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Discovery of Ll-CATH: a novel cathelicidin from the Chong’an Moustache Toad (Leptobrachium liui) with antibacterial and immunomodulatory activity

BMC Veterinary Research volume 20, Article number: 343 (2024) Cite this article

Abstract

Background

Cathelicidins are vital antimicrobial peptides expressed in diverse vertebrates, crucial for immunity. Despite being a new field, amphibian cathelicidin research holds promise.

Results

We isolated the cDNA sequence of the cathelicidin (Ll-CATH) gene from the liver transcriptome of the Chong’an Moustache Toad (Leptobrachium liui). We confirmed the authenticity of the cDNA sequence by rapid amplification of cDNA ends and reverse transcription PCR, and obtained the Ll-CATH amino acid sequence using the Open Reading Frame Finder, an online bioinformatics tool. Its translated protein contained a cathelin domain, signal peptide, and mature peptide, confirmed by amino acid sequence. The comparative analysis showed that the mature peptides were variable between the amphibian species, while the cathelin domain was conserved. The concentration of Ll-CATH protein and the expression of its gene varied in the tissues, with the spleen showing the highest levels. The expression levels of Ll-CATH in different tissues of toads was significantly increased post infection with Aeromonas hydrophila. Chemically synthesized Ll-CATH effectively combated Proteus mirabilis, Staphylococcus epidermidis, Vibrio harveyi, V. parahaemolyticus, and V. vulnificus; disrupted the membrane of V. harveyi, hydrolyzed its DNA. Ll-CATH induced chemotaxis and modulated the expression of pro-inflammatory cytokine genes in RAW264.7 macrophages.

Conclusions

This study unveiled the antibacterial and immunomodulatory potential of amphibian cathelicidin, implying its efficacy against infections. Ll-CATH characterization expands our knowledge, emphasizing its in a bacterial infection therapy.

Peer Review reports

Background

Antimicrobial peptides (AMPs) are a renowned group of peptides which are widely prevalent in various plant and animal species. They play a vital role in the organism’s defense against a range of pathogenic microorganisms like bacteria, parasites, fungi, and even viruses [1]. These short, typically cationic, and amphiphilic peptides offer host protection by directly interacting with protein targets found on bacterial membrane, damaging its integrity and resulting in death. With the ability to bypass the bacteria membrane, AMPs can then bind to intracellular targets and induce cell damage [2, 3]. In addition to their antimicrobial effects, recent research has highlighted the immunomodulatory functions of AMPs, including their ability to promote bacterial clearance [4, 5].

A class of AMPs called cathelicidins, are found only in vertebrates and they were first isolated from bovine neutrophils [6]. Since then, there have been a rise of discoveries of cathelicidins in various species, including fish [7, 8], amphibians [9], reptiles [10], birds [11, 12], and mammals [13]. Cathelicidins form part of the innate immune system of the host, which are made up of a signal peptide at the N-terminal, a cathelin domain that is conserved, as well as an antimicrobial domain towards the C-terminal. The latter varies in length and sequence [14]. Upon proteolytic cleavage from its C-terminal, cathelicidins are able to exhibit their broad-spectrum antimicrobial activity [15]. Many studies have delved in depth on the potent and effective broad-spectrum antimicrobial activity against various microorganisms [16,17,18]. Furthermore, they offer a wide range of immunomodulatory functions such as promoting wound healing, apoptosis, chemoattraction, and activation of immune cells [19,20,21,22].

In amphibians, numerous homologs of cathelicidins have been discovered in different families such as Lf-CATH1 and 2 from the Fragile Wart Frog, Limnonectes fragilis [23], cathelicidin-PP from the Puer Treefrog, Zhangixalus puerensis [9, 17], and Cathelicidin-DM from the Asian Black-spined Toad, Duttaphrynus melanostictus [24]. Studies conducted on amphibians cathelicidin reveal their vast antimicrobial activities, for instance, synthetic Lf-CATH1 and 2 displayed potent antimicrobial activity against a broad spectrum of drug-resistant strains of microorganisms including a number of clinical drug-resistant strains [23]. Despite the intense focus on antimicrobial activities in amphibian cathelicidin, the host immunoregulatory function of amphibian cathelicidins has received limited attention and remains largely unexplored.

Thus, our study aims to provide a comprehensive approach to identify, characterise, and chemically synthesise a novel amphibian cathelicidin named Ll-CATH which is derived from the Chong’an Moustache Toad, Leptobrachium liui (Pope, 1947) (Anura: Megophryidae). This anuran species is highly endemic as they are only found in streams located in the mountainous regions of eastern China. Furthermore, our investigation aims to evaluate both the antibacterial activities and immunomodulatory properties of Ll-CATH in providing an effective shield against foreign pathogens.

Results

Identification and characterization of Ll-CATH

We performed a BLAST search using MEGA software and identified the L. liui cathelicidin cDNA sequence from the liver transcriptome. It is currently deposited in the GenBank database under the accession number OQ411022. The cDNA consists of an open reading frame measuring 516 nt, which is predicted to encode a 171-amino acid polypeptide. The estimated molecular weight of the polypeptide is 19.5 kDa, and it has an isoelectric point of 9.08. Analysis of the sequence revealed that the polypeptide is composed of a signal peptide (1–20 aa), a cathelin domain (21–136 aa), and a mature peptide (137–171 aa) (Fig. 1). The mature peptide of Ll-CATH has an estimated molecular weight of 3.9 kDa and an isoelectric point of 9.99. When compared to other amphibian cathelicidins, the amino acid sequence alignment shows high variability in the functional mature peptide, while the cathelin domain remains conserved. Furthermore, the cathelin domain of cathelicidins contains four conserved cysteines (Fig. 1).

Fig. 1
figure 1

Multiple sequence alignments of Ll-CATH amino acid sequences with other amphibians. The shading threshold is set at 60%. Gray shading represents similar residues, black indicates identical residues, and hyphens (-) represent alignment gaps. The signal peptide, cathelin domain, and mature peptide are indicated above the alignment. The predicted cleavage site for the signal peptide or mature peptide is marked with a downward arrow (↓). The four conserved cysteine residues are denoted by an asterisk (*)

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The phylogenetic tree analysis revealed that Ll-CATH amino acid sequence is classified within the amphibian cathelicidin cluster, with its closest relative being the cathelicidin from Xenopus laevis (Fig. 2).

Fig. 2
figure 2

Phylogenetic analysis of cathelicidin amino acid sequences using the neighbor-joining method. Fork values represent the percentage of trees supporting each grouping after bootstrapping (1000 replicates; shown only when > 60%). Scale bar indicates the number of substitutions per base

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Tissue-specific patterns of protein concentration and gene expression

To assess the tissue-specific distribution of cathelicidin, its protein concentration was determined by enzyme-linked immunosorbent assay (ELISA), while gene expression levels were analyzed using reverse transcription (RT)-real-time quantitative PCR (qPCR). The concentrations of cathelicidin varied significantly among the seven tissues examined. The concentration of cathelicidin in L. liui exhibited tissue-specific variation. The highest mean cathelicidin concentration was observed in the spleen, lung, and skin, while the lowest was found in the heart (Fig. 3A). Moreover, the expression of Ll-cath, the gene encoding cathelicidin, showed distinct tissue-specific patterns with varying levels of expression. The spleen exhibited the highest expression, followed by the lung and skin, while the heart displayed the lowest expression. Notably, the expression level in the spleen was approximately 335.9-fold higher compared to that in the heart (Fig. 3B).

Fig. 3
figure 3

Tissue-specific patterns of (A) cathelicidin concentration and (B) Ll-cath expression level in different tissues of healthy Leptobrachium liui. The Ll-cath expression levels were normalized to the Ll-gapdh. Means with diferent letters differ signifcantly (Tukey’s post hoc test, P < 0.05, a > b > c > d > e)

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Alterations in Ll-cath expression post Aeromonas hydrophila infection

At 12 h post-infection (hpi) with A. hydrophila, a significant upregulation of the Ll-cath was observed in all examined tissues. The intestine exhibited the highest level of upregulation, with a fold-change of 20.0, while the kidney displayed the lowest level of upregulation, with a fold-change of 2.1 (Fig. 4).

Fig. 4
figure 4

Changes in Ll-cath expression following Aeromonas hydrophila infection. The Ll-cath expression levels were normalized to Ll-gapdh. ** P < 0.01, *** P < 0.001

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In vitro antibacterial activity of Ll-CATH.

Ll-CATH demonstrated varying degrees of antibacterial activity, with the most potent activity observed against Vibrio vulnificus, displaying a minimal inhibitory concentration (MIC) of 3.75 μg/mL. The MIC values for Staphylococcus epidermidis and Vibrio harveyi were 12.5 μg/mL and 50 μg/mL, respectively. For Proteus mirabilis and Vibrio parahaemolyticus, the MIC values were 100 μg/mL. However, Ll-CATH did not exhibit significant bactericidal activity against Escherichia coli, Salmonella enterica, A. hydrophila, Listeria monocytogenes, and Streptococcus iniae (Table 1).

Table 1 The MIC of Ll-CATH in antibacterial assay
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Impact of Ll-CATH on V. harveyi cell membrane integrity and genomic DNA (gDNA)

The lactate dehydrogenase (LDH) assay revealed that Ll-CATH exerted a detrimental effect on the integrity of the V. harveyi cell membrane. The greatest impact was observed at a concentration of 50 μg/mL, resulting in a 1.61-fold increase in the release of LDH compared to the control group (Fig. 5A). To assess the influence of Ll-CATH on DNA hydrolysis, V. harveyi gDNA was treated with Ll-CATH and subsequently analyzed using agarose gel electrophoresis. The results indicated that incubation with either low or high concentrations of Ll-CATH led to the disappearance of gDNA bands (Fig. 5B, Fig. S1).

Fig. 5
figure 5

Impact of Ll-CATH on Vibrio harveyi cell membrane integrity and genomic DNA (gDNA). A Influence of Ll-CATH on V. harveyi cell membrane integrity. Bovine serum albumin (BSA) served as a negative control. LDH release was presented as the fold-change relative to the negative control, which was assigned a value of 1. B Hydrolytic activity of Ll-CATH on bacterial gDNA. gDNA was subjected to electrophoresis on a 1.0% agarose gel. BSA was utilized as a negative control. The original gel electrophoresis image is shown in Fig. S1. ** P < 0.01, *** P < 0.001

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Impact of Ll-CATH on RAW264.7 cells viability.

The CCK-8 assay results revealed that Ll-CATH exhibited no cytotoxic effects on RAW264.7 cells and did not alter cellular viability. Interestingly, Ll-CATH demonstrated a notable promotion of cell proliferation in RAW264.7 cells at concentrations of 1.0 μg/mL and higher (Fig. 6).

Fig. 6
figure 6

Impact of Ll-CATH on RAW264.7 cell viability. The viability of RAW264.7 cells was evaluated using the CCK-8 assay after treatment with different concentrations of Ll-CATH (0.1, 1.0, and 10.0 μg/mL) for 24 h. Bovine serum albumin (BSA) was utilized as a negative control. * P < 0.05, ** P < 0.01

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Effect of Ll-CATH on chemotaxis of RAW264.7 cells

To investigate the potential effect of Ll-CATH on chemotactic activity, an in vitro transwell migration assay was conducted using RAW264.7 cells. The findings demonstrated that Ll-CATH effectively stimulated the chemotaxis of RAW264.7 cells. In the Ll-CATH treatment groups (0.1, 1.0, and 10.0 μg/mL), the percentage of migrated cells increased by 1.27-, 3.04-, and 5.78-fold, respectively, compared to the bovine serum albumin (BSA) treatment group (Fig. 7).

Fig. 7
figure 7

Effect of Ll-CATH on RAW264.7 cell chemotaxis. A Microscopy results. B Migration index. * P < 0.05

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Effect of the Ll-CATH on cytokine gene expression in RAW264.7 cells

We examined the potential influence of Ll-CATH treatment on the expression of cytokine genes in RAW264.7 cells. Following an 8-h treatment with 10.0 μg/mL Ll-CATH, the tnf-α expression in RAW264.7 cells exhibited a significant downregulation compared to the BSA-treated control group (Fig. 8A). Similarly, after an 8-h treatment with 0.1 μg/mL Ll-CATH, the il-1β expression in RAW264.7 cells demonstrated a significant downregulation compared to the BSA-treated control group (Fig. 8B).

Fig. 8
figure 8

Effect of Ll-CATH on (A) tnf-α and (B) il-1β expression in RAW264.7 cells. RAW264.7 cells were treated with 1.0 μg/mL Ll-CATH for 8 h. The expression of cytokine genes was assessed using qPCR, and transcript levels were normalized to 18S rRNA. * P < 0.05, ** P < 0.01

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Discussion

Cathelicidins are a family of AMPs found in many vertebrate species including humans, and are relatively understudied in amphibians. In this study, we successfully identified cDNA sequences of a cathelicidin gene in L. liui. From our results, the predicted amino acid sequence of Ll-CATH encompassed a signal peptide, a cathelin domain, and a mature peptide. The cathelin domain encompasses four conserved cysteines which forms two disulfide bonds, a consistent trait with other amphibian cathelicidins described in previous studies [25, 26]. Furthermore, phylogenetic analysis indicated that Ll-CATH shows the closest relationship to cathelicidin from X. laevis. In different species of amphibians, the amino acid sequences of cathelicidins show high variability and are species-specific [17]. Cathelicidins can play a huge role in taxonomic classification and even for molecular phylogenetic analysis.

We note that Ll-cath demonstrated constitutive expression in various healthy tissues of L. liui, with higher expression levels observed in the spleen and lung. These findings were consistent with previous studies conducted on cathelicidins in A. loloensis [27], L. fragilis [23], and D. melanostictus [24]. Additionally, our results demonstrated a consistent correlation between the expression levels of Ll-cath and the concentrations of the corresponding peptides across different tissues in L. liui. The similar result also existed on LEAP2 in the previous study of this species [28]. Meanwhile, in line with other studies [17, 29], our results indicated a significant upregulation of Ll-cath expression in all tested tissues following infection with A. hydrophila. For example, cathelicidin-PP expression in Z. puerensis exhibited significant upregulation in the skin, spleen, lung, and gut upon E. coli infection [17]. These collective findings strongly suggest the crucial role of cathelicidins in the innate immunity of amphibians.

Cathelicidins have been previously recognized for their wide-ranging antimicrobial activity [17, 25, 27, 29,30,31]. For example, cathelicidin-RC1 extracted from L. catesbeianus exhibits powerful antimicrobial properties against 38 bacterial strains and 10 fungal species [25]. In this study, chemically synthesized Ll-CATH demonstrated substantial antibacterial efficacy against P. mirabilis, S. epidermidis, V. harveyi, V. parahaemolyticus, and V. vulnificus. It was observed that Ll-CATH exhibits antimicrobial activities against marine bacteria, a phenomenon also observed in the Chinese tiger frog (Hoplobatrachus rugulosus) [16]. The cell membranes of marine bacteria typically consist of a high concentration of negatively charged molecules, such as phosphate and sulfate [32]. The cationic structure of the amphibian cathelicidin enables it to effectively interact with these negatively charged molecules, resulting in the disruption of the bacterial cell membrane and subsequent bactericidal activity. Evaluation through the LDH release assay revealed that Ll-CATH induced an increase in LDH release in V. harveyi, indicating impairment of the bacterial cell membrane integrity. Similar observations of membrane damage through LDH assays and electron microscope analyses have been reported for other anuran-derived cathelicidins [16, 17, 25, 27, 33]. When the cell membrane is disrupted, intracellular contents will leak out and ultimately result in the bacterial death [34]. Additionally, certain AMPs, such as Hoplobatrachus rugulosus cathelicidin [16] and P. nigromaculatus brevinin-2 [35], have been found to hydrolyze bacterial DNA. In this study, Ll-CATH demonstrated a significant ability to hydrolyze bacterial gDNA, thus making it a powerful protector in the host immune system.

The immunomodulatory activity of cathelicidins has been extensively documented in the animal kingdom. These cathelicidins exhibit rapid, potent, and effective properties against a wide range of pathogens [16, 36, 37]. However, the understanding of their effects in amphibians remains limited. In our study, we demonstrate that Ll-CATH can influence chemotaxis in RAW264.7 cells, aligning with the findings observed in the fish cathelicidin [38]. We hypothesized that cathelicidins, similar to chemokines, play a part in recruiting immune cells to infection sites. Pro-inflammatory cytokines such as tnf-α and il-1β play a crucial role in initiating the innate immune response and reflect cell activation in macrophages [39, 40]. However, excessive levels of these cytokines can potentially harm the host [41]. Notably, our study revealed that Ll-CATH treatment decreased tnf-α and il-1β expression in RAW264.7 cells, and could serve to indicate that Ll-CATH plays an important role in preventing the overstimulation of pro-inflammatory immune responses in amphibians. Ll-CATH showed a significant ability to inhibit the production of TNF-α at its highest effective concentration. This suggests that, under certain experimental conditions, Ll-CATH can effectively interfere with the biosynthetic process of TNF-α, thereby reducing its release. However, when we reduced the concentration of Ll-CATH to the lowest effective concentration, its inhibitory effect was greatly reduced and it could only exert a limited inhibitory effect on il-1β production. This difference may be due to different modes of interaction between Ll-CATH and the two cytokines, or to different degrees of interference with intracellular signalling pathways at different concentrations. Therefore, we need to further investigate the inhibitory mechanism of Ll-CATH on these two cytokines in order to better understand its mode of action and optimise its conditions of use.

Conclusion

In summary, we had successfully characterized a novel cathelicidin gene, Ll-cath, from the Chong’an Moustache Toad L. liui. The Ll-CATH exhibited unique features, with a conserved precursor structure but bears similarity to other mature peptides found in cathelicidins. Importantly, Ll-CATH demonstrated broad-spectrum antibacterial activity, highlighting its potential as an effective antimicrobial agent. Our findings indicated that Ll-CATH exerted its antibacterial effects through the disruption of bacterial cell membranes and the hydrolysis of bacterial gDNA. Additionally, Ll-CATH showed the ability to induce chemotaxis in immune cells and modulate the expression of pro-inflammatory cytokine genes. These results highlighted the immunomodulatory potential of amphibian cathelicidins and could hold great potential as an alternative to antibiotics. Nevertheless, we believe that delving deeper into their molecular mechanisms for future studies could hold the key in understanding the multifunctional nature of cathelicidins.

Materials and methods

Animal collection and experimental conditions

In mid-November 2020, we captured twelve male adult L. liui individuals weighing between 30–40 g each from the Zhejiang Jiulongshan National Nature Reserve (28.370° N, 118.887° E) in Suichang Country, Zhejiang Province, China. The toads were captured by hand and subsequently divided into control and infection groups in a random manner for the purposes of this study. To provide suitable housing, each L. liui specimen was housed individually in food-grade polypropylene plastic bins (300 × 200 × 120 mm) containing 2.4 L of pathogen-free pure water. The water in the system was recirculated and filtered, and the temperature was maintained between 9–12 °C. Before commencing the actual experiments, the toads were given a period of 2 weeks to acclimate to the laboratory conditions.

All the samples were collected with permission in accordance with the local license. All experimental procedures were performed in experimental units of the Laboratory of Amphibian Diversity Investigation, Lishui University, China, and approved by the Ethics Committee of Lishui University (Permit No. AREC-LSU202011-001), according to the OIE standards for use of animals in research by ARRIVE guidelines.

Ll-cath cDNA sequencing analyses

The Ll-cath cDNA was obtained from the liver transcriptome (SRR23238843). In order to determine the molecular mass and theoretical isolelectric point, we used ProtParam at the ExPasy web site [42]. We then analysed the signal peptide using SignalP-5.0 [43]. Multiple alignments were conducted using the alignment program ClustalW [44]. Phylogenetic and molecular evolutionary analyses were carried out using MEGA version 7 software where a Poisson model was used [45].

Ll-cath expression profiles in L. liui

After a 2-week acclimatization period, four healthy L. liui individuals were euthanized with MS-222 (400 ppm) and immediately dissected on ice. Seven tissues (spleen, lung, kidney, intestine, skin, heart, and liver) were isolated and stored separately at -80 °C. To investigate the effects of harmful microorganisms on Ll-cath expression, we conducted an A. hydrophila infection following established protocols [46]. Briefly, 100 μL A. hydrophila (1 × 105 colony forming units (CFU)/mL) was injected into four healthy L. liui individuals in the infection group. The control group received an injection of the same volume of 0.65% saline. At 12 hpi, five tissues (kidney, intestine, spleen, liver, and skin) were separately extracted from each specimen and stored at -80 °C.

Quantification of cathelicidin concentrations

Cathelicidin concentrations in seven different tissues of healthy individuals were quantified using the Frog CAMP ELISA Kit (#SU-B85015; Quanzhou Kenuodi Biotechnology, Quanzhou, China) based on the protocol provided. Firstly, we prepared the tissues homogenates and pelleted out insoluble tissue fragments. Next, the supernatant was repeatedly centrifuged to eliminate additional organelles. To determine the final protein concentration in each sample, we used the Total Protein Quantitative Assay Kit (#A045–2–2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to manufacturer’s protocol. The cathelicidin concentration was then expressed as mass per mg of total protein.

qPCR analysis

Total RNA was extracted using Trizol reagent (Sangon Biotech, Shanghai, China), and cDNA synthesis was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser (#RR047A, TaKaRa, Dalian, China). Primers (Table 2) were designed using Primer3web v 4.1.0 and qPCR was conducted using the TB Green® Premix Ex Taq™ (#RR420A, TaKaRa, Dalian, China) on the Real-time PCR Detection System (CFX96, Bio-Rad, Hercules, CA, USA). From the data, we obtained the cycle threshold (Ct) values of Ll-cath and Ll-gapdh. The relative expression of Ll-cath to Ll-gapdh was calculated using the 2−ΔΔCt method [47]. Melt curve analysis was performed to ensure the quality and specificity of the amplified products.

Table 2 Sequences of oligonucleotide primers used in this study
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Antibacterial assay

The Ll-CATH mature peptide (SRPCNCRCCYVARGNGRCLLRPGCFTVAARPNRSV) was synthesized chemically with a purity of over 95% (GL Biochem, Shanghai, China). To evaluate the antibacterial activity of Ll-CATH, we used the following bacteria: A. hydrophila, S. epidermidis, P. mirabilis, L. monocytogenes, E. coli, S. enterica, S. iniae, V. harveyi, V. parahaemolyticus, and V. vulnificus. The MIC of the peptides in two-fold dilution concertation series was determined the bacterial growth inhibition assays as, as previously described [35, 46]. The bacterial sedimentation absorbance at

DNA degradation assay

The hydrolysis of V. harveyi gDNA by Ll-CATH was assessed based on studied methods [46, 48]. Briefly, V. harveyi gDNA was extracted using an Ezup Column Bacteria Genomic DNA Purification Kit (#B518255, Sangon Biotech). Subsequently, gDNA was mixed with Ll-CATH peptide at concentrations of 25, 50, and 100 μg/mL for 30 min, respectively. The mixture was loaded onto a 1.0% agarose gel and gel electrophoresis was performed. The intensity of the nucleic acid bands was analyzed using ImageJ v1.53t software (NIH, Bethesda, MD, USA).

LDH release assay

To assess bacterial cell membrane damage, we followed the protocol from LDH Release Assay Kit (#C0016, Beyotime, Shanghai, China). V. harveyi (1 × 109 CFUs) were combined with Ll-CATH at concentrations of 25, 50, and 100 μg/mL for 2 h, respectively. Samples were then centrifuged and loaded onto a 96-well plate. LDH detection working solution was added to each well, incubated at room temperature for 30 min and absorbance at 490 nm was measured.

Cell counting kit (CCK)-8 assay

The CCK-8 kit (#E-CK-A362, Elabscience Biotechnology, Wuhan, China) was used to assess cell viability. RAW264.7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% foetal bovine serum (FBS) and 100 U/mL of penicillin and streptomycin at 37 °C and 5% CO2. RAW264.7 cells (a mouse macrophage cell line) were seeded at a density of approximately 5 × 103 cells per well in 96-well plates and exposed to various concentrations of Ll-CATH (0.1, 1.0, and 10.0 μg/mL) for 24 h. A control group treated with BSA at 10.0 μg/mL was also included. Subsequently, CCK-8 solution was added to each well, and the plates were incubated for 4 h before measuring the absorbance at 450 nm using a spectrophotometer.

Chemotaxis assay

We then conducted cell chemotaxis assay using a 24-well transwell chamber (Corning, NY, USA) following a cited protocol [49]. Ll-CATH (0.1, 1.0, and 10.0 μg/mL) was added to the transwell chamber, which was overlaid with a nitrocellulose filter membrane with a pore size of 5 μm. Next, RAW264.7 cells (7.5 × 105 cells) were added to the upper chamber and incubated at 37 °C for 24 h. The cells that migrated to the lower chamber were subjected to the addition of Wright-Giemsa stain, followed by their enumeration under a magnification of 40 × 10. The migration assays were performed in triplicate, with three samples analyzed in each replicate, followed by the calculation of the percentage of cells that migrated to the lower chamber.

Cytokine gene expression assay

RAW264.7 cells were exposed to Ll-CATH at concentrations of 0.1, 1.0, and 10.0 μg/mL for 8 h. Subsequently, the cells were collected, and total RNA was extracted for RT-qPCR analysis. The expression of tnf-α and il-1β was normalized to 18S rRNA expression.

Data analysis

The data were analyzed and presented as mean ± standard deviation (SD). Statistical analysis was performed using SPSS v13.0 (SPSS Inc., Chicago, USA). KolmogorovSmirnov and Bartlett’s tests were used to determine the normality and homogeneity of the data respectively. We normalized the data using log10 and conducted a one-way analysis of variance (ANOVA) and statistical significance was determined at a threshold of p < 0.05.

Availability of data and materials

The cathelicidin (Ll-CATH) cDNA sequence was submitted to GenBank under accession number OQ411022. The datasets used and/or analysed during the current study are available from the first or corresponding authors on reasonable request.

Abbreviations

ANOVA:

Analysis of variance

AMPs:

Antimicrobial peptides

BSA:

Bovine serum albumin

CATH:

Cathelicidins

CFU:

Colony forming units

Ct:

Cycle threshold

gDNA:

Genomic DNA

Hpi:

Hours post-infection

LDH:

Lactate dehydrogenase

MIC:

Minimal inhibitory concentration

qPCR:

Real-time quantitative PCR

RT:

Reverse transcription

SD:

Standard deviation

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Acknowledgements

The authors would like to thank ZHONG Junjie and XIANG Ziyong for help during the animal collection.

Funding

The current study was supported by grants from County-School Cooperation Project in Suichang County (2024-HZ12), Research Project of the Lishui Science and Technology Bureau (2022SJZC006), and Key Research and Development Project of Lishui City (2021ZDYF09).

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Author notes

  1. Jie Chen and Chi-Ying Zhang contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Amphibian Diversity Investigation, College of Ecology, Lishui University, Lishui, 323000, China

    Jie Chen, Li Ma & Guo-Hua Ding

  2. College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou , Zhejiang, 311121, China

    Chi-Ying Zhang

  3. Administration Center of Zhejiang Jiulongshan National Nature Reserve, Suichang, Zhejiang, 323300, China

    Yu Wang

  4. College of Medicine, Lishui University, Lishui, 323000, China

    Le Zhang

  5. Department of Biological Science, National University of Singapore, Singapore, 117558, Singapore

    Rachel Wan Xin Seah

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  1. Jie Chen
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  3. Yu Wang
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  4. Le Zhang
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  6. Li Ma
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Contributions

Jie Chen: Conceptualization, Data curation, Formal analysis, Methodology, Visualization, Writing—original draft, Writing—review & editing. Chi-Ying Zhang: Data curation, Investigation, Methodology, Writing—original draft, Writing—review & editing. Yu Wang: Investigation, Methodology, Resources. Le Zhang: Funding acquisition, Investigation, Methodology. Rachel Wan Xin Seah: Writing—original draft, Writing—review & editing. Li Ma: Conceptualization, Funding acquisition. Guo-Hua Ding: Conceptualization, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Visualization, Writing—original draft, Writing—review & editing.

Corresponding author

Correspondence to Guo-Hua Ding.

Ethics declarations

Ethics approval and consent to participate

All the samples were collected with permission in accordance with the local license. All experimental procedures were performed in experimental units of the Laboratory of Amphibian Diversity Investigation, Lishui University, China, and approved by the Ethics Committee of Lishui University (Permit No. AREC-LSU202011-001), according to the OIE standards for use of animals in research by ARRIVE guidelines.

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The authors declare no competing interests.

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Chen, J., Zhang, CY., Wang, Y. et al. Discovery of Ll-CATH: a novel cathelicidin from the Chong’an Moustache Toad (Leptobrachium liui) with antibacterial and immunomodulatory activity. BMC Vet Res 20, 343 (2024). https://doi.org/10.1186/s12917-024-04202-9

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Keywords

BMC Veterinary Research

ISSN: 1746-6148

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