMP induced ROS accumulation in fluconazole-resistant C. tropicalis, and ROS levels showed a time-dose dependence (Fig. 4F). These results suggest that the presence of GmAMP induced ROS production, which contributed to the crucial factor in the antimicrobial effect of GmAMP.

Hemolytic and cytotoxicity

To evaluate mammalian cytotoxicity, we assessed the safety of GmAMP on human red blood cells and RAW 264.7 cells. The hemolysis experiment was performed using 2% human red blood cells. At a concentration of 200 µM, GmAMP exhibited slight hemolysis with a hemolysis rate of 35.64% (Fig. 5A). Moreover, GmAMP showed no obvious cytotoxicity to RAW 264.7 cells, and the cell viability of mouse macrophage RAW 264.7 remained above 80% at 200 µM concentration of GmAMP (Fig. 5B).

Therapeutic effect on fluconazole-resistant C. tropical infection in vivo

The G. mellonella larvae infection model was used to investigate the treatment efficacy of GmAMP in vivo. In the peptide toxicity test (Fig. 6B), all larvae survived, suggesting that GmAMP did not exhibit significant toxicity within the 32 mg/kg dosage range. Survival of larvae infected with fluconazole-resistant C. tropicalis was increased by injecting various concentrations of GmAMP, with 75% survival in the 32 mg/kg group (P < 0.05), whereas the control group had 40% survival rate at 5 days post infection (Fig. 6C). These results were also reflected by the fungal burden of G. mellonella larvae. The number of colonies per larva was significantly reduced in all treatment groups after 24 h of treatment with GmAMP, whereas 32 mg/kg GmAMP reduced the fungal burden from 5.27 × 10⁸ to 6.37 × 10⁶ CFU per larva (Fig. 6D). Consequently, GmAMP has the potential for clinical application as it effectively treats fluconazole-resistant C. tropicalis infection and reduces the fungal burden in vivo.