Abstract
Cathelicidins, a class of gene-encoded effector molecules of vertebrate innate immunity, provide a first line of defense against microbial invasions. Although cathelicidins from mammals, birds, reptiles and fishes have been extensively studied, little is known about cathelicidins from amphibians. Here we report the identification and characterization of two cathelicidins (cathelicidin-RC1 and cathelicidin-RC2) from the bullfrog Rana catesbeiana. The cDNA sequences (677 and 700 bp, respectively) encoding the two peptides were successfully cloned from the constructed lung cDNA library of R. catesbeiana. And the deduced mature peptides are composed of 28 and 33 residues, respectively. Structural analysis indicated that cathelicidin-RC1 mainly assumes an amphipathic alpha-helical conformation, while cathelicidin-RC2 could not form stable amphipathic structure. Antimicrobial and bacterial killing kinetic analysis indicated that the synthetic cathelicidin-RC1 possesses potent, broad-spectrum and rapid antimicrobial potency, while cathelicidin-RC2 exhibited very weak antimicrobial activity. Besides, the antimicrobial activity of cathelicidin-RC1 is salt-independent and highly stable. Scanning electron microscopy (SEM) analysis indicated that cathelicidin-RC1 kills microorganisms through the disruption of microbial membrane. Moreover, cathelicidin-RC1 exhibited low cytotoxic activity against mammalian normal or tumor cell lines, and low hemolytic activity against human erythrocytes. The potent, broad-spectrum and rapid antimicrobial activity combined with the salt-independence, high stability, low cytotoxic and hemolytic activities make cathelicidin-RC1 an ideal template for the development of novel peptide antibiotics.
Highlights
Living in an environment surrounded by diverse microbial pathogens, multicellular organisms are permanently under threats [1]
Total RNA was extracted from the lung of R. catesbeiana and cDNA library was constructed by a cDNA library construction kit
By PCR-based cDNA cloning method, two cDNAs encoding two different cathelicidins were obtained from the cDNA library (GenBank accession numbers: KF766530-KF766531)
Summary
Living in an environment surrounded by diverse microbial pathogens, multicellular organisms are permanently under threats [1] Their successful survival depends on prompt and effective immune response to microbial invasions. AMPs are a group of gene-encoded, small, and cationic peptides that possess microbicidal activities against microorganisms [3]. They are evolutionarily ancient weapons and widely distributed throughout the life kingdom [1,4]. AMPs target to the microbial membrane by electrostatic adsorption and subsequently result in cell rupture This process occurs so quickly that it can be completed in several minutes, as a result it is unlikely for microbes to evolve resistance. Due to the high efficiency and low possibility to induce resistance, AMPs have attracted great attentions as a new generation of antibiotics
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