Screening and computational characterization of novel antimicrobial cathelicidins from amphibian transcriptomic data

As cold-blooded organisms living in damp and dark environments, amphibians have evolved robust defense mechanisms to protect themselves from predators and infections. Among the wide repertoire of bioactive compounds they produce are antimicrobial peptides (AMPs), which are required as part of innate immunity. One important class of AMPs is cathelicidins, known for their broad-spectrum activity against pathogens and their immunoregulatory roles. However, despite their promising biomedical potential and the increasing availability of omics data, few cathelicidins have been studied in amphibians, mostly through conventional experimental techniques. Here, we present 210 novel cathelicidin sequences from amphibian transcriptomes, identified through a comprehensive computational pipeline, which employed HMMER and BLAST tools to screen cathelicidin domains. These sequences reveal a typical tripartite domain architecture that was confirmed by SignalP and InterProScan analysis. Phylogenetic inference with IQ-TREE classified the sequences into six categories based on evolutionary relationships. Compared to cathelicidins from other vertebrates, amphibian mature peptides exhibit longer average lengths (around 50 amino acids), fewer aromatic and hydrophobic residues, and reduced thermal stability. Furthermore, these amphibian cathelicidins were characterized for their physicochemical and biological properties, revealing significant antimicrobial potential with lower hemolytic capability, especially in anurans, which suggests a balance between their antimicrobial and hemolytic activities predicted through AMPlify, ampir, AmpGram, and HemoPI. Secondary structure estimations, including three-dimensional modeling using AlphaFold2, indicate that amphibian cathelicidins predominantly feature $\alpha$-helices and coils. Some representative models also display a high $\alpha$-helix composition with amphipathic topology, facilitating interactions with simulated bacterial membranes as assessed by the PPM approach. Thus, these findings highlight the functional role of cathelicidins in amphibian immunity and their promising biomedical applicability, emphasizing the importance of applying computational methods to expand the scope and reveal the diverse landscape of cathelicidins across vertebrates.