Full length articleAntimicrobial and host cell-directed activities of Gly/Ser-rich peptides from salmonid cathelicidins
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
References (60)
- et al.
AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense
Trends Immunol.
(2009) - et al.
The expanding scope of antimicrobial peptide structures and their modes of action
Trends Biotechnol.
(2011) - et al.
Characterisation of cathelicidin gene family members in divergent fish species
Mol. Immunol.
(2008) - et al.
The salmonid cathelicidins: a gene family with highly varied C-terminal antimicrobial domains
Comp. Biochem. Physiol. B Biochem. Mol. Biol.
(2009) - et al.
Structural and functional characterization of CATH_BRALE, the defense molecule in the ancient salmonoid, Brachymystax lenok
Fish. Shellfish Immunol.
(2013) - et al.
Bacterial DNA indicated as an important inducer of fish cathelicidins
Mol. Immunol.
(2008) - et al.
Identification and characterization of a novel cathelicidin from ayu, plecoglossus altivelis
Fish. Shellfish Immunol.
(2011) - et al.
Cod cathelicidin: isolation of the mature peptide, cleavage site characterisation and developmental expression
Dev. Comp. Immunol.
(2011) - et al.
Hagfish intestinal antimicrobial peptides are ancient cathelicidins
Peptides
(2003) - et al.
Functional characterization of codCath, the mature cathelicidin antimicrobial peptide from Atlantic cod (Gadus morhua)
Peptides
(2011)
Immunomodulatory effect of cathelicidins in response to a beta-glucan in intestinal epithelial cells from rainbow trout
Dev. Comp. Immunol.
Antimicrobial activity of Bac7 fragments against drug-resistant clinical isolates
Peptides
Dual mode of action of Bac7, a proline-rich antibacterial peptide
Biochim. Biophys. Acta
SMAP-29: a potent antibacterial and antifungal peptide from sheep leukocytes
FEBS Lett.
Anaphylatoxin-like molecules generated during complement activation induce a dramatic enhancement of particle uptake in rainbow trout phagocytes
Dev. Comp. Immunol.
Measurement of invasion by gentamicin resistance
Methods Enzymol.
Evolution of the primate cathelicidin. correlation between structural variations and antimicrobial activity
J. Biol. Chem.
Circular dichroism and 1H nuclear magnetic resonance studies on the solution and membrane structures of GAP-43 calmodulin-binding domain
J. Biol. Chem.
Discovery and characterization of two isoforms of moronecidin, a novel antimicrobial peptide from hybrid striped bass
J. Biol. Chem.
Mammalian defensins in immunity: more than just microbicidal
Trends Immunol.
Host defense peptides: front-line immunomodulators
Trends Immunol.
Cathelicidin modulates the function of monocytes/macrophages via the P2X7 receptor in a teleost, plecoglossus altivelis
Fish. Shellfish Immunol.
Chapter 2 Use of fish cell lines in the toxicology and ecotoxicology of fish. Piscine cell lines in environmental toxicology
The human cathelicidin LL-37 modulates the activities of the P2X7 receptor in a structure-dependent manner
J. Biol. Chem.
Mechanistic and functional studies of the interaction of a proline-rich antimicrobial peptide with mammalian cells
J. Biol. Chem.
Antimicrobial mechanisms of fish leukocytes
Dev. Comp. Immunol.
Antimicrobial responses of teleost phagocytes and innate immune evasion strategies of intracellular bacteria
Dev. Comp. Immunol.
Cathelicidin-BF suppresses intestinal inflammation by inhibiting the nuclear factor-kappaB signaling pathway and enhancing the phagocytosis of immune cells via STAT-1 in weanling piglets
Int. Immunopharmacol.
Ecotoxicology and innate immunity in fish
Dev. Comp. Immunol.
Roles of antimicrobial peptides such as defensins in innate and adaptive immunity
Ann. Rheum. Dis.
Cited by (21)
Intervention of antimicrobial peptide usage on antimicrobial resistance in aquaculture
2022, Journal of Hazardous MaterialsCitation Excerpt :Antimicrobial peptides can play a critical role in killing/controlling the microorganisms as well as modulating the immune response so that they have been used as alternatives of antibiotics in aquaculture (Tassanakajon et al., 2018). Current studies on antimicrobial peptides in aquaculture mainly focus on their function by reporting that antimicrobial peptides enable the build-up of efficient immune responses in shrimps (Tassanakajon et al., 2018), enhance phagocytic uptake and the intracellular killing of live microorganisms for trout cultivation (D'Este et al., 2016), and show broad defending activities against diverse microorganism (Muncaster et al., 2018). Will antimicrobial peptides have positive effect on controlling ARGs?
Mammalian antimicrobial peptides
2022, Antimicrobial Peptides: Challenges and Future PerspectivesAn overview of antimicrobial peptides
2022, Viral, Parasitic, Bacterial, and Fungal Infections: Antimicrobial, Host Defense, and Therapeutic StrategiesIn vitro immunomodulatory activities of peptides derived from Salmo salar NK-lysin and cathelicidin in fish cells
2019, Fish and Shellfish ImmunologyCitation Excerpt :It is know that the synthetic C-terminal domain of rainbow trout rtCATH-1 and rtCATH-2 displayed antibacterial activity against Gram-positive or Gram-negative bacteria and low cytotoxic effects on host cells [28,33]. Additionally, purified codCATH showed antifungal activity [34] and some synthetic CATH-derived peptides increased phagocytic uptake by bacterial-stimulated head kidney lymphocytes and intracellular killing of live E. coli [35]. Besides, Atlantic salmon cathelicidin 1 and 2 (asCATH1 and asCATH2) stimulated the transcription of the chemokine interleukin-8 in peripheral blood leukocytes (PBL) [36].
Fundamental Uses of Peptides as a New Model in Both Treatment and Diagnosis
2024, Recent Patents on BiotechnologyApplications of antimicrobial peptides (AMPs) as an alternative to antibiotic use in aquaculture - A mini-review
2023, Annals of Animal Science