The first identified cathelicidin from tree frogs possesses anti-inflammatory and partial LPS neutralization activities

Abstract

As of February 2017, approximately 7639 amphibian species have been described in the AmphibiaWeb database. However, only 20 cathelicidin-like antimicrobial peptides have been identified to date from 10 amphibian species. Half of these peptides were identified from genome sequences and have not yet been functionally characterized. In this study, a novel cathelicidin-like peptide designated cathelicidin-PP was purified from the skin of tree frog Polypedates puerensis. Cathelicidin-PP is a 32 residue peptide of sequence ASENGKCNLLCLVKKKLRAVGNVIKTVVGKIA. Circular dichroism spectroscopy indicated that cathelicidin-PP mainly adopts a β-sheet structure in membrane-mimetic solutions. Cathelicidin-PP exhibits potent antimicrobial activity against bacteria and fungi, especially Gram-negative bacteria. Meanwhile, it shows low cytotoxicity toward mammalian cells. Scanning electron microscopy analysis indicated that cathelicidin-PP kills bacteria through the disruption of the bacterial cell membrane integrity. Furthermore, cathelicidin-PP exerts significant anti-inflammatory functions by inhibiting the lipopolysaccharide (LPS)-mediated generation of nitric oxide and pro-inflammatory cytokines, tumor necrosis factor-α, interleukin-1β, and interleukin-6. The MAPKs (ERK, JNK, and p38) and NF-κB signaling pathways are involved in the anti-inflammatory effect. Cathelicidin-PP caused partial neutralization of LPS in a dose-dependent manner. Quantitative PCR indicated that infection of tree frogs with bacteria causes increased expression of cathelicidin-PP in immune-related tissues. Taken together, cathelicidin-PP is the first identified cathelicidin-like peptide from tree frogs. Our findings demonstrate that in addition to direct bactericidal capacity, cathelicidin-PP also possesses immunomodulatory properties, including partial neutralization of LPS, and inhibiting the production of inflammatory cytokines.

Keywords: Anti-inflammation; Cathelicidin; LPS neutralization; Polypedates puerensis; Tree frog.

Fig. 1

Purification of cathelicidin-PP from the skin of P. puerensis and MALDI–TOF MS. a The filtrate of the skin secretion of P. puerensis by 10 kDa cutoff was divided by a Wondasil C₁₈ RP-HPLC column. b The eluted peak (arrow in a) containing antimicrobial activity was further purified by C₁₈ RP-HPLC column. The purified cathelicidin-PP is indicated by an arrow. c MALDI-TOF mass spectrometry analysis of cathelicidin-PP

Fig. 2

The cDNA sequence of cathelicidin-PP precursor. Deduced amino acid sequence is shown below the cDNA sequence. The cathelicidin-PP precursor contains the signal peptide (gray), followed by a cathelin domain ending with a pair of basic residues (in bold), and the mature peptide (black). The stop codon is indicated by an asterisk. Amino acid numbers or nucleotide numbers are shown after the sequences

Fig. 3

Multi-sequence alignment of cathelicidin-PP precursor with other amphibian cathelicidins. The symbols under the alignment indicate the following: asterisk identical sites; colon conserved sites; dot less conserved sites. Dashes are inserted to optimize the alignment. The two conserved cysteine residues involved in disulfide bridges are gray shaded. GenBank accession numbers for the analyzed sequences are shown in Fig. 4

Fig. 4

Phylogenetic analysis of known amphibian cathelicidins. The tree was constructed using the neighbor-joining method based on 21 amphibian cathelicidin precursors, including cathelicidin-PP. The numbers on the branches represent the percent bootstrap support. Cathelicidin-PP is indicated by a triangle

Fig. 5

Scanning electron microscopy of bacteria treated with or without cathelicidin-PP. a, b Control, E. coli cells treated with PBS. c, d E. coli cells treated with cathelicidin-PP (1 × MIC, 0.69 μM) dissolved in PBS. White arrow indicates damage to the plasma membranes of bacteria or the intracellular inclusions efflux

Fig. 6

The CD spectra of cathelicidin-PP in different solutions. a SDS/H₂O solution (5, 10, 20, 40 mM). b LPS/H₂O solution (50, 100, 200, 400 ng/ml). Cathelicidin-PP was dissolved in different solutions to an ultimate concentration of 0.2 mg/ml

Fig. 7

Effects of cathelicidin-PP on iNOS transcription and NO production induced by LPS. a iNOS mRNA. b Nitrite production. Data are mean ± SEM. Values from three independent experiments. *p < 0.05, **p < 0.01, significantly different compared with the control that was treated with serum-free RPMI 1640 and 100 ng/ml LPS

Fig. 8

Effects of cathelicidin-PP on pro-inflammatory cytokine transcription and secretion induced by LPS. a TNF-α mRNA. b TNF-α secretion. c IL-1β mRNA. d IL-1β secretion. e IL-6 mRNA. f IL-6 secretion. Data are mean ± SEM. Values from three separate experiments. *p < 0.05, **p < 0.01, significantly different compared with the control that was incubated with serum-free RPMI 1640 and 100 ng/ml LPS

Fig. 9

Effects of cathelicidin-PP on LPS-induced inflammatory response pathways. a Western blot of phosphorylation of ERK, JNK, p38, and NF-κB p65 in peritoneal macrophages. The cells were incubated with LPS (100 ng/ml) and different concentrations of cathelicidin-PP (0, 5, 10, and 20 μg/ml). After incubation for 30 min, the cells were collected, and the cytoplasmic or nuclear proteins were extracted for Western blot analysis. b Ratio of P-ERK1 (44 kDa), P-ERK2 (42 kDa), P-JNK1 (54 kDa), P-JNK2 (46 kDa), P-p38, and P-p65 to β-actin. Band densities were analyzed using Quantity One software (Bio-Rad, Richmond, CA, USA). Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, ratios of peptide-treated groups are significantly different from that induced by 100 ng/ml LPS alone

Fig. 10

LPS-neutralizing activity of cathelicidin-PP. A chromogenic LAL assay was used to evaluate neutralizing activity. Data are mean ± SEM. Values from three separate experiments. *p < 0.05, **p < 0.01, significantly different compared with the control (PBS)

Fig. 11

Fold increase of cathelicidin-PP in immune-related tissues at different time courses after immune challenge with E. coli. a Fold increase of cathelicidin-PP in skin. b Fold increase of cathelicidin-PP in spleen. c Fold increase of cathelicidin-PP in gut. d Fold increase of cathelicidin-PP in lung. Expression levels in different tissues were calculated relative to the level of cathelicidin-PP in corresponding uninfected tissue, which was arbitrarily defined as 1. Values for infection treatment are significantly different from control values. *p < 0.05, **p < 0.01, significantly different compared to the control (n = 5)