Evidence of an Antimicrobial-Immunomodulatory Role of Atlantic Salmon Cathelicidins during Infection with Yersinia ruckeri

Abstract

Cathelicidins are a family of antimicrobial peptides that act as effector molecules of the innate immune system with broad-spectrum antimicrobial properties. These evolutionary conserved cationic host-defence peptides are integral components of the immune response of fish, which are generally believed to rely heavily on innate immune defences to invading pathogens. In this study we showed that Atlantic salmon cathelicidin 1 and 2 (asCATH1 and asCATH2) stimulated peripheral blood leukocytes increasing the transcription of the chemokine interleukin-8. Further, functional differences were identified between the two cathelicidins. In the presence of serum, asCATH1 displayed greatly diminished host haemolytic activity, while the constitutively expressed asCATH2 had no haemolytic activity with or without serum. These findings support our hypothesis that fish cathelicidins exert their primary antimicrobial action at the site of pathogen invasion such as epithelial surfaces. Further, we hypothesise that like their mammalian counterparts in the presence of serum they act as mediators of the innate and adaptive immune response via the release of cytokines thus indirectly protecting against a variety of pathogens. We highlight the importance of this immunomodulatory role from the involvement of asCATHs during an infection with the fish pathogen Yersinia ruckeri. While we were able to demonstrate in vitro that asCATH1 and 2, possessed direct microbicidal activity against the fish pathogen, Vibrio anguillarum, and a common gram negative bacterium, Escherichia coli, little or no bactericidal activity was found against Y. ruckeri. The contribution of either asCATH in the immune response or as a potential virulence factor during yersiniosis is highlighted from the increased expression of asCATH1 and 2 mRNA during an in vivo challenge with Y. ruckeri . We propose that Atlantic salmon cathelicidins participate in the interplay between the innate and adaptive immune systems via the release of cytokines enabling a more effective response to invading pathogens.

Distinctive Structural Hallmarks and Biological Activities of the Multiple Cathelicidin Antimicrobial Peptides in a Primitive Teleost Fish

“…In addition to the direct antimicrobial effects, mammalian CAMPs possess other modulatory activities, such as LPS-neutralizing (20,21) and IL-8-stimulating activities (22,23,28,38,39). Furthermore, the LPS-binding activities are often positively correlated with their antibacterial activities (21).…”

“…Human CAMP LL-37 was used as a reference. Antibacterial activity assay was performed by 2-fold microtiter broth dilution method as previously described with minor modifications (27,28). Briefly, 80 ml serial 2-fold dilutions of peptides prepared in water were added to each corresponding well of a 96-well microtiter plate (Thermo Scientific).…”

“…Hemolytic activities of trout CAMPs, at the concentration of 60 mM, to trout and human erythrocytes were evaluated as previously described (27,28). The absorbance was measured at 405 nm using an ELx800 microplate reader (BioTek).…”

“…Trout peripheral blood leukocytes (PBLs) were isolated and stimulated with CAMPs at the concentration of 10 mM as previously described (28). Total RNA was isolated from the PBLs at 0, 4, 8, 16, and 24 h postincubation.…”

“…In human and Atlantic salmon (Salmo salar), CAMPs have been associated with the ability to stimulate IL-8 expression (22,23,28,38,39). As shown in Fig.…”