Gene Cloning, Peptide Identification And Functional Characterization Of Novel Cathelicidin From Equus Asinus

In current work, Ea-CATH1 and Ea-CATH2 were identified from a constructed lung cDNA library of donkey (Equus asinus) as members of cathelicidin-derived antimicrobial peptides, using a nested-PCR-based cloning strategy. Composed of 25 (KRRGSVTTRYQFLMIHLLRPKKLFA), 26 (KGRGSETTRYQFVPVHFFPWNKLSDF) residues, respectively, Ea-CATH1(3060.75 Da) and Ea-CATH2(3144.54 Da) are smaller than most of other cathelicidins,and have no sequence homology to other cathelicidins identified to date. They are both basic peptides, containing 7 and 4 basic amino acids respectively.Chemically synthesized Ea-CATH1 exerted potent antimicrobial activity against most of 32 strains of bacteria and fungi tested, especially the clinically isolated (IS) drug-resistance strains, and MIC values against Gram-positive bacteria are mostly in the range of 0.3–2.4μg/ml. Ea-CATH1 shows an extraordinary serum stability and no hemolytic activity against human erythrocytes in a dose up to 20μg/ml.CD spectra shows that Ea-CATH1 mainly adopts anα-helical conformation in 50% TFE/H2O solution, but a random coil in aqueous solution. In vitro, Ea-CATH1 with concentration of 0.6μg/ml completely kills 106CFU/ml Staphylococcus aureusATCC2592 in two hours, which is far better than ampicillin. SEM observation of S. aureus ATCC2592 treated by Ea-CATH1 demonstrated that Ea-CATH could cause rapid disruption of the bacterial membrane, and in turn lead to the cell lysis. This might explain the much faster killing kinetics of Ea-CATH1 than conventional antibiotics revealed by killing kinetics data.In the presence of CaCl2, Ea-CATH1 exerted hemagglutination activity, which might potentiate an inhibition against the bacterial polyproteins interaction with the host erythrocyte surface, thereby possibly restrict bacterial colonization and spread. Ea-CATH1 of 20μg/ml dissolved in PBS around physiological pH value shows high activity of degranulation of mast cell, indicating that it maybe a host defense effector molecule. In conclusion, Ea-CATH1 is a promising lead compound for antimicrobial drug design.