Gene cloning, expression and characterization of avian cathelicidin orthologs, Cc-CATHs, from Coturnixcoturnix

• Published in: The FEBS journal 2011-05, Vol.278 (9), p.1573
• Main Authors: Feng, Feifei, Chen, Chen, Zhu, Wenjuan, He, Weiyu, Guang, Huijuan, Li, Zheng, Wang, Duo, Liu, Jingze, Chen, Ming, Wang, Yipeng, Yu, Haining
• Format: Article
• Language: English
• Subjects: Amino acids; Antibiotics; Birds; Cloning; Drug resistance; Gene expression; Peptides
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2011.08080.x

Cathelicidins comprise a family of antimicrobial peptides sharing a highly conserved cathelin domain, which play a central role in the early innate host defense against infection. In the present study, we report three novel avian cathelicidin orthologs cloned from a constructed spleen cDNA library of Coturnixcoturnix, using a nested-PCR-based cloning strategy. Three coding sequences containing ORFs of 447, 465 and 456bp encode three mature antimicrobial peptides (named Cc-CATH1, 2 and 3) of 26, 32 and 29 amino acid residues, respectively. Phylogenetic analysis indicated that precursors of Cc-CATHs are significantly conserved with known avian cathelicidins. Synthetic Cc-CATH2 and 3 displayed broad and potent antimicrobial activity against most of the 41 strains of bacteria and fungi tested, especially the clinically isolated drug-resistant strains, with minimum inhibitory concentration values in the range 0.3-2.5μm for most strains with or without the presence of 100mm NaCl. Cc-CATH2 and 3 showed considerable reduction of cytotoxic activity compared to other avian cathelicidins, with average IC50 values of 20.18 and 17.16μm, respectively. They also exerted a negligible hemolytic activity against human erythrocytes, lysing only 3.6% of erythrocytes at a dose up to 100μg·mL-1. As expected, the recombinant Cc-CATH2 (rCc-CATH2) also showed potent bactericidal activity. All these features of Cc-CATHs encourage further studies aiming to estimate their therapeutic potential as drug leads, as well as coping with current widespread antibiotic resistance, especially the new prevalent and dangerous 'superbug' that is resistant to almost all antibiotics. Three novel avian cathelicidin orthologues were cloned encoding three antimicrobial peptides of 26, 32, 29 amino acid residues length. Synthetic Cc-CATH2 and3 displayed broad and potent antimicrobial activity. Cc-CATH2 and3 showed much reduced cytotoxicity, and a negligible hemolytic activity, lysing only 3.6% of erythrocytes at doses up to 100μg·mL-1. Recombinant Cc-CATH2 showed potent bactericidal activity as expected. [PUBLICATION ABSTRACT]