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Functional diversity of cathelicidin peptides in the frog lung: An integrated transcriptomic and functional analysis
Pawarit Khamlaiwong1, Chutima Karnmongkol1, Rujiraphorn Musigapan2
1Department of Biotechnology, Faculty of Agricultural Technology, Kalasin University, Kalasin, 46000, Thailand.
None:
Amphibians have evolved sophisticated innate immune mechanisms shaped by dual aquatic-terrestrial lifestyles, yet the pulmonary cathelicidin repertoire remains incompletely characterized. This study investigated the diversity, and comparative activity profiles of lung-expressed cathelicidin in the tiger frog (Hoplobatrachus rugulosus) through an integrated transcriptomic and biochemical approach. RNA sequencing of lung tissue identified four cathelicidin paralogs-Hr-CATH1 to Hr-CATH4 (AS34, SN34, AR37 and DL37)- representing the complete cathelicidin complement co-expressed in this tissue, including both previously reported sequences and paralogs not yet functionally characterized. RT-qPCR analysis confirmed the expression of all four transcripts and revealed distinct expression patterns among the paralogs. Bioinformatic analysis revealed conserved signal peptide and cathelin-like pro-regions, alongside divergent mature peptide domains, resulting in substantial variation in physicochemical properties, from highly cationic (AS34, +9) to anionic (DL37, -4). Circular dichroism spectroscopy showed membrane-induced α-helical adoption across all peptides. Biological activity assays demonstrated that AS34 exhibited potent broad-spectrum antimicrobial potency consistent with membrane disruption and DNA binding, whereas SN34 displayed moderate selective activity and AR37 and DL37 lacked direct antimicrobial effects. Notably, all four peptides suppressed lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages. AS34 further downregulated pro-inflammatory gene expression (TNF-α, IL-1β, IL-6, iNOS, COX-2). All peptides established low to moderate hemolytic efficacy at high concentration. Collectively, these findings suggest functional diversity among lung-expressed cathelicidins contributing to a multifaceted pulmonary defense in H. rugulosus, and position AS34 as a promising scaffold for developing novel antimicrobial and anti-inflammatory therapeutics.
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