RESUMEN
Antimicrobial peptides (AMPs) are an important component of innate immunity in vertebrates. The cathelicidin family of AMPs is well characterized in mammals and has also been reported in several fish species. In this study we investigated the regulation of cathelicidin expression in a gadoid and a salmonid cell-line in order to dissect the signalling pathways involved. For this, fish cells were treated with microbial lysates, purified microbial components and commercial signalling inhibitors and expression of cathelicidin was assessed with quantitative real-time PCR (qPCR). We found that cathelicidin expression was induced in both cell lines in response to microbial stimuli, but the response patterns differed in these evolutionary distant fish species. Our data suggest that in salmonids, pattern recognition receptors such as TLR5 may be involved in the stimulation of cathelicidin expression and that the signalling cascade can include PI3-kinase and cellular trafficking compartments. A detailed knowledge of the regulating factors involved in AMP-related defence responses, including cathelicidin, could help in developing strategies to enhance the immune defence of fish.
Asunto(s)
Catelicidinas/genética , Proteínas de Peces/genética , Gadus morhua/inmunología , Regulación de la Expresión Génica , Inmunidad Innata , Salmón/inmunología , Transducción de Señal , Animales , Candida albicans/fisiología , Catelicidinas/metabolismo , Línea Celular , Proteínas de Peces/metabolismo , Gadus morhua/genética , Gadus morhua/microbiología , Bacterias Gramnegativas/fisiología , Lactobacillus/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Salmón/genética , Salmón/microbiología , Especificidad de la EspecieRESUMEN
Cathelicidins are among the best characterized antimicrobial peptides and have been shown to have an important role in mammalian innate immunity. We recently isolated a novel mature cathelicidin peptide (codCath) from Atlantic cod and in the present study we functionally characterized codCath. The peptide demonstrated salt sensitivity with abrogation of activity at physiological salt concentrations. In low ionic strength medium we found activity against marine and non-marine Gram-negative bacteria with an average MIC of 10 µM, weak activity against a Gram-positive bacterium (MIC 80 µM), and pronounced antifungal activity (MIC 2.5 µM). The results suggest the kinetics and mode of action of codCath to be fast killing accompanied by pronounced cell lysis. Extracellular products (ECPs) of three marine bacteria caused breakdown of the peptide into smaller fragments and the cleaved peptide lost its antibacterial activity. Proteolysis of the peptide on the other hand was abolished by prior heat-treatment of the ECPs, suggesting a protease involvement. We observed no cytotoxicity of the peptide in fish cells up to a concentration of 40 µM and the selectivity of activity was confirmed with bacterial and mammalian membrane mimetics. We conclude that the potent broad-spectrum activity of codCath hints at a role of the peptide in cod immune defense.
Asunto(s)
Antiinfecciosos/metabolismo , Catelicidinas/metabolismo , Gadus morhua/inmunología , Animales , Péptidos Catiónicos Antimicrobianos , Bacterias/efectos de los fármacos , Bacterias/enzimología , Catelicidinas/genética , Hongos/efectos de los fármacos , Humanos , Inmunidad Innata , Pruebas de Sensibilidad Microbiana , Proteolisis , Sales (Química)/química , Agua de MarRESUMEN
Cathelicidin antimicrobial peptides are multifunctional peptides that are important in the innate immune system of mammals. Cathelicidins have been identified in several fish species. In this study we have isolated cathelicidin from Atlantic cod (Gadus morhua) and identified the cleavage site from the cathelin propart. This is the first isolation of a cathelicidin from teleost fish. The mature cathelicidin was found to be a 67-residues peptide, highly cationic with a pI of 13. Reversed phase chromatographic fractions containing the purified peptide had pronounced antimicrobial activity and the activity of the mature peptide was confirmed using a synthetic peptide. We examined the expression of cathelicidin during cod larvae early development using real-time PCR and detected expression that varied in the course of the first 68 days post hatching (dph). Two groups of larvae having a different food regime were compared. Cathelicidin expression was found to differ between the two groups and this could be linked to their food input. The presence and rapid adjustment of cathelicidin expression in the larvae indicate that the immune system of cod is active from early on in development and responds to external stimuli by the production of antimicrobial peptides.