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

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

Relative expression of asCATH1 and 2 in the gill (A) and spleen (B) of Atlantic salmon post-infection with <i>Y. ruckeri</i>.

<p>Gene expression was measured by quantitative real-time PCR. Expression at 8, 24, 48, 72 and 96 h post-infection was compared to expression at 0 h. Each bar represents the mean ± SE of at least 4 fish sampled at each time point. * indicates a significant upregulation of cathelicidin expression compared to expression at 0 h (p<0.05, as assessed by one-way analysis of variance and Dunnett's post-test).</p

Antibacterial activity of asCATH1 and 2.

<p>Bacteria were incubated with varying concentrations of cathelicidin peptides for 18 h before absorbance was read at 600 nm to determine bacterial growth. MIC values are shown as the range of concentrations which produced a 50% reduction in growth compared to a control without cathelicidin.</p

Oligonucleotide primers used in real-time pcr experiments.

<p>Oligonucleotide primers used in real-time pcr experiments.</p

Kinetic profile of IL-8, IL-1 and IL-18 gene expression in Atlantic salmon peripheral blood leukocytes stimulated with asCATH1 (A,C,E) and asCATH2 (B,D,E).

<p>Gene expression was assessed by real-time PCR at 0, 2, 5, 8 and 16 h post incubation with asCATH1 or asCATH2 at 2.5 µM (A,B), 5 µM (C,D) and 10 µM (E,F). Expression was normalised to the mean expression of four stable reference genes. Data shown are the means ± SE of quadruplicate PBL samples assayed in duplicate by quantitative real-time PCR and presented as fold induction compared to 0 h. * indicates a significant fold induction (P<0.05) compared to 0 h while the dotted line represents the minimum fold change (2 fold) deemed biologically significant.</p

Antibacterial activity of asCATH1 and 2 against three species of bacteria - <i>Y. ruckeri</i> (A), <i>V. anguillarum</i> (B) and <i>E. coli</i> (C).

<p>Bacteria were grown to mid-log phase then incubated for 18 h with varying concentrations of the Atlantic salmon cathelicidins. LL-37 was included as a control for bacterial killing and <i>E. coli</i> ATCC 25922 was included as a control bacterium. Absorbance was read at 600 nm and percentage growth was calculated by comparison to the no inhibition control lane in the assay. The MIC was defined as 50% inhibition, which is indicated by a dotted line on each plot. Data shown are the mean ± SE of triplicate wells.</p

Haemolytic activity of the Atlantic salmon cathelicidins to Atlantic salmon erythrocytes.

<p>Erythrocytes were incubated with varying concentrations of the cathelicidins for 2 h and the absorbance of the well supernatants was read at 405 nm to detect released haemoglobin. Percentage haemolysis was calculated from 100% lysis controls, which were incubated with 0.2% Triton-X 100. Data shown are the means ± SD of duplicate wells.</p