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BACKGROUND: The liver-expressed antimicrobial peptide 2 (LEAP2) family is an important group of antimicrobial peptides (AMPs) involved in vertebrate defence against bacterial infections. However, research on LEAP2 in amphibians is still in its infancy.
RESULTS: This study aimed to explore the role of LEAP2 in the Chinese spiny frog (Quasipaa spinosa). The cDNA of the LEAP2 gene (QsLEAP2) was cloned from a Chinese spiny frog. The QsLEAP2 protein comprises a signal peptide, a prodomain, and a mature peptide. Sequence analysis indicated that QsLEAP2 is a member of the amphibian LEAP2 cluster and closely related to the LEAP2 of the African clawed frog (Xenopus laevis). Expression of QsLEAP2 was detected in various tissues, with the liver exhibiting the highest expression. Following infection with Aeromonas hydrophila, QsLEAP2 expression was significantly upregulated in the spleen, lungs, kidneys, liver, and gut. The synthetic mature peptide QsLEAP2 exhibited selective antimicrobial activity against several bacterial strains in vitro. It disrupted bacterial membrane integrity and hydrolysed bacterial genomic DNA, exhibiting bactericidal effects on specific bacterial species. Furthermore, QsLEAP2 induced chemotaxis in RAW264.7 murine leukemic monocytes/macrophages, enhancing their phagocytic activity and respiratory bursts. Docking simulations revealed an interaction between QsLEAP2 and QsMOSPD2.
CONCLUSIONS: These findings provide new insights into the role of LEAP2 in the amphibian immune system.
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40082911
???displayArticle.pmcLink???PMC11905587 ???displayArticle.link???BMC Vet Res ???displayArticle.grants???[+]
2023zdyf11 Key R & D Plan Projects of Lishui, 2024tpy24 Lishui City Rural Science and Technology special commissioner project
Fig. 1. Multiple sequence alignment and three-dimensional structure prediction of QsLEAP2. (A) Multiple sequence alignment of the QsLEAP2 protein and its homologues. The shading threshold was set at 70%; similar residues are highlighted in grey, identical residues are in black, and alignment gaps are denoted by “-”. (B) AlphaFold2 was used to model the three-dimensional structure of QsLEAP2
Fig. 2. Phylogenetic analysis of LEAP2 amino acid sequences was performed with MEGA X using the neighbour-joining method. Branch point values show the percentage of trees with that grouping from a bootstrap analysis (1000 replicates; shown only if over 60%). The scale bar represents substitutions per base
Fig. 3. Expression of QsLEAP2: (A) Tissue-specific expression, with significant differences indicated by letters (one-way ANOVA, P < 0.05). (B-F) Changes in QsLEAP2 expression post-Aeromonas hydrophila infection, normalised to Qs18S rRNA. Data are mean ± SEM, analysed by one-way ANOVA, n = 4, *P < 0.05
Fig. 4. Impact of QsLEAP2 on Shigella flexneri cell membrane and genomic DNA. (A) QsLEAP2 affects S. flexneri cell membrane integrity, with LDH release shown as fold-change relative to a negative control (BSA, set at 1). Data are mean ± SEM from four experiments, analysed by one-way ANOVA (*P < 0.05). (B) Hydrolytic activity of QsLEAP2 on bacterial genomic DNA, with BSA as a negative control; one of four independent experiments is presented. Marker: 1 Kb DNA Marker P (Sangon; lot: B600022)
Fig. 5. The impact of QsLEAP2 on RAW264.7 cell chemotaxis. Results are presented as mean ± SEM; n = 4; *P < 0.05
Fig. 6. The effect of QsLEAP2 on the phagocytic activity of RAW264.7 cells. The mean fluorescence intensity (MFI) was reported as a fold-change in comparison to the BSA-treated control group, which was arbitrarily set to a value of 100. Results are depicted as the mean ± SEM; n = 4; *P < 0.05
Fig. 7. The impact of QsLEAP2 on the respiratory burst of RAW264.7 cells. The respiratory burst was quantified by assessing the optical density at a wavelength of 620 nm. Results are presented as the mean ± SEM, with n = 4. *P < 0.05
Fig. 8. Molecular docking of QsLEAP2 and QsMOSPD2 conducted using HDOCK. (A) Domain architecture of QsMOSPD2 protein. The blue area represents the transmembrane region of QsMOSPD2. (B-C) In silico protein–protein docking. The interaction between QsLEAP2 and QsMOSPD2 is represented in the model using both cartoon and surface versions
Fig. 4. (B) The hydrolytic activity of QsLEAP2 on the bacterial genomic DNA was assessedthrough electrophoresis. BSA was employed as a negative control
