CATH_BRALE

Antibiotic-resistant bacteria are severe threats to aquaculture industry. Boosting and modulating host immune responses has been proved to be an effective strategy to combat with bacterial infections and there is an urgent need for novel immunomodulators. Cathelicidins is an important family of host defense peptides (HDPs) that possess direct antimicrobial activities and potent immunomodulatory properties. Several cathelicidins have been identified and characterized from diverse fish species. Considering the relatively conserved immune systems between different fish species, it is reasonable to speculate that cathelicidins from different fish species possess immunomodulating functions on the other fish species. In the present study, two fish-derived cathelicidins (CATH_BRALE and codCath1) were selected to investigate their protective effect on zebrafish with bacterial infections. They exhibited potent and broad-spectrum antimicrobial activities against the tested aquatic Gram-positive and Gram-negative pathogenic bacteria, with MIC values ranging 2.34-18.75 μg/ml for CATH_BRALE and 2.34-37.5 μg/ml for codCath1. And their antimicrobial effect is so rapid that they killed the bacteria within 60 min. Unlike conventional antibiotics, they kill bacteria by inducing bacterial membrane permeabilization and cell disruption. Besides direct antimicrobial activity, CATH_BRALE and codCath1 exhibited potent immunomodulatory functions by both inhibiting bacteria induced zebrafish pro-inflammatory cytokine gene (TNF-α, IL-1β, and IL-6) expression and stimulating zebrafish chemokine gene IL-8 expression. In vivo challenge test proved that they could significantly decrease the bacterial numbers and enhance the survival rates of zebrafish. All the results above imply the great potential of CATH_BRALE and codCath1 as novel peptide immunomodulators in fish aquaculture industry.

Thick-lipped lenok, Brachymystax lenok is one of the ancient fish species in China and northeast Asia countries. Due to the overfishing, the population of lenok has been declined significantly. Cathelicidins are innate immune effectors that possess both bactericidal activities and immunomodulatory functions. This report identifies and characterizes the salmonoid cathelicidin (CATH_BRALE) from this ancient fish. It consists of open reading frame (ORF) of 886 bp encoding the putative peptide of 199 amino acids. Sequence alignment with other representative salmonid cathelicidins displayed two distinctive features of current lenok cathelicidin: high level of arginine, resulting in high positive charge and glycine residues, which is significantly different from most acknowledged types of cathelicidins; and the six-aminoacid tandem repeated sequence of RPGGGS detected in a variable number of copies among fish cathelicidins, suggesting the existence of a genetically unstable region similar to that found in some mammalian cathelicidins. Expression of CATH_BRALE is predominantly found in gill, with lower levels in the gastrointestinal tract and spleen. The homology modeled structure of CATH_BRALE exhibits structural features of antiparallel b-sheets flanked by a-helices that are representative of small cationic cathelicidin family peptides. CATH_BRALE possesses much stronger antimicrobial activity against gram-negative bacteria than that of the human ortholog, LL-37. The growth of two typical fish bacterial pathogens, gram-negative bacterium of Aeromonas salmonicida and Aeromonas hydrophila was substantially inhibited by synthetic CATH_BRALE, with both MICs as low as 9.38 mM.