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BACKGROUND: The liver-expressed antimicrobial peptide 2 (LEAP2) is essential in host immunity against harmful pathogens and is only known to act as an extracellular modulator to regulate embryonic development in amphibians. However, there is a dearth of information on the antimicrobial function of amphibian LEAP2. Hence, a LEAP2 homologue from Leptobrachium liui was identified, characterized, and chemically synthesized, and its antibacterial activities and mechanisms were determined.
RESULTS: In this study, LEAP2 gene (Ll-LEAP2) cDNA was cloned and sequenced from the Chong'an Moustache Toad (Leptobrachium liui). The predicted amino acid sequence of Ll-LEAP2 comprises a signal peptide, a mature peptide, and a prodomain. From sequence analysis, it was revealed that Ll-LEAP2 belongs to the cluster of amphibian LEAP2 and displays high similarity to the Tropical Clawed Frog (Xenopus tropicalis)'s LEAP2. Our study revealed that LEAP2 protein was found in different tissues, with the highest concentration in the kidney and liver of L. liui; and Ll-LEAP2 mRNA transcripts were expressed in various tissues with the kidney having the highest mRNA expression level. As a result of Aeromonas hydrophila infection, Ll-LEAP2 underwent a noticeable up-regulation in the skin while it was down-regulated in the intestines. The chemically synthesized Ll-LEAP2 mature peptide was selective in its antimicrobial activity against several in vitro bacteria including both gram-positive and negative bacteria. Additionally, Ll-LEAP2 can kill specific bacteria by disrupting bacterial membrane and hydrolyzing bacterial gDNA.
CONCLUSIONS: This study is the first report on the antibacterial activity and mechanism of amphibian LEAP2. With more to uncover, the immunomodulatory functions and wound-healing activities of Ll-LEAP2 holds great potential for future research.
Fig. 1. Multiple alignment of the amino acid sequences of Ll-LEAP2 and its homologs. The threshold for shading was 70%; similar residues are marked in grey, identical residues are marked in black, and alignment gaps are marked as “-”. The four conserved cysteine residues in the mature peptide are indicated by “*”. A cartoon picture of the experimental animal was painted by DING Zimu
Fig. 2. Amino acid sequences and predicted secondary structures of Ll-LEAP2 and other amphibian LEAP2 mature peptides
Fig. 3. Phylogenetic reconstruction of amino acid sequences of LEAP2 based on neighbour-joining method. The values at the forks indicate the percentage of trees in which this grouping occurred after bootstrapping (1000 replicates; shown only when > 60%). The scale bar shows the number of substitutions per base
Fig. 4. Mean values (+ SE) for (A) LEAP concentration and (B) its gene relative expression of different tissues in healthy Leptobrachium liui. Means with different letters differ significantly (Tukey’s post hoc test, a > b > c > d > e)
Fig. 5. Mean values (+ SE) for the relative expression of Ll-LEAP2 between control and infection groups in different tissues. **: P < 0.01, ***: P < 0.001
Fig. 6. Effects of Ll-LEAP2 on the integrity of cell membrane in Aeromonas hydrophila. The BSA treatment was used as the negative control group. LDH release represents fold-change relative to the control group, which was assigned a value of 1. Means with different letters indicate significant differences (Tukey’s post hoc test, a > b)
Fig. 7. Hydrolytic effect of Ll-LEAP2 on bacterial genomic DNA. A Various concentrations of Ll-LEAP2 were incubated with 800 ng of genomic DNA of Aeromonas hydrophila at room temperature for 30 min, then genomic DNA was analyzed by electrophoresis on a 1.0% agarose gel. The BSA treatment was used as the negative control group. One of three independent experiments is shown. B Mean values (+SE) for the intensity of nucleic acid bands in different treatments. Means with different letters indicate significant differences (Tukey’s post hoc test, a > b > c)
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