For homology and structural analysis, the following programs and tools were utilized: Protein calculator,¹ XtalPred,² SignalP 4.1,³ SpiderP,⁴ Pepfold,⁵ and HeliQuest.⁶ For prediction of AMPs, the following programs and tools were utilized: database of antimicrobial peptides (ADAM),⁷ AmpGram,⁸ the support vector machine in CAMPR3,⁹ and the database of antimicrobial activity and structure of peptides (DBAASP).¹⁰

The peptides used in the study were synthesized by Biostem (Ansan, Korea) using the solid phase method. The peptides were synthesized with purity >95% and were verified by high-performance liquid chromatography and mass spectroscopy. Both peptides were reconstituted in distilled water to a final concentration of 1 mM, and the aliquots were stored at −80°C before further experiments.

The bacterial and fungal strains used in this study were obtained from the American Type Culture Collection (ATCC), the Korean Culture Center of Microorganisms (KCCM), or the Culture Collection of Antimicrobial Resistant Microbes (CCARM). Escherichia coli KCCM 11234, Pseudomonas aeruginosa ATCC 9027, Bacillus cereus KCCM 21366, Staphylococcus aureus KCCM 11335, and MDR P. aeruginosa isolates CCARM 2007 and 2095 were grown in tryptic soy agar (TSA, Difco Laboratories, Detroit, MI, United States) at 37°C. candida albicans ATCC 10231 was grown in yeast extract peptone dextrose broth (YPDB, Difco Laboratories) at 37°C. Fusarium oxysporum isolate KCCM 60555 was cultured on potato dextrose agar (PDA, Difco Laboratories) plates at 25°C for 5 days and collected using a cell scraper. HaCaT (CLS 300493, Eppelheim, Germany) was purchased from the Cell Lines Service (CLS) and cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, United States) supplemented with 1% penicillin and streptomycin (Gibco). Adipose-derived mesenchymal stem cell (hADMSC) was obtained from CEFO Co. (Seoul, Korea) and was maintained in CEFOgro™ Human MSC Growth Medium (CEFO Co.). Both cell lines were cultured at 37°C under humidified air with 5% CO₂.

The colony-forming unit (CFU) assays were used to determine the antimicrobial activity of peptides. Cultures of mid-log phase bacterial and fungal cells were diluted to 2 × 10⁶ CFU/mL and 2 × 10⁴ cells/mL, respectively, and were mixed with an equal volume of peptides. After incubating for 3 h, the samples were spread onto agar plates and cultured overnight. The relative colony formation was calculated based on the number of counted colonies on the control and sample plates.

Outer membrane permeability of Gram-negative bacteria was assessed by 1-N-phenyl-naphtylamine (NPN; Sigma-Aldrich, St. Louis, MO, United States) uptake assay. NPN buffer was diluted in 5 mM HEPES buffer (Sigma-Aldrich) to reach a final concentration of 10 μM. Bacteria culture was washed with HEPES buffer and was diluted to reach 1 × 10⁸ CFU/mL. For each well, 100 μL of bacteria suspension was transferred, followed by the addition of 50 μL of NPN and peptides. The fluorescence intensity was measured for 15 min at 460 nm with excitation at 355 nm using Infinite F200 Pro multimode microplate reader (Tecan, Switzerland).

For cytoplasmic membrane permeabilization assay, 3,3’-Dipropylthiadicarbocyanine iodide (DiSC₃(5); Sigma-Aldrich) dye was used. Bacterial culture was diluted in HEPES buffer containing 0.4 μM DiSC₃(5) and 0.1 mM EDTA to a final concentration of 1 × 10⁷ CFU/mL. Bacterial cells of 100 μL were transferred into a 96-well plate and incubated for 30 min at 37°C. The fluorescence was measured immediately after an equal volume of peptide was added at excitation wavelength of 622 nm and emission wavelength of 670 nm.

To determine membrane integrity after peptide treatment, fungal cells were harvested and mixed with peptides at the desired concentration. Melittin, a well-known AMP in bee venom, was used as a positive control. After incubating for 3 h, cells were washed with PBS and were treated with 50 μg/mL PI for 30 min at 4°C. Samples were washed three times with PBS to remove unbound dye from the cells. In case of C. albicans, PI uptake was measured by BD LSRFortessaTM Flow Cytometer (BD Biosciences, San Jose, CA, United States). For PI staining of F. oxysporum, 10 μL of the each sample was observed with a C1Si confocal microscope (Nikon, Japan).

To evaluate the cytotoxic effects of the peptides, hADMSC and HaCaT cells were seeded in 96-well plates at a density of 1 × 10⁵ cells/mL. Different concentrations of the peptides were treated to the cells were incubated for 24 h. Quanti-Max WST-8 Cell Viability assay solution (Biomax, Seoul, Korea) was added to each well, followed by incubation for 1 h in cell incubator. Absorbance was determined at 450 nm using a microplate reader (Molecular Devices), and the relative cell viability was calculated based on the control.

All experiments were conducted in triplicate and the results are expressed as mean ± standard error of the mean (SEM). The statistical significance of the data was evaluated by performing a one-way ANOVA test followed by Tukey’s post-test using GraphPad Prism 9.3.1 (GraphPad Software, La Jolla, CA, United States). p-values of <0.05 were considered statistically significant.

In order to identify novel AMPs, the A. bruennichi transcriptome obtained in a previous study was analyzed (Figure 1A). RNA sequencing followed by de novo assembly was performed to annotate transcripts expressed in the A. bruennichi venom gland. Sequences showing homology with known toxin peptides in the Uniprot database were identified. As a result, TBIU001927 showed high similarity to the U1-lycotoxinLs1k (E-value of 7e-24, 53% identity) and U29-aranetoxin-Av1a (E-value of 5e-55, 76% identity) sequences (Figure 1B). In addition, TBIU029815 was found to have significant homology to the U4-aranetoxinAv1a (E-value of 9e-05, 37% identity) and U4-aranetoxinAv1G sequences (Figure 1C). Sequences that showed homology were spider-derived peptides that were annotated for their toxin activity. Finally, mature 64-mer and 33-mer peptide sequences were identified from TBIU001927 and TBIU029815, respectively, by predicting signal peptides or propeptide regions from precursors.

AMPs are the main components of animal venom and function by protecting the host from infection and aiding in hunting prey. Because TBIU01927 and TBIU029815 were identified as putative toxin peptides expressed in the A. bruennichi venom gland, further in silico analyses were conducted. Secondary structure analysis showed that the C-terminals of both transcripts had an α-helical structure. Accordingly, the segment from each transcript with high sequence homology was secured from the α-helix regions and was named Aranetoxin-Ab2a (AATX-Ab2a) and Aranetoxin-Ab3a (AATX-Ab3a) (Figure 2A). Amino acid composition and physiochemical analysis were subsequently performed. It was calculated that both peptides had a net charge above +2.9 and had helices with a hydrophobic face (Figure 2B). Moreover, the peptides were predicted as AMPs when analyzed with machine learning-based prediction tools (Table 1). As AATX-Ab2a and AATX-Ab3a showed general characteristics of AMP, the synthesis of these peptides was carried out for functional validation.

To confirm the antimicrobial activity of AATX-Ab2a and AATX-Ab3a, colony-forming assays were performed. Common pathogenic bacterial and fungal strains were treated with peptides ranging in concentration from 1 to 64 μM. The peptides inhibited the bacterial growth of Gram-positive strains, B. cereus and S. aureus, in a dose-dependent manner (Figures 3A,B). The colony formation of Gram-negative strains, E. coli and P. aeruginosa, also showed a significant decrease after AATX-Ab2a and AATX-Ab3a treatment (Figures 3C,D). Because the peptides exerted strong antibacterial effects on Gram-negative bacteria, MDR P. aeruginosa (MDR-PA) isolates were also tested against peptides. The CCARM 2007 and CCARM 2095 isolates, which are resistant to multiple antibiotics of the cephalosporin, ureidopenicillin, and quinolone groups, were used in this study. The results showed that MDR-PA CCARM 2007 and CCARM 2095 were susceptible to AATX-Ab2a and AATX-Ab3a, with a complete inhibition being observed at 64 μM (Figures 3E,F).

Next, the anti-fungal effects of the peptides were tested on yeast C. albicans and filamentous fungus F. oxysporum, which are representative animal and plant pathogens, respectively. The growth of the two species was significantly inhibited by peptide treatment, showing 90% inhibition of growth when treated with 16 μM or higher concentration of peptides (Figures 4A,B). The results demonstrated the strong inhibitory effects of AATX-Ab2a and AATX-Ab3a on a broad range of pathogens, including Gram-positive, Gram-negative, MDR-PA, and fungal species. Accordingly, the mechanism of action of the peptides was further investigated.

NPN uptake assays were performed to measure the permeabilization of the outer membrane of Gram-negative bacterial strains. NPN emits strong fluorescent signals in hydrophobic conditions, as is the case when entering the inner portion of the membrane by pore formation. Fluorescence was recorded for 15 min for every 1 min after treating 16 μM of peptides (Figures 5A,B). The results showed a significant increase in fluorescence signal in both E. coli and P. aeruginosa. AATX-Ab2a and AATX-Ab3a induced rapid outer membrane disruption on MDR-PA as well, which was similar to or higher than that of 16 μM melittin treatment.

The fluorescent dye DiSC₃(5) was used to assess the disruption of the bacterial cytosolic membrane. This dye fluoresces when leaked out of cells by membrane depolarization. DiSC₃(5) was stabilized in the cytoplasm before the addition of the peptides. Likewise, treatment with 16 μM AATX-Ab2a and AATX-Ab3a induced an instant spike of signal in all of the bacterial cells (Figures 5C–F). Gram-negative bacteria were more susceptible to the peptides as the fluorescence intensity was similar to that of melittin treatment. Specifically, CCARM 2007 and CCARM 2095 were shown to be more susceptible to peptide treatment than E. coli and P. aeruginosa, showing higher fluorescence intensity. The overall results indicated that the two peptides are potent AMPs that can increase the permeability of both the outer and cytosolic membranes of bacteria.

The effects of the peptides on fungal cell integrity were determined using propidium iodide (PI). PI is a red fluorescent dye that binds to nucleic acids and enters cells only when the plasma membrane is damaged. C. albicans was incubated with either PBS or 16 μM of AATX-Ab2a, AATX-AB3a, or melittin for 2 h, followed by flow cytometry analysis (Figure 6). Although almost every cell in the control group showed an intact membrane, 14.75% of C. albicans cells were stained with PI when treated with melittin. AATX-Ab2a and AATX-Ab3a treatments increased the proportion of PI-positive cells to 21.03 and 17.31%, respectively, indicating that the peptides induced a stronger membrane disruption than that of melittin. The effects of peptides on F. oxysporum were observed with confocal microscopy. After incubation with the peptides, red fluorescence was observed in both AATX-Ab2a- and AATX-Ab3a-treated groups, which was comparable to that of the melittin-treated group (Figures 7A,B). The overall results indicated that AATX-Ab2a and AATX-Ab3a interact with both bacterial and fungal membranes, increasing membrane permeability and causing cell damage.

In order to be used as antibiotic agents, candidate molecules should have low toxicity toward human cells and tissue. Thus, cell viability assays were performed to determine whether AATX-Ab2a and AATX-Ab3a are toxic to human cells. Immortalized human epithelial keratinocytes and human primary mesenchymal stem cells were used in this study. The cells were treated with AATX-Ab2a and AATX-Ab3a for 24 h, and cell viability was measured using WST-8. In both cell lines, no significant cytotoxicity was observed up to a concentration of 16 μM, and cell viability was maintained above 80% at 32 μM (Figure 8). AATX-Ab2a showed a stronger effect on cell viability than AATX-Ab3a at the highest concentration of 64 μM. It was demonstrated that AATX-Ab2a and AATX-Ab3a exhibited low cyto-toxicity to normal cells at concentrations with effective antibacterial and antifungal activities.