Antimicrobial peptides : immunomodulatory and therapeutic potential for use in Atlantic salmon (Salmo salar)

The continued use of antibiotics will undoubtedly lead to increases in antibiotic resistant bacteria and decreased effectiveness of antibiotic therapeutics. Therefore, a need exists for new therapeutic agents which effectively treat bacterial outbreaks but limit the ability of microbes to become resistant over time. Antimicrobial peptides are innate defense peptides produced by multicellular organisms to combat a wide variety of pathogens present in the environment. They are naturally produced by the host, exhibit remarkably diverse structures and bioactivity both in and amongst species, and function through a variety of bactericidal mechanisms which limit bacterial resistance. These characteristics make them a potential source for the development of new anti-infective agents. The purpose of this thesis was to assess the antimicrobial activity and stimulatory potential of natural and synthetically derived peptides for use in Atlantic salmon (Salmo salar). In this research, four peptides (P9-4, P11-5, P11-6, and Protegrin-1) were shown to be effective at inhibiting salmon pathogens Yersinia ruckeri and Aeromonas hydrophila, with minimum inhibitory concentrations (MIC) of less than 20 μM in saline conditions. Additionally, all peptides were effective against Escherichia coli (MIC between 2.5 μM-40 μM). The antimicrobial activity and haemolytic ability of these peptides was greatly reduced in the presence of serum, with limited haemolysis observed in erythrocytes incubated with 640 μM of each peptide. Furthermore, P9-4 and P11-5 were shown to significantly increase transcription of the chemokine interleukin-8 (IL-8) in serum cultures of Atlantic salmon peripheral blood leukocytes (PBL) following six hour in vitro stimulation. Results from these experiments suggest that the potent antimicrobial and conversely haemolytic ability, of peptides commonly seen in conventional media becomes more modest when subjected to more realistic biologically relevant conditions. Additionally, two peptides were capable of influencing expression of the chemokine IL-8 which plays a role in chemotaxis of immune cells. This provides preliminary evidence for the use of these peptides as immunostimulants. The ability of antimicrobial peptides to modulate the functions of immune cells in Atlantic salmon was explored. Functional assays were used to assess the direct stimulatory capacity of antimicrobial peptides on the induction of phagocytosis, cell proliferation, and respiratory burst which play an important role in the immune system of teleost fish. Synthetic antimicrobial peptides were selected as well as the two known Atlantic salmon cathelicidins (asCATH1 and asCATH2) and examined in these experiments. The synthetic peptides K6L2W3 and HHC-10 were shown to significantly improve phagocytic ability, phagocytic index, and respiratory burst in head kidney leukocytes (HKLs). Additionally, HHC-10 was shown to significantly improve cell proliferation. Of the natural antimicrobial peptides, asCATH2 significantly improved phagocytosis, cell proliferation and respiratory burst whereas asCATH1 did not. Previous work involving these cathelicidins suggested that functional difference exists between these peptides and that asCATH2 may play a multifaceted biological role during infections. This work provides supplemental evidence to support that claim. The findings in this thesis show that some antimicrobial peptides possess potent antimicrobial abilities while others act to modulate cells to improve pathogen destruction and infection clearance. This is in agreement with previous research indicating that peptides exhibit a structure-activity relationship and diverse mechanisms of action. Moreover, this research further supports the potential of antimicrobial peptides as a natural blueprint for new drug development with applications in aquaculture and showcases the versatility of these peptides in teleost fish