Antimicrobial activity and immunomodulation of four novel cathelicidin genes isolated from the tiger frog Hoplobatrachus rugulosus.

Cathelicidin is a family of antimicrobial peptides in vertebrates that plays an important role in resistance and immunization against pathogenic microorganisms. In the present study, the full-length cDNA sequences of four novel cathelicidins (cathelicidin-1 to cathelicidin-4) in the tiger frog Hoplobatrachus rugulosus, encoding 153, 188, 132, and 160 amino acids, respectively, were firstly cloned by rapid amplification of the cDNA ends (RACE) technique. Sequence comparison and phylogenetic tree analysis indicated that the structures of the four cathelicidins are highly diverse. Afterwards, the tissue distribution profiles and antimicrobial patterns of cathelicidins in H. rugulosus were determined by real-time PCR. The four cathelicidins showed tissue-specific distribution patterns in the healthy frogs, and the transcriptional levels of cathelicidins exhibited a tissue- and time-dependency profile in the frogs challenged with pathogenic bacteria Aeromonas hydrophila for 72 h. The synthetic peptides of cathelicidin-1 and cathelicidin-2 exhibited broad-spectrum in vitro antimicrobial activity, and cathelicidins exerted antimicrobial activities through excessive induction of reactive oxygen species and direct disruption of the microbial membrane structure. In addition, the intraperitoneal injection of cathelicidin proteins significantly increased the marine medaka Oryzias melastigma resistance to bacterial challenges. The existence of multiple cathelicidins, their distinct tissue distribution patterns, and the inducible expression profiles suggest a sophisticated, highly redundant, and multilevel network of antimicrobial defense mechanisms in tiger frogs. This study provides evidence that cathelicidins have antimicrobial and immunomodulatory activities, and cathelicidins derived from H. rugulosus have potential therapeutic applications against pathogenic infections in aquaculture.