Elsevier

Fish & Shellfish Immunology

Volume 59, December 2016, Pages 456-468
Fish & Shellfish Immunology

Full length article
Antimicrobial and host cell-directed activities of Gly/Ser-rich peptides from salmonid cathelicidins

https://doi.org/10.1016/j.fsi.2016.11.004Get rights and content

Highlights

  • Salmonid cathelicidin-derived peptides show medium-sensitive antimicrobial activity.

  • They are not cytotoxic to fish cells and promote proliferation of trout fibroblasts.

  • Peptide STF (1–37) potentiates phagocytosis and respiratory burst in trout leukocytes.

  • STF (1–37) and β-glucan act synergistically to immunostimulate trout leukocytes.

Abstract

Cathelicidins, a major family of vertebrate antimicrobial peptides (AMPs), have a recognized role in the first line of defense against infections. They have been identified in several salmonid species, where the putative mature peptides are unusually long and rich in serine and glycine residues, often arranged in short multiple repeats (RLGGGS/RPGGGS) intercalated by hydrophobic motifs. Fragments of 24–40 residues, spanning specific motifs and conserved sequences in grayling or brown, rainbow and brook trout, were chemically synthesized and examined for antimicrobial activity against relevant Gram-positive and Gram-negative salmonid pathogens, as well as laboratory reference strains. They were not active in complete medium, but showed varying potency and activity spectra in diluted media. Bacterial membrane permeabilization also occurred only under these conditions and was indicated by rapid propidium iodide uptake in peptide-treated bacteria. However, circular dichroism analyses indicated that they did not significantly adopt ordered conformations in membrane-like environments. The peptides were not hemolytic or cytotoxic to trout cells, including freshly purified head kidney leukocytes (HKL) and the fibroblastic RTG-2 cell line. Notably, when exposed to them, HKL showed increased metabolic activity, while a growth-promoting effect was observed on RTG-2 cells, suggesting a functional interaction of salmonid cathelicidins with host cells similar to that shown by mammalian ones. The three most active peptides produced a dose-dependent increase in phagocytic uptake by HKL simultaneously stimulated with bacterial particles. The peptide STF(1–37), selected for further analyses, also enhanced phagocytic uptake in the presence of autologous serum, and increased intracellular killing of live E. coli. Furthermore, when tested on HKL in combination with the immunostimulant β-glucan, it synergistically potentiated both phagocytic uptake and the respiratory burst response, activities that play a key role in fish immunity. Collectively, these data point to a role of salmonid cathelicidins as modulators of fish microbicidal mechanisms beyond a salt-sensitive antimicrobial activity, and encourage further studies also in view of potential applications in aquaculture.

Introduction

Antimicrobial peptides (AMPs) are an important component of the innate immune system of vertebrates, contributing to the first line of defense against microbial pathogens. Several different families of AMPs have been described, and their members often display broad-spectrum antimicrobial activities and in many cases also immunomodulatory functions [1], [2], [3]. Families of AMPs isolated in fish include some that are also present in other classes of vertebrates, such as defensins, cathelicidins, and hepcidins, as well as others that are peculiar to fish [4].

Cathelicidins are characterized by a conserved N-terminal proregion that contains a cathelin-like motif, with the antimicrobial activity being located in the variable C-terminal region [5]. This region is generally quite cationic and, when released from the proregion, generally adopts an amphipathic active conformation on interaction with bacterial membranes. These two features promote the initial interaction and subsequent insertion into bacterial membranes, whose integrity is eventually compromised.

To date, cathelicidins have been identified in several salmonid species [6], [7], [8], [9], [10], the smelt Osmerus mordax [8], the ayu Plecoglossus altivelis [11], and the Atlantic cod Gadus morhua [7], [12]. An ancient ancestor cathelicidin has also been found in the jawless hagfish Myxine glutinosa [13]. Two different cathelicidins, rtCATH1 and rtCATH2 are present in the rainbow trout Oncorhynchus mykiss [6] as well as in most other Salmonidae [8], and two additional cathelicidin genes have been recently identified in rainbow trout [14]. The C-terminal antimicrobial domains in the paralogous CATH1 and CATH2 peptides have rather different sequences, while there is a convincing homology between orthologous peptides from different salmonid species. Both CATH1 and CATH2 C-terminal regions are unusually long with respect to most other vertebrate cathelicidins, and rich in serine/glycine residues. They show an initial QKIRTRR sequence that is highly conserved in Salmonidae, Osmeridae and Gadidae families [8], while the downstream sequences are quite divergent and characterized by the presence of short multiple repeats, such as RLGGGS or RPGGGS, sometimes intercalated by hydrophobic motifs (LIG, IAGA, AGFI).

Peptides derived from the long C-terminal cathelicidin regions of O. mykiss [6], [14], [15] and other fish [9], [11], [16], [17] were shown to be antibacterial against several species of Gram-positive and Gram-negative bacteria, including fish pathogens. However, in these studies antibacterial assays were either carried out in diluted media or with unspecified conditions, so that the often discordant data does not definitely demonstrate that the principal biological function of the C-terminal peptides is a direct antibacterial activity. In this respect, atlantic salmon cathelicidins have been shown to be ineffective in vitro against a pathogen which upregulated their expression in vivo during bacterial infection, thus suggesting an immunomodulatory role of these molecules [16]. Their ability to stimulate cytokine gene expression in salmon leukocytes [16], and similar effects displayed by trout cathelicidins on leukocytes [14] and epithelial cells [18], support a role for salmonid cathelicidins in fish immunity beyond that simply being endogenous antibiotics.

In this study we have investigated the functions of selected fragments from the C-terminal antimicrobial domain of salmonid cathelicidins, in terms of both the direct antimicrobial and immunomodulatory activities. Peptide fragments from brown trout, grayling, rainbow trout and brook trout cathelicidins were evaluated against a bacterial panel including both reference strains and trout pathogens. The more active peptides were further characterized for their effects on host cell viability as well as for the ability to modulate phagocyte functions, such as phagocytosis and respiratory burst activity. In the case of a brown trout peptide, these effects have also been examined in combination with the fish immunostimulant β-glucan. Results obtained extend and enhance previous findings on the biological functions of these important immune effectors beyond direct microbial killing, and support potential applications of these compounds in aquaculture.

Section snippets

Reagents

l-α-phosphatidylglycerol (PG, egg yolk) and cardiolipin (dPG, bovine heart) were from Avanti polar lipids (Alabaster, AL, USA), lipopolysaccharide (LPS, Salmonella minnesota) and propidium iodide (PI) were from Sigma-Aldrich (St. Louis, MO, USA). Dehydrated media for microbiological assays (Mueller-Hinton broth, tryptic soy broth and agar technical) were obtained from Difco laboratories (Detroit, MI, USA). Solutions, media and supplements used for cell culture and leukocyte purification were

Analysis and synthesis of C-terminal peptides from CATH1 and CATH2 cathelicidins

The C-terminal peptides from fish cathelicidins CATH1 and CATH2 were selected for synthesis according to the predicted amino acid sequences encoded by exon IV of the respective cathelicidin genes (AMP domain, see Fig. 1) [8]. Due to the length of the putative AMPs, fragments including different motifs or conserved sequences from both types of cathelicidins were selected for functional evaluation of their relative importance in antimicrobial activity. Three peptides, one from CATH1 of Salmo

Conclusions

Overall the data presented in this study point to a role of the salmonid cathelicidins in activation of phagocyte-mediated microbicidal mechanisms rather than as canonical AMPs principally showing direct antimicrobial activity. Salmonid CATHs are characterized by unique, intrinsically disordered sequences, and kill or inhibit bacterial pathogens only in low-salt conditions unlikely to occur physiologically. Given the prominent function of phagocytic leukocytes in fish defense against infection,

Acknowledgements

This study was carried out with the financial support of the project “INNOV-H2O”, Italy-Slovenia Cross-Border Cooperation Programme 2007–2013 (96/2009) funded by the European Regional Development Fund for Territorial Cooperation and National public funds, and FRA 2014 project of the University of Trieste. The authors are grateful to Prof. Marco Galeotti and Prof. Emilio Tibaldi (Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine) for their help in providing

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