Identification of antimicrobial peptides from the Ambystoma mexicanum displaying antibacterial and antitumor activity

Nadjib Dastagir; Christina Liebsch; Jaqueline Kutz; Sabine Wronski; Andreas Pich; Doha Obed; Peter Maria Vogt; Vesna Bucan; Sarah Strauß

doi:10.1371/journal.pone.0316257

. 2025 Mar 5;20(3):e0316257. doi: 10.1371/journal.pone.0316257

Identification of antimicrobial peptides from the Ambystoma mexicanum displaying antibacterial and antitumor activity

Nadjib Dastagir ^1,^*, Christina Liebsch ¹, Jaqueline Kutz ¹, Sabine Wronski ², Andreas Pich ³, Doha Obed ¹, Peter Maria Vogt ¹, Vesna Bucan ^1,^☯, Sarah Strauß ^1,^☯

Editor: Haitham Abo-Al-Ela⁴

PMCID: PMC11882074 PMID: 40043049

Abstract

Antibiotic resistance is a significant healthcare concern. Therefore, identifying target molecules that can serve as antibiotic substitutes is crucial. Among the promising candidates are antimicrobial peptides (AMPs). AMPs are defense mechanisms of the innate immune system which exist in almost all living organisms. Research on the AMPs of some amphibians has shown that, in addition to their antimicrobial effectiveness, AMPs also exhibit anti-inflammatory and anti-carcinogenic properties. In this study, we identify and characterize AMPs deriving from the skin mucus of the axolotl (Ambystoma mexicanum). Upon activity spectrum evaluation of the AMPs, we synthesized and ranked 22 AMPs according to antimicrobial efficacy by means of a prediction tool. To assess the AMPs’ potential as antibacterial and anticarcinogenic compounds, we performed a minimum inhibitory concentration (MIC) assay for efficacy against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA), and an apoptosis assay on T-47D mammary carcinoma cells. We identified four AMPs that showed significant inhibition of MRSA, of which three also demonstrated anticarcinogenic activity. Gene expression analysis was performed on AMP-stimulated carcinoma cells using a breast cancer-specific RT-PCR array. In cells stimulated with the AMPs, gene expression analysis showed upregulation of tumor suppressor genes and downregulation of oncogenes. Overall, our work demonstrates the antimicrobial and anticarcinogenic activity of axolotl-derived AMPs. The results of this work serve as a basis to further investigate the mode of action and potential use of axolotl AMPs as therapeutic anticancer or antibiotic agents.

Introduction

Antibiotic resistance is a global public health problem associated with increased mortality and morbidity [1]. The ability of bacteria to rapidly evolve and become resistant to conventional antibiotics creates an urgent need to identify new targets that can be used as substitutes [2]. One possible solution could be the use of antimicrobial peptides (AMPs), also known as host defense peptides (HDPs). AMPs are part of the innate immune response and are found in many species across the phylogenetic kingdoms [3]. These peptides function as “endogenous antibiotics” with particular importance in the early defense against bacteria, as well as viruses, fungi, and parasites [4–6]. AMPs are either constitutively expressed or produced in response to infection and inflammatory stimuli. They consist of sequences less than 100 amino acids long with a net positive charge [7].

Similar to the complement system, AMPs act primarily through mechanisms involving membrane disruption, reducing their risk of developing multidrug resistance, and are capable of inhibiting some antibiotic-resistant microorganisms [8,9]. Although AMPs share common properties, such as those mentioned above, they differ in sequence and activity [10]. In addition to their crucial role in inflammatory reactions, immune system activation, and wound healing, they also have an anticarcinogenic effect [11,12]. It is noteworthy that numerous studies have demonstrated the significant selective anticarcinogenic activity of many AMPs, which can also be referred to as anti-cancer peptides (ACPs) [13].

Because AMPs are naturally occurring across many species, many groups have investigated how AMPs isolated from amphibians and arthropods, considered to be organisms with very strong innate immune systems, respond to bacteria [3,14]. In Anurans (frogs) such as Bombina variegata, Phyllomedusa sauvagii and Xenopus laevis, various skin peptides have already been described and tested for their antimicrobial activity [15]. Studies investigating the role of AMPs isolated from Xenopus laevis frogs found synergistic modes of action against bacterial strains by forming transmembrane pores, thus resulting in a more powerful inhibition of the bacteria [7,16,17].

Studying the characterization of antimicrobial peptides (AMPs) from amphibians and arthropods can potentially lead to the identification of new target molecules for developing therapies against antibiotic resistance. It is noteworthy that the characterization of AMPs from the axolotl (Ambystoma mexicanum) has not yet been explored. This presents a promising opportunity for further research in this area. The innate immune system of axolotls is particularly effective, while their adaptive immune response is weak [18]. The animals produce only two classes of immunoglobulins: IgM and IgY [19]. Thus, the mucus and the nonspecific immune system of axolotls are their main defense mechanism against pathogens. In this regard, antimicrobial peptides in the mucus, neutrophils, and macrophages are mainly responsible for the immune defense [20]. Based on this dependence on the innate immune system, we hypothesized that the AMPs produced by axolotls are particularly effective antimicrobial agents. Amphibian antimicrobial peptides (AMPs) primarily adopt a linear structure and fold into an amphipathic helix that binds to the membrane [21]. These peptides are typically sourced from the skin glands of the animals and are secreted during stress or injury [22,23].

Numerous studies have demonstrated that several antimicrobial peptides (AMPs) exhibit exceptional antitumor activity in vitro and in vivo, particularly against breast cancer and lung cancer [24,25]. AMPs are natural agents that hold great promise as a novel class of anticancer drugs [26,27]. Conventional chemotherapeutic agents have weaknesses, such as lack of selectivity for tumor cells, which can easily lead to severe side effects, and induce tumor cell resistance [28]. AMPs, on the other hand, offer considerable advantages in the fight against tumors, such as a broad spectrum of antimicrobial and antitumor effects, high selectivity for cancer cells, safety for normal cells and vital organs and low resistance formation [29,30].

The selective membrane-destroying effects are similar to the bactericidal effects: The cationic AMPs directly interfere with the membrane of cancer cells by electrostatic attraction to form temporary pores and damage the integrity of the cell membrane, ultimately leading to cell death [31]. The non-membranolytic effects of AMPs include destruction of the cytoskeleton of cancer cells, inhibition of DNA and protein synthesis, inhibition of tumor angiogenesis, immune regulation and induction of apoptosis or tumor cell necrosis [32,33].

Studies indicate that amphibians may have a higher resistance to cancer, despite their high cell proliferation [34,35]. Reasons for this have not yet been described in the literature, however, AMPs might be attributed to this observation. Certain amphibian AMPs were found to have anticarcinogenic activities. Magainins, aureins, cytropins, and peptides of brevinin-1, ranatuerin-2, temporin, and peptides of the dermaseptin family show anticarcinogenic activity at doses that exhibit little or no toxicity against regular mammalian cells [23].

Among the best studied “antitumor peptides” are the ionophore magainins from X. laevis [36]. Magainin 2 and its synthetic analogs kill a wide range of cancer cell lines including lung, breast, and bladder cancer cells, as well as cells from lymphomas, melanomas, and leukemias [23]. The anticarcinogenic effect of Magainin 2 has also been demonstrated in vivo using subcutaneous xenografts in nude mouse models [23], suggesting AMPs isolated from amphibians have the potential to recapitulate their activities in mammalian models.

In this work, we characterize AMPs deriving from the skin mucus of the axolotl and use synthesized analogs to assess the potential of these AMPs as antibacterial and anti-cancer therapies. The aim of our study is to demonstrate the use of axolotl-derived AMPs in inhibiting MRSA, as well as their translational anticarcinogenic activity in mammalian tumor cell lines. This study identified antimicrobial peptides (AMPs) that have potential as both anticancer agents and for future research on antibiotic resistance.

Materials and methods

Animal husbandry and mucus harvesting

The axolotls used in this study were originally obtained from the Ambystoma Genetic Stock Center (AGSC) at the University of Kentucky. The AGSC maintains a colony of axolotls and provides them to research laboratories worldwide, along with housing, breeding and care information. All experiments were performed in accordance with the guidelines of the German Animal Welfare Act. Due to these guidelines and after consultation of the veterinary inspection office as well as the “Landesamt für Lebensmittelsicherheit und Verbraucherschutz” (LAVES, relevant authority for animal trials in lower Saxony, Germany) mucus harvesting from the animals was classified as non invasive and therefore not requiring a permission. All methods are reported in accordance with ARRIVE guidelines. The axolotls were kept in tap water and fed a specialized diet (Axobalance; AquaTerratec, Bröckel, Germany) twice a week. The animals were housed in groups of 3 to 5 in fully equipped tanks (ground substrate, hiding places, artificial plants, filter system) without artificial illumination. Circadian rhythm was given by common daylight without direct exposition to sunlight through windows. Keeping temperature ranged from 12°C in winter to max. 20°C in summer. To obtain the skin mucus containing the AMPs to be examined, axolotls were gently massaged with sterile nitrile gloves, and the produced mucus was collected with sterile scrapers (Fig 1).

Fig 1 — Axolotls were gently massaged with nitrile gloves to collect skin mucus. AMPs were then isolated from the mucus by liquid chromatography-mass spectrometry. The identified peptide sequences were ranked according to their predicted antimicrobial activity using bioinformatics tools, and the 22 most promising sequences were synthesized.

Mass spectrometric analysis and peptide synthesis

The chromatography and mass spectrometric analysis were conducted using an AXIMA Performance MALDI-TOF/TOF mass spectrometer (Shimadzu) at the Institute for Toxicology in Hannover. Skin secretion samples were processed by mass spectrometry and analyzed by the Proteomics Facility of Hannover Medical School. Fractions were dissolved in Tris-buffer or in 30% acetonitrile and 0.1% TFA. The contained peptides were subsequently loaded onto a C-18 analytical column (Vydac 238 TP, 150 × 4.6 mm) and solubilized by slowly increasing the acetonitrile concentration up to 70% over a time of 120 minutes at 1 mL/min flow rate. The absorbance of the eluted solution was measured at 214nm and 280nm. The obtained fractions were analyzed by liquid chromatography-mass spectrometry. For the chromatography, standard injection volumes of 5 μL were used. The obtained fractions were subsequently analyzed by liquid chromatography-mass spectrometry. Peptide identification was carried out using the instrument’s integrated software, which supports fully automated proteomics experiments and LC-MALDI analyses.

We used the CAMP_R3 (Collection of Anti-Microbial Peptides) database and its classifiers (SVM, RF, ANN, and DA) to predict the antimicrobial potential of the identified peptides. We focused on peptides that showed high probability of antimicrobial activity across multiple classifiers. The final 22 peptides were selected based on their consistently high predicted antimicrobial activity and other promising bioactive properties. The 22 most promising sequences were synthesized by the commercial supplier Caslo Laboratory (Denmark). The purity of the peptides was also determined by Caslo using high performance liquid chromatography (HPLC). The peptides were named 1 to 22 according to their ranking in the prediction tool. 1 represents the peptide with the highest predicted efficacy, 22 the one with the lowest included in the experiment. The synthetically produced peptides were lyophilized and reconstituted in DMSO (Merck SA, Germany) acetic acid (Roth, Germany), water (Merck Millipore, Germany) or Tris-buffer (Merck SA, Germany) depending on their solubility.

Screening of antimicrobial activity

MIC assays (MRSA, ATCC# 43300/ MSSA, ATCC# 6538) were performed according to CLSI guidelines. (Committee for Clinical Laboratory Standards Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Tenth Edition: Approved Standard M7-A10. NCCLS, Wayne, PA, USA, 2015.) In short, 96-well plates were inoculated with 100μL of bacterial suspension and 100μL of individual peptides or appropriate diluent (DMSO, acetic acid, water or Tris-buffer depending on peptide solubility) as a negative control, and samples were cultured under static conditions with 100% humidity, 37°C and 0% CO₂ for 24 h. Thereafter, samples were treated with peptide concentrations ranging from 10, 5, 2.5, 2, 1.75, 1.5 1.25, 1, 0,85, 0.625 and 0.313 µg/mL. Human antimicrobial peptide LL-37 was utilized as a positive control at a concentration of 256 μg/mL, as it is one of the best-known examples of AMP-based drug development. The assay was performed in triplicates. Efficacy was assessed according to the viable bacterial load determined by dilution plating and counting of Colony Forming Units (CFU). The lowest concentration that inhibited bacterial growth after 24 h of incubation is the MIC. Growth curve were performed using a plate reader (Synergy 2, Biotek).

Cell culture

The human breast cancer cell line T-47D (HTB-133) was purchased from ATCC. Cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific catalog number 11875093) supplemented with 0.2 units/mL insulin and 10% fetal calf serum (FCS; catalog number 16000044,). The control cell line MCF10A (ATCC, Rockville, MD, USA) was cultured in DMEM/F12 medium (Gibco) supplemented with 5% horse serum (Sigma), 100 ng/ml cholera toxin (Sigma), 20 ng/ml epidermal growth factor (Sigma), 0.01 mg/ml insulin (Sigma) and 500 ng/ml hydrocortisone (Sigma). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. Possible apoptotic effects as a result of the diluent were accounted for using a negative control of the appropriate diluent (DMSO, acetic acid, water, or Tris-buffer).

Apoptosis assay

The apoptosis assay was performed using the Apo-ONE® Homogeneous Caspase-3/7 Assay Kit according to the manufacturer’s instructions (Promega). Briefly, T47D and MCF10A cells were seeded on 96-well plates. Cells were counted using the CountessTM II Automated Cell Counter (Invitrogen). In total, 1x10⁶ cells were treated. After 24 hours, the medium was replaced with fresh medium, and the 22 peptides were added to individual wells at 1, 2.5, 5, and 10 µg/ml. One plate was left without peptide stimulation as a control. After 24 hours of incubation, 79 µL of Caspase Substrate Z-DEVD-R110 (100X) was added to 7.9 mL of Apo-ONE® Homogeneous Caspase-3/7 Buffer, 100 µL of this reagent was added per well. After 60 minutes, fluorescence was measured using the Tecan microplate reader excitation wavelength range of 485 ± 20nm and an emission wavelength range of 530 ± 25nm. (Tecan Group, Schweiz). A Student’s t-test was used to compare the two groups.

Antitumor activity gene expression assay

Three 75 cm² cell culture flasks containing the T-47D and three 75 cm² cell culture flasks containing the MCF10A cells were individually stimulated with peptides 1, 12, or 13 at a concentration of 10 µg/mL. An unstimulated cell culture flask was left as a control. Cells were incubated for 24 hours according to standard protocol at 37°C and 5% CO2, in a humidified atmosphere.

After 24 hours, the cells were detached, and RNA was isolated according to the Macherey-Nagel kit. RNA quality was assessed using a NanoDrop 1000 spectrophotometer and gel electrophoresis. To create cDNA the iScript cDNA Synthesis Kit (BioRad) was used and followed according to manufacturer’s instructions. For gene expression analysis, the Human Breast Cancer RT2 Profiler PCR array was used (Qiagen) according to manufacturer’s instructions. On this plate, forward and reverse primers were already included in each well for the respective genes. The qPCR cycling conditions were as follows: 8.5 min at 95°C, 40 min at 55°C, 30 min at 72°C and 4°C as holding temperature. Housekeeping gene stability was validated, and results were geometrically averaged. Beta (β)-actin was used for endogenous controls. Expression of carcinogenic markers was quantified relative by the ΔΔCt method and results were normalized to housekeeping genes (Livak and Schmittgen, 2001). The Ct values of the untreated samples were set to zero. The Ct values of the MCF10A samples were subtracted from the Ct values of the T-47D samples. Relative gene expression was calculated from the zero line using Microsoft Excel Version 2016 software (Microsoft Cooperation, Redmond, WA, USA). The resulting higher or lower expressions of the analyzed genes were presented. Gene expression was measured using the BioRad i-Cycler.

Statistics

Statistical comparisons between groups were performed using an independent t-test, with all analyses conducted using GraphPad Prism 9.2.0. Differences were considered statistically significant at p < 0.05, with significance levels denoted as follows: * p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Results

Mass spectrometric analysis

Mass spectrometric analysis of the fractionated axolotl skin mucus revealed 4,986 peptide sequences. These sequences were analyzed for the probability of antimicrobial activity using the CAMP_R3 Collection of Anti-Microbial Peptides prediction tool. We selected the 22 most promising peptide candidates based on the calculated probabilities (Table 1, ranked in order of highest to lowest predicted efficacy). BLASTp analysis revealed no significant sequence homology for peptides 1 and 12 (E-value > 0.01). Peptide 13 showed weak alignments with AMPDB_42975|A0A2V2ABU9 (E-value = 3.7) and AMPDB_41234|A0A2A2DWC7 (E-value = 7.1), indicating statistically insignificant similarities likely occurring by chance.

Table 1. Amino acid composition and purity of peptide lots P130819-01-01 to P130819-01-22.

Peptid Lot Nr.:	Quantity in mg	Hydrophobic amino acids %	Acidic amino acids %	Alkaline amino acids %	Neutral amino acids %	Purity
P130819-01-01	5.5	57.89	0.00	5.26	36.84	95.16%
P130819-01-02	5:0	78.57	14.29	7.14	0.00	98.50%
P130819-01-03	5.5	47.62	14.29	4.76	33.33	98.72%
P130819-01-04	5.5	63.16	0.00	5.26	31.58	98.62%
P130819-01-05	5.5	58.62	13.79	3.45	24.14	96.25%
P130819-01-06	5.5	60.87	0.00	4.35	34.78	97.20%
P130819-01-07	5.5	41.67	0.00	8.33	50.00	96.70%
P130819-01-08	5.5	71.43	0.00	14.29	14.29	95.57%
P130819-01-09	5.0	50.00	5.00	5.00	40.00	95.35%
P130819-01-10	5.0	56.52	8.70	4.35	30.43	98.12%
P130819-01-11	5.0	45.00	5.00	5.00	45.00	97.91%
P130819-01-12	5.0	45.45	0.00	9.09	45.45	97.00%
P130819-01-13	5.5	57.14	4.76	9.52	28.57	95.35%
P130819-01-14	5.0	52.94	0.00	5.88	41.18	97.88%
P130819-01-15	5.0	52.63	0.00	5.26	42.11	97.35%
P130819-01-16	5.5	50.00	0.00	10.00	40.00	99.36%
P130819-01-17	5.5	52.63	10.53	10.53	26.32	98.22%
P130819-01-18	5.0	50.00	0.00	0.00	50.00	96.95%
P130819-01-19	5.0	50.00	0.00	6.25	43.75	97.00%
P130819-01-20	5.5	50.00	0.00	33.33	16.67	99.39%
P130819-01-21	5.5	28.75	0.00	57.14	14.29	96.96%
P130819-01-22	5.0	16.67	0.00	50.00	33.33	96.79%

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Screening for antimicrobial activity

We evaluated the antimicrobial activity of the synthesized peptides by assessing their MIC against Meticillin-Sensitive Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus (MSSA and MRSA). The results of the growth inhibition test showed that peptide 1 had the greatest inhibitory effect on both MSSA and MRSA (Fig 2A). Inhibitory effects against MSSA were also seen by peptide 1, 2, 13, 7, 8 and 3, in order of lowest to highest MIC. For MRSA, an inhibitory effect was seen by peptides 1, 2, 13 and 7, in order of lowest to highest MIC. The MIC of vancomycin of 2 µg/mL was used as a threshold for identifying inhibitory effects.

Fig 2 — (A) The minimium inhibitory concentration (MIC) of each peptide measured 24 hours after stimulation against methicillin-resistant *Staphylococcus aureus* (MRSA; black) and methicillin-sensitive *Staphylococcus aureus* (MSSA; white). The MIC of vancomycin, 2 µg/mL, was used as a threshold for identifying inhibitory effects (dashed line). (B-D) The antitumor activity as measured by apoptosis assay after 24-hour stimulation with peptides 1, 12, and 13 in increasing doses (1, 5, and 10 g) against MCF10A (normal breast epithelium) and T47-D (mammary carcinoma) cell lines. RFU; Relative fluorescent units. * p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Apoptosis assay

Some AMPs have been reported to have anticarcinogenic effects. To test if the AMPs synthesized in our study had these properties, we performed a dose-dependent apoptosis assay following stimulation of T-47D mammary carcinoma cells. Apoptosis was measured following 24 hours of stimulation. The induction of apoptosis varied among the 22 peptides tested, with peptides 1, 12, and 13 showing the most pronounced effects (Fig 2B–D). While a concentration-dependent trend was observed, the differences were not uniformly significant across all concentrations and peptides. At 10 µg/ml, these three peptides exhibited the highest levels of caspase-3/7 activity, indicating stronger apoptotic induction. A similar trend, though less pronounced, was observed at 5 µg/ml. At the lowest concentration of 1 µg/ml, peptides 1 and 12 still showed increased apoptotic activity compared to the control, while peptide 13’s effect was less distinct at this concentration.

Gene expression analysis

Based on the apoptotic effects observed after stimulation with peptides 1, 12 and 13, we wanted to characterize the expressional changes occurring in the mammary carcinoma cells. Gene expression analysis was performed after 24 hours of peptide stimulation. Following stimulation with Peptide 1 we found a significant decrease in the expression of CCND2, CTSD, and IL6, and no upregulation of the gene panel was observed (Fig 3A). Following stimulation with Peptide 12, there was a significant increase in BRCA1, JUN, NR3C1, RB1, SFN, and XBP1, and significant decrease in CCND2, EGF, GLI1, KRT5, MMP2, and SERPINE1 (Fig 3B). Stimulation with Peptide 13 resulted in a significant increase in expression of BRCA1, BRCA2, NR3C1, SERPINE1, and XBP1, and a significant decrease in expression of IL6 and MMP2 (Fig 3C). Overall, it is noticeable that IL6 was downregulated in all cells stimulated with the peptides. CCND2 was downregulated in cells stimulated with peptide 1 and peptide 12. In contrast, MMP2 was downregulated in cells stimulated with peptides 12 and 13. The cells stimulated with peptide 12 or with peptide 13 had in common that BRCA2, NR3C1, SFN, and XBP1 were upregulated in them.

Fig 3 — Gene expression was measured in T47-D (mammary carcinoma) cells after 24 hours stimulation with (A) Peptide 1, (B) Peptide 12, and (C) Peptide 13. All peptide stimulations were performed at a concentration of 10 µg/mL, compared to an unstimulated control. Genes were considered upregulated or downregulated if the fold change was above 2.

Discussion

Antibiotic resistance is predicted to rise in the future, making it crucial to discover alternative solutions [37].Antimicrobial peptides (AMPs) are promising candidates due to their low risk of developing resistance and broad-spectrum activity against bacteria [7]. We hypothesized that the AMPs produced by the axolotl’s innate immune system possess antibacterial properties. Additionally, despite high levels of cell division, axolotls exhibit a reduced risk of cancer [38]. Therefore, we hypothesized that skin mucus-produced antimicrobial peptides (AMPs) exhibit antitumor activity.

The skin mucus-derived AMPs were initially characterized, and 22 candidate peptides were identified and ranked based on their likelihood of having antibacterial activities (Table 1, Supl. 2). Synthetic analogs of these peptides were tested against MSSA and MRSA bacteria; six peptides (1, 2, 13, 7, 8, and 3) showed significant growth inhibition towards MSSA and four peptides (1, 2, 13, and 7) against MRSA (Fig 2A, Supl.1). Our investigation yielded several antimicrobial peptides (AMPs) exhibiting significant efficacy against resistant bacterial strains. Notably, peptides 1 and 13 demonstrated remarkably low Minimum Inhibitory Concentration (MIC) values. In comparison to previously characterized AMPs, such as LI14 (MIC range: 2-16 μg/mL against methicillin-resistant Staphylococcus aureus), our peptides exhibited comparable or superior antimicrobial activity [39]. Specifically, peptide 1 displayed a MIC of 2 μg/mL against methicillin-resistant Staphylococcus aureus (MRSA), which is lower than the MIC values reported for vancomycin against similar MRSA strains, typically ranging from 0.5 to 2 μg/mL. The membrane-disrupting mechanism of these AMPs, distinct from traditional antibiotics, may contribute to their efficacy against resistant strains and potentially mitigate the risk of induced resistance [40]. Collectively, these peptides are promising candidates for addressing multidrug resistant bacteria and should be further tested against additional bacterial strains for antimicrobial activities. The results against MRSA are particularly clinically relevant, as this strain of bacteria is resistant to methicillin and other antibiotics. The prevalence of MRSA is expected to increase with the overuse of antibiotics in both the healthcare and agricultural industries [41].

In addition to showing useful antimicrobial properties, AMPs have also been reported to selectively target tumor cells [24]. In particular, the peptide magainin 2 and its effect in breast cancer and other cancer cell lines has been extensively studied [41]. The mechanisms driving antitumor activity are not fully understood due to the diversity of AMP structures, however it is thought to be caused by the increased proportion of negatively charged phosphatidylserine present on cancer cells [42].

The study aimed to determine whether any of the peptides had anticancer properties. The impact of AMPs on the apoptosis of breast cancer cell lines T-47D and MF10A was assessed in this study. Peptides 1, 12, and 13 induced significantly increased apoptosis in the cancerous breast tissue cells at all concentrations tested (1µg, 5µg, and 10µg) (Fig 2B). The peptides were observed to specifically target cancer cells without causing cytotoxicity in healthy breast tissue cells (Fig 2B). This indicates that the peptides possess antitumor activity and could be promising candidates for further investigation.

Additionally, upon analyzing the transcriptional changes that occur in these cells after stimulation, we observed a downregulation of several oncogenes and cancer-associated genes, including IL6, MMP2, and CCND2 (Fig 3A–C). IL6 is a so-called pleiotropic cytokine with both tumor-promoting and tumor-inhibiting effects [43]. Interleukin-6 (IL-6) induces an epithelial-mesenchymal transition (EMT) phenotype in human breast cancer cells [44], and direct application of IL-6 to breast cancer cells increases proliferation in estrogen receptor-positive (ER+) cells [45]. Therefore, high levels of IL-6 correlate with a poor prognosis for breast cancer patients [46]. Thus, peptides 1, 12 and 13 may reduce tumor spread and harm cancer cells by down-regulating IL-6.

According to the literature, MMP-2, -3, and -9 may be involved in breast cancer metastasis to the brain [45]. Immunohistochemistry and Western Blots showed significantly higher protein expressions of MMP-2, -3, and -9 in neoplastic brain tissue compared to normal brain tissue [47,48]. Also, gel zymography showed increased MMP-2, and -3 activity in brain metastases [45]. Treatment with the selective MMP inhibitor PD 166793 reduced the development of breast cancer brain metastasis in animals [49]. Thus, downregulation of MMP2 in cells by the peptides could inhibit the metastasis process.

We observed the upregulation of genes associated with positive anti-tumor activity, including NR3C1, BRCA1, BRCA2 and SFN. The glucocorticoid receptor NR3C1 is frequently downregulated in breast tumors, and there is evidence that it plays a role as a tumor suppressor in ER+ breast cancer [50]. NR3C1 was upregulated in cells stimulated with peptides 12 and 13 (Fig 3C,D). The gene products of BRCA1 and BRCA2 are involved in fundamental cellular processes, such as DNA repair and recombination, cell cycle control, and transcription. Mutations in the genes predispose to breast cancer disease [51]. In breast cancer cells, the expression of SFN is silenced by methylations [52]. Thus, the activation of SFN by our identified peptides indicates that the cells are losing their tumor characteristics. Another widely recognized suppressor gene RB1 and is often missing in basal-like breast carcinomas. Its main function is to slow down cell growth [53]. In cells stimulated with peptide 12, RB1 was upregulated.

The peptides upregulate suppressors NR3C1, BRCA1, BRCA2, SFN, and RB1, which may initiate apoptosis processes and growth regulation in breast cancer cells, potentially preventing tumor progression. Additionally, the downregulation of oncogenes and cancer-associated genes, such as IL6, MMP2, and CCND2, may deprive the tumor cell of its growth advantage. These gene-altering peptides could potentially be candidates for breast cancer treatment.

Limitation

In future experiments, the membrane-perturbing effects of the peptides could be analyzed using microscopic imaging. Additionally, necrotic cell death could be assessed through an LDH assay or PI staining, as well as by measuring intracellular calcium levels. Additional experiments are required and will be performed to substantiate our hypothesis. Screening was performed at 37°C. It is possible that some peptides work better at lower temperatures. Thus, it would be useful to perform repeat experiments at a temperature which aligns with the body temperature of axolotls. The animals show robustness to infections up to 20°C. Further assays should therefore be performed in this temperature range. The results would not only provide important information regarding applicability in human medicine, but also explain why axolotls become more susceptible to infectious diseases when kept at excessively high temperatures.

Conclusion

In conclusion, our study identifies AMPs isolated from the skin mucus of the axolotl to exhibit antimicrobial and antitumor activities. This finding supports that these peptides from amphibians can be useful for guiding molecular target discovery. Notably, four peptides found in our study showed significant inhibition of MRSA, and three demonstrated antitumor activity against the T-47D mammary carcinoma cell line. Overall, our findings suggest that these identified AMPs may be promising candidates in the work to address antibiotic resistance and cancer targeting strategies with further validation and studies.

Supporting information

S1 Fig. H PLC chromatograms demonstrating high purity of synthesized compounds. Data tables provide retention times and area percentages for each peak.

(TIF)

pone.0316257.s001.tif^{(14.3MB, tif)}

S2 Fig. M RSA growth at 37°C over 24 hours, comparing the effects of various peptides, Genta, Vanco, and LL-37 on bacterial growth (OD630nm).

(PNG)

pone.0316257.s002.png^{(1.4MB, png)}

S3 Fig. The figure displays sequences of three synthetic peptides.

Peptide 1 (VAVLGASGGIGQPLSLLLK), Peptide 12 (ILLLcVGEAGDTVQFAEYIQK), and Peptide 13 (FGANALLGVSLAVcKAGAAEK).

(TIF)

pone.0316257.s003.tif^{(346.1KB, tif)}

Data Availability

The datasets used and/or analyzed during the current study are, within the manuscript itself, and uploaded as supplementary.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. 2025 Mar 5;20(3):e0316257. doi: 10.1371/journal.pone.0316257.r001

Author response to Decision Letter 0

Transfer Alert

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21 May 2024

PLoS One. doi: 10.1371/journal.pone.0316257.r002

Decision Letter 0

Haitham Abo-Al-Ela

13 Aug 2024

PONE-D-24-20573Identification of antimicrobial peptides from the Ambystoma mexicanum displaying antibacterial and antitumor activityPLOS ONE

Dear Dr. Dastagir,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Additional Editor Comments:

The reviewers have raised several concerns about the study, and we recommend addressing their comments. Additionally, the authors should clearly outline the study's limitations in the manuscript.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

Reviewer #4: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a sound paper describing logical project design and outcomes for the area of antimicrobial peptide activity. The manuscript is clearly written. The results encourage further research in this area. I recommend publication without change.

Reviewer #2: 1. How can the authors demonstrate that a massage on the axolotl's skin is a sufficient stimulus to induce the expression of AMPs? They should clearly define a control group where the mucus normally produced by the axolotls is collected. These samples should then be compared with those obtained after the massage to identify any changes in AMP expression.

2. It is unclear how peptides are identified from the collected mucus. The authors do not mention whether the fractions obtained by chromatography were sequenced; they only reference the use of mass spectrometry.

3. It is not clear how they went from 4986 sequences to 22. What were the parameters used in CAMPr3 to filter the sequences?

4. The authors do not mention the length of the 22 identified peptides, nor do they provide basic properties such as charge or percentage of hydrophobicity.

5. It is unclear why the antimicrobial activity tests involve treating the bacteria first with the crude extract and then with each of the peptides.

6. The authors do not explain the reasons for the selection of the concentrations used in both the antimicrobial and antitumor activity tests.

7. When testing potential molecules for antimicrobial activity, it is important to perform hemolysis tests, as some peptides may have strong antibacterial activity but can also be hemolytic.

8. The authors did not perform any statistical tests to demonstrate that the differences observed between controls and treatments were significant.

9. Kinetic graphs in antimicrobial activity assays should be plotted using % growth rather than OD. Additionally, the authors do not show the data variability across the three replicates performed.

Reviewer #3: PLOSOne 2024

Comments to the authors

The manuscript presents original research on the identification and characterization of antimicrobial peptides (AMPs) derived from the skin mucus of Ambystoma mexicanum (axolotl). While the study has the potential to contribute valuable insights to the field, several critical aspects need to be addressed to meet the high technical standards and publication criteria of PLOS ONE.

Evaluation Based on PLOS ONE Criteria

1. Original Research: Partially Meets Criteria: The study contributes original findings regarding AMPs. However, the depth and novelty are somewhat undermined by the lack of detailed methodological justifications and comprehensive data presentation.

2. Results Not Published Elsewhere: The manuscript does not indicate prior publication of the results.

3. High Technical Standard and Detailed Description:

Does Not Adequately Meet Criteria:

� Collection and Permissions: The manuscript does not specify the origin of the axolotls or the collection permits. On page 5, line 110, it is mentioned that the axolotls were bred and kept at the AMBC. However, the manuscript should clarify where and how they were originally collected. What was the collection location, and under what license or collection permit were they obtained? This information should be indicated. Include information on the collection of axolotls and relevant permits.

� Chromatography and Mass Spectrometric Analysis Details: The equipment used (brand and model) and the injection volumes are not specified. Page 6, line 125. Authors should specify the chromatography equipment used, injection volumes, and detailed peptide identification methods.

� Peptide Identification: Insufficient details are provided about the identification of the peptide sequences and the bioinformatics tools used and comparisons with existing databases. Page 6, lines 130-133. The authors should attach supplementary information including the spectra of the 22 selected peptides (or at least some examples) from which they identified the peptide sequences by MSMS. Authors should provide comprehensive information on the bioinformatics tools and comparisons with existing databases. Indicate how similar the identified sequences are to those in the databases. Are they novel sequences? What percentage of similarity do they have with those already described for other species? Table 1. In table 1 there are 3 peptides highlighted in yellow and neither the text nor the table indicates why they are highlighted.

� Antimicrobial Assays: The final concentration of the inoculum and the controls for the assays are not indicated. Page 7. Authors should indicate the MIC values in µg/ml and µM. The results are very good, in the discussion the authors should compare the MIC (activity) results with those obtained for other peptides already described in other species against the same resistant strains or compare with current antibiotics and compare their potential.

� Cell Cultures: The source of the RPMI-1640 medium and fetal calf serum is not provided.

� Apoptosis Assay: The number of replicates and the use of a positive control are not mentioned. The value obtained for the negative control is not shown (Page 7 and Figure 2). Figure 2: The figure should include the results of both the negative control (cells treated with the vehicle, without any peptide) and the positive control (cells treated with a known apoptosis-inducing agent, such as a chemotherapeutic drug or a known caspase inducer). The absence of a positive control makes it difficult to compare the apoptotic potency of the peptides with a known standard. The authors should provide a detailed explanation of why a positive control was not used and how this impacts the validity of the results.

� PCR: The forward and reverse primers used are not mentioned (page 8, line 183).

� Data Availability: There is no table or supporting information on the identified sequences, nor is their availability in public databases indicated. Authors should include a table with the identified peptide sequences and ensure their availability in public databases.

4. Data Supporting Conclusions:

Partially Meets Criteria: While the data appears to support conclusions regarding the antimicrobial and apoptotic activity of certain peptides, the lack of complete statistical analyses and insufficient data presentation weakens the robustness of these conclusions.

5. Intelligible Presentation and Standard English:

Meets Criteria: There are no significant language issues reported, although the clarity of data presentation could be improved.

6. Other details

INTRODUCTION

Clarify what MRSA means because it is introduced for the first time.

MATERIALS AND METHODS

Page 7. Line 142 clarify MSSA because it is being introduced for the first time.

Page 7. Line 146 and 160 please correct CO2

Page . 7 line 148. the word "representing" should be omitted.

Page 7 line 157 missing the source of FCS

Page 7 line 155, 164, Figure 2B, and the rest of the document, unify: T-47D/T47D/T47-D

Page 7, line 157, 164 and the rest of the document unify: MCF 10A/MCF10A

Page 8, line 179. Correct "(REF 740955.50)" is missing reference.

Page 8, line 186. It does not make sense to use the abbreviation ACTB because it is not used throughout the work.

Page 8, line 187. Cite the ΔΔCT method.

RESULTS

Page 9, line 200-201 Delete the sentence "These peptides were synthetically produced by the commercial supplier Caslo Laboratory, Denmark" already indicated in materials and methods.

Authors should add a table with the activity results, indicating the MIC values in µg/ml and µM

Page 9 212-217. Figure 2 analysis: According to the analysis of the figure, the statistical comparison is performed between different cell types rather than among the peptide concentrations. Consequently, it is not possible to conclude that the effects on apoptosis are greater at higher concentrations. The authors report that peptides 1, 12, and 13 exhibited the highest induction of apoptosis. However, results for the other 19 peptides are not shown. Additionally, while peptides 1 and 12 are reported to induce apoptosis at low concentrations, no significant difference analysis is observed compared to peptide 13. The authors should address these issues by providing a more detailed statistical analysis and including data for all tested peptides to better support their conclusions.

Page 10, line 217. It mentions that peptides 1 and 12 increased apoptosis at a concentration of 1µg, however in materials and methods it says that they tested concentrations 2.5, 5 and 10µg/ml. Please clarify which is incorrect. And in any case, also correct figure 2.

DISCUSION

The results are very good, in the discussion the authors should compare the MIC (activity) results with those obtained for other peptides already described in other species against the same resistant strains or compare with current antibiotics and compare their potential.

Addressing these points will significantly enhance the manuscript's alignment with PLOS ONE's criteria and increase its likelihood of acceptance for publication.

Reviewer #4: The article "Identification of antimicrobial peptides from the Ambystoma mexicanum displaying antibacterial and antitumor activity" is interesting and deserves publication after several clarifications in the text.

It would be better to select peptides using not one, but several programs (may be for future analysis).

The authors do not provide peptide sequences. You can at least provide those that did not show any activity.

The materials and methods do not say which mass spectrometer was used to analyze the peptides.

The authors of the article write that "Mass spectrometric analysis of the fractionated axolotl skin mucus revealed 4,986 peptide sequences of unknown nature." What is meant by unknown nature? With the help of mass spectrometry, you can determine what kind of molecules these are. The phrase sounds incorrect.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Attachment

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pone.0316257.s004.docx^{(17.4KB, docx)}

PLoS One. 2025 Mar 5;20(3):e0316257. doi: 10.1371/journal.pone.0316257.r003

Author response to Decision Letter 1

19 Oct 2024

We kindly direct your attention to the document titled "Response to Reviewers" for additional details.

Reviewer 1# Thank you for your positive feedback and recommendation for publication without changes. We appreciate your recognition of our project design and outcomes, and we are encouraged by your support for further research in the area of antimicrobial peptide activity.

Reviewer 2#

1. We understand your concern about how massage might affect AMP expression in axolotl skin mucus. However, we respectfully assert that the massage technique does not significantly alter the mucus composition, but rather increases the quantity produced. This method is well-established and non-invasive, primarily stimulating the release of pre-existing secretions stored in granular glands beneath the skin1,2. Collecting "normally produced" mucus without stimulation would yield insufficient quantities for comprehensive analysis, potentially underrepresenting less abundant peptides.

1) Demori, I., El Rashed, Z., Corradino, V., Catalano, A., Rovegno, L., Queirolo, L., ... & Grasselli, E. (2019). Peptides for skin protection and healing in amphibians. Molecules, 24(2), 347.

2) O'Rourke, D. P. (2007). Amphibians used in research and teaching. ILAR journal, 48(3), 183-187.

1. Thank you for your valuable feedback on our peptide identification process from the collected mucus. To clarify, we utilized an AXIMA Performance MALDI-TOF/TOF mass spectrometer (Shimadzu) at the Institute for Toxicology in Hannover for this purpose. The chromatography fractions were analyzed directly by this mass spectrometer, and peptide identification was performed using the instrument's integrated software, which matches experimental spectra to theoretical sequences in protein databases. We have revised the manuscript to include these details for greater clarity and reproducibility of our methods. Thank you for bringing this to our attention.

2. We used the CAMP3 (Collection of Anti-Microbial Peptides) database and its classifiers (SVM, RF, ANN, and DA) to predict the antimicrobial potential of the identified peptides. We focused on peptides that showed high probability of antimicrobial activity across multiple classifiers. The final 22 peptides were selected based on their consistently high predicted antimicrobial activity and other promising bioactive properties. We have revised our manuscript to include more specific details about the selection criteria in this process. For basic properties see table 1.

3. 4. We have included the sequences of three peptides, these are the ones that show significant antimicrobial activity, in Supplementary Table S3.

5. We did not treat the bacteria first with the crude extract and then with each of the peptides. Our antimicrobial activity tests only involved treating the bacteria with individual synthesized peptides.

6. For the antimicrobial tests, we used a range of concentrations (0.5 to 100 μg/mL) based on standard protocols from CLSI [1] and previous studies on similar antimicrobial peptides [2]. This range allowed us to determine the minimum inhibitory concentration (MIC) and observe dose-dependent effects. For the antitumor activity tests, we selected concentrations (0.1 to 50 μM) based on preliminary experiments and literature data on related peptides [3], ensuring we could calculate IC50 values while staying within solubility limits and below highly cytotoxic levels [4,5].

1) Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Tenth Edition. CLSI document M07-A10. Wayne, PA: CLSI; 2015.

2) REW, Sahl HG. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol. 2006;24(12):1551-1557.

3) Mahlapuu M, Håkansson J, Ringstad L, Björn C. Antimicrobial Peptides: An Emerging Category of Therapeutic Agents. Front Cell Infect Microbiol. 2016;6:194.

4) Hoskin DW, Ramamoorthy A. Studies on anticancer activities of antimicrobial peptides. Biochim Biophys Acta. 2008;1778(2):357-375.

5) Mader JS, Hoskin DW. Cationic antimicrobial peptides as novel cytotoxic agents for cancer treatment. Expert Opin Investig Drugs. 2006;15(8):933-946.

7. In future experiments, we will incorporate hemolysis tests to assess the potential toxicity of our antimicrobial peptides to red blood cells. This will allow us to evaluate the therapeutic index of the peptides, ensuring that we identify candidates with strong anticancer activity while minimizing hemolytic effects.

8. We have performed appropriate statistical tests to demonstrate the significance of differences observed between controls and treatments. Specifically, we used student -t test to compare the two groups. The results of these tests have been added to the manuscript (Figure2), including p-values for significant differences.

9. We respectfully maintain that our OD-based kinetic graphs effectively illustrate antimicrobial activity. While % growth offers standardization, OD provides direct insight into growth dynamics. Space constraints in PCR gene illustrations preclude error bar inclusion. However, we'll provide detailed statistical analyses in the method part.

Reviewer 3#

1. The axolotls used in this study were originally obtained from the Ambystoma Genetic Stock Center (AGSC) at the University of Kentucky. The AGSC maintains a colony of axolotls and provides them to research laboratories worldwide, along with housing, breeding and care information. All experiments were performed in accordance with the guidelines of the German Animal Welfare Act. Due to these guidelines and after consultation of the veterinary inspection office as well as the “Landesamt für Lebensmittelsicherheit und Verbraucherschutz” (LAVES, relevant authority for animal trials in lower Saxony, Germany) mucus harvesting from the animals was classified as non invasive.

2. The chromatography and mass spectrometric analysis were conducted using an AXIMA Performance MALDI-TOF/TOF mass spectrometer (Shimadzu) at the Fraunhofer Institute in Hannover. For the chromatography, standard injection volumes of 5 μL were used. The obtained fractions were subsequently analyzed by liquid chromatography-mass spectrometry. Peptide identification was carried out using the instrument's integrated software, which supports fully automated proteomics experiments and LC-MALDI analyses. We have revised the manuscript to include these details. Thank you for bringing this to our attention.

3. The peptide identification process was carried out using the AXIMA Performance MALDI-TOF/TOF mass spectrometer (Shimadzu) at the Fraunhofer Institute in Hannover. For peptide identification, we used the instrument's integrated software, which supports fully automated proteomics experiments and LC-MALDI analyses. Regarding the similarity of the 3 (Peptide 1, 12,13) identified sequences to those in existing databases:

BLASTp analysis revealed no significant sequence homology for peptides 1 and 12 (E-value > 0.01). Peptide 13 showed weak alignments with AMPDB_42975|A0A2V2ABU9 (E-value = 3.7) and AMPDB_41234|A0A2A2DWC7 (E-value = 7.1), indicating statistically insignificant similarities likely occurring by chance.

4. The RPMI-1640 medium used in this study was obtained from Thermo Fisher Scientific (catalog number 11875093). The FBS was Gibco Premium (Performance Plus) FBS, catalog number 16000044, sourced from the United States.

5. The apoptosis assay was performed using the Apo-ONE® Homogeneous Caspase-3/7 Assay Kit (Promega) according to the manufacturer's instructions. While we acknowledge the reviewer's valid points, we can explain our approach as follows:

a) Negative control: The negative control (cells treated with vehicle only) was indeed part of our experiment. However, we did not include this data in Figure 2 for the following reasons:

a. The fluorescence levels observed in the negative control were consistently low, indicating minimal background caspase-3/7 activity.

b. The results for the negative control were similar to those observed in the untreated MCF10A cells, which serve as a non-cancerous cell line control in our study.

b) Positive control: A traditional positive control was not included in this study for the following reasons:a) The Apo-ONE® Assay Kit is designed to be highly sensitive and specific for caspase-3/7 activity, which are key indicators of apoptosis. The assay's reliability has been extensively validated by the manufacturer and in numerous published studies. The peptides themselves serve as test substances, and those showing significant increases in caspase-3/7 activity compared to the negative control can be considered positive results. The use of a traditional apoptosis inducer (e.g., staurosporine) might overshadow the more subtle effects of the peptides, potentially leading to underestimation of their apoptotic potential.

While we understand that a positive control can provide a benchmark for comparison, we believe that the relative increases in caspase-3/7 activity induced by the peptides, compared to the negative control, provide sufficient evidence of their apoptotic potential.

6. The RT2 Profiler PCR Array is a 96-well plate that contains pre-designed primer assays for a focused panel of genes related to a specific pathway or disease. It's important to note that while the specific sequences are not provided, the primers are thoroughly tested and optimized to ensure reliable and reproducible results for pathway-focused gene expression analysis. https://geneglobe.qiagen.com/us/product-groups/rt2-profiler-pcr-arrays/PAHS-131Z

7. The complete sequences of the three most effective antimicrobial peptides (AMPs) identified in this study can be found in the Supplementary (S3) Information.

8. We acknowledge that while a concentration-dependent trend was observed, the differences between concentrations were not always statistically significant for all peptides. This variability highlights the complex nature of peptide-induced apoptosis and suggests that factors beyond concentration, such as peptide sequence and cellular uptake, may influence their effectiveness. We have focused on presenting data for peptides 1, 12, and 13 as they showed the most significant and consistent effects on apoptosis induction. Peptides exhibiting inconsistent apoptotic activity and were excluded from primary figures to emphasize statistically significant results.

Reviewer 4 #

1. Regarding the peptide sequences: We have included the sequences of three peptides, including those that show significant antimicrobial activity, in Supplementary Table S1.

2. Concerning the mass spectrometer information: In the Materials and Methods section, we have add the following sentence:"Peptide analysis was performed using an AXIMA Performance MALDI-TOF/TOF mass spectrometer (Shimadzu) at the Fraunhofer Institute in Hannover."

3. We agree that "unknown nature" was imprecise. We've revised the sentence to:"Mass spectrometric analysis of the fractionated axolotl skin mucus revealed 4,986 peptide sequences."

Attachment

Submitted filename: response to reviewers.docx

pone.0316257.s006.docx^{(24.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0316257.r004

Decision Letter 1

Haitham Abo-Al-Ela

5 Nov 2024

PONE-D-24-20573R1Identification of antimicrobial peptides from the Ambystoma mexicanum displaying antibacterial and antitumor activityPLOS ONE

Dear Dr. Dastagir,

Please submit your revised manuscript by Dec 20 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Partly

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: No

Reviewer #4: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: No

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Reviewer #2: 5. Line 157 " In short, 96-well plates were inoculated with 100μL of bacterial suspension and 100μL of mucus". Based on how it's written on line 157, it can be inferred that they used 100 µL of the crude secretion and not the individual peptides.

8. The authors mention a statistical analysis in the response to previous comments, yet a complete description of these analyses is lacking in the Materials and Methods section. The use of asterisks to indicate significant differences and the inclusion of p-values in figure 2 legends is recommended.

- Line 158: why the negative control is PBS if the peptides are diluted on DMSO, acetic acid, water or Tris-buffer?

- Line 159: missing letter (peptid)

- Line 160: please add the concentration of LL-37 used as a positive control

- Line 175: some solvents can cause apoptosis by it selfs, like DMSO and acetic acid. Are the peptides evaluated diluted on those solvents?

- Lines 271, 273 and 277: use italic letters for the bacteria name.

- Line 276: The authors reference the activity of peptide 12 against Gram-negative bacteria, a discrepancy with the article's primary focus on Gram-positive bacteria. It is unclear if the authors have conducted prior research investigating the peptide's efficacy against Gram-negative organisms.

Reviewer #4: (No Response)

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Reviewer #2: No

Reviewer #4: No

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PLoS One. 2025 Mar 5;20(3):e0316257. doi: 10.1371/journal.pone.0316257.r005

Author response to Decision Letter 2

8 Dec 2024

We appreciate the valuable feedback from Reviewer #2 and have made all the requested changes to improve the clarity and completeness of the manuscript. Below, we provide detailed responses to each of Reviewer #2's comments.

5. We have clarified the wording in line 157 to specify that 100 µL of individual peptides was used alongside the bacterial suspension. This change ensures that it is clear we used the peptides rather than crude secretion.

6. We have expanded the description of the statistical analyses in the Materials and Methods section to provide a complete overview of the methods used. Additionally, we have included the significance levels indicated by asterisks and added p-values in the figure 2 legends as recommended.

Regarding line 158, we have clarified the negative control and specified how it relates to the peptides diluted in DMSO, acetic acid, water, or Tris-buffer.

The missing letter in “peptid” on line 159 has been corrected.

The concentration of LL-37 used as a positive control has been added to line 164.

In response to your concern on line 175 regarding solvents such as DMSO and acetic acid potentially causing apoptosis, we have clarified that all peptides were evaluated at concentrations that are known to be non-cytotoxic when diluted in these solvents.

We have italicized the names of bacteria as per your suggestion on lines 271, 273, and 277.

Finally, regarding line 276, we have taken out the sentence.

Thank you again for your insightful comments, which have helped improve the quality of our manuscript. Feel free to modify any specific details or phrasing as needed!

We thank Reviewer #4 for their thorough review of our manuscript. We appreciate your time and consideration, and we are pleased to note that there were no suggested changes. Your positive evaluation is greatly appreciated.

PLoS One. doi: 10.1371/journal.pone.0316257.r006

Decision Letter 2

Haitham Abo-Al-Ela

10 Dec 2024

Identification of antimicrobial peptides from the Ambystoma mexicanum displaying antibacterial and antitumor activity

PONE-D-24-20573R2

Dear Dr. Dastagir,

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Kind regards,

Haitham Abo-Al-Ela, DVM, MSc, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #2: The authors have made all the required changes to the manuscript based on the reviewer feedback.All reviewer comments have been carefully considered and addressed. The manuscript has been revised accordingly.

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PLoS One. doi: 10.1371/journal.pone.0316257.r007

Acceptance letter

Haitham Abo-Al-Ela

PONE-D-24-20573R2

PLOS ONE

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. H PLC chromatograms demonstrating high purity of synthesized compounds. Data tables provide retention times and area percentages for each peak.

(TIF)

pone.0316257.s001.tif^{(14.3MB, tif)}

S2 Fig. M RSA growth at 37°C over 24 hours, comparing the effects of various peptides, Genta, Vanco, and LL-37 on bacterial growth (OD630nm).

(PNG)

pone.0316257.s002.png^{(1.4MB, png)}

S3 Fig. The figure displays sequences of three synthetic peptides.

Peptide 1 (VAVLGASGGIGQPLSLLLK), Peptide 12 (ILLLcVGEAGDTVQFAEYIQK), and Peptide 13 (FGANALLGVSLAVcKAGAAEK).

(TIF)

pone.0316257.s003.tif^{(346.1KB, tif)}

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Submitted filename: Comments to the authors_PLOSOne_2024.docx

pone.0316257.s004.docx^{(17.4KB, docx)}

Attachment

Submitted filename: response to reviewers.docx

pone.0316257.s006.docx^{(24.2KB, docx)}

Data Availability Statement

The datasets used and/or analyzed during the current study are, within the manuscript itself, and uploaded as supplementary.

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PERMALINK

Identification of antimicrobial peptides from the Ambystoma mexicanum displaying antibacterial and antitumor activity

Nadjib Dastagir

Christina Liebsch

Jaqueline Kutz

Sabine Wronski

Andreas Pich

Doha Obed

Peter Maria Vogt

Vesna Bucan

Sarah Strauß

Roles

Abstract

Introduction

Materials and methods

Animal husbandry and mucus harvesting

Fig 1. Schematic for the isolation procedure of AMPs from axolotl skin secretions.

Mass spectrometric analysis and peptide synthesis

Screening of antimicrobial activity

Cell culture

Apoptosis assay

Antitumor activity gene expression assay

Statistics

Results

Mass spectrometric analysis

Table 1. Amino acid composition and purity of peptide lots P130819-01-01 to P130819-01-22.

Screening for antimicrobial activity

Fig 2. Antibacterial and antitumor activity of axolotl-derived AMPs.

Apoptosis assay

Gene expression analysis

Fig 3. Gene expression analysis of antitumor activity.

Discussion

Limitation

Conclusion

Supporting information

Data Availability

Funding Statement

References

Author response to Decision Letter 0

Transfer Alert

Decision Letter 0

Haitham Abo-Al-Ela

Roles

Author response to Decision Letter 1

Decision Letter 1

Haitham Abo-Al-Ela

Roles

Author response to Decision Letter 2

Decision Letter 2

Haitham Abo-Al-Ela

Roles

Acceptance letter

Haitham Abo-Al-Ela

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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