Lf-CATH1

Antimicrobial peptides (AMPs) are a group of gene-encoded small peptides that play pivotal roles in the host immune system of multicellular organisms. Cathelicidins are an important family of AMPs that exclusively exist in vertebrates. Many cathelicidins have been identified from mammals, birds, reptiles and fish. To date, however, cathelicidins from amphibians are poorly understood. In the present study, two novel cathelicidins (OL-CATH1 and 2) were identified and studied from the odorous frog Odorrana livida. Firstly, the cDNAs encoding the OL-CATHs (780 and 735 bp in length, respectively) were successfully cloned from a lung cDNA library constructed for the frog. Multi-sequence alignment was carried out to analyze differences between the precursors of the OL-CATHs and other representative cathelicidins. Mature peptide sequences of OL-CATH1 and 2 were predicted (33 amino acid residues) and their secondary structures were determined (OL-CATH1 showed a random-coil conformation and OL-CATH2 demonstrated a-helical conformation). Furthermore, OL-CATH1 and 2 were chemically synthesized and their in vitro functions were determined. Antimicrobial and bacterial killing kinetic analyses indicated that OL-CATH2 demonstrated relatively moderate and rapid antimicrobial potency and exhibited strong anti-inflammatory activity. At very low concentrations (10 μg/mL), OL-CATH2 significantly inhibited the lipopolysaccharide (LPS)-induced transcription and production of pro-inflammatory cytokines TNF-a, IL-1b and IL-6 in mouse peritoneal macrophages. In contrast, OL-CATH1 did not exhibit any detectable antimicrobial or anti-inflammatory activities. Overall, identification of these OL-CATHs from O. livida enriches our understanding of the functions of cathelicidins in the amphibian immune system. The potent antimicrobial and anti-inflammatory activities of OL-CATH2 highlight its potential as a novel candidate in anti-infective drug development.

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.

Cathelicidins are an important family of antimicrobial peptides (AMPs), which play pivotal roles in vertebrate immune responses against microbial infections. They are regarded as potential drug leads for the development of novel antimicrobial agents and three related drugs have been developed into clinical trials. Thus, it is meaningful to identify more cathelicidins from vertebrate species. Cathelicidins from ranid frogs possess special structural characteristics and activities, but to date only 12 ranid frog cathelicidins have been identified. In the present study, two novel cathelicidins (PN-CATH1 and 2) were identified from the black-spotted frog, Pelophylax nigromaculata. PN-CATHs possess low sequence similarity with the known cathelicidins. They exhibited moderate, but broad-spectrum and rapid antimicrobial activities against the tested bacteria. They kill bacteria by mainly inducing bacterial membrane disruption and possibly generating intracellular ROS formation. They also possess potent anti-biofilm and persister cell killing activity, indicating their potential in combating infections induced by biofilms-forming bacteria. Besides direct antimicrobial activity, they exhibited potent anti-inflammatory activity by effectively inhibiting the LPS-induced production of pro-inflammatory cytokines in mouse macrophages, which could be partly ascribed to their direct LPS-neutralizing ability. Furthermore, PN-CATHs demonstrated powerful in vitro free radical scavenging activities. Ultraviolet radiation significantly increased their in vivo gene expression in frog skin. Meanwhile, they possess weak cytotoxic activity and extremely low hemolytic activity. PN-CATHs represent the first discovery of cathelicidins family AMPs with both potent anti-infective and antioxidant activities. The discovery of PN-CATHs provides potential peptide leads for the development of novel anti-infective and antioxidant drugs.