Thesis:

Diseño, generación y aplicación de péptidos sintéticos y recombinantes derivados de flagelina de vidrio anguillarum como efectores de inmunoestimulación en peces.

The increase in diseases affecting farmed fish has placed Chile’s salmon industry at risk of losing its international competitiveness. The main current challenge is to deepen the understanding of the fish immune response in order to identify and develop molecules that can act as immunological modulators. The use of immunostimulants is an effective strategy to enhance immunocompetence and disease resistance in fish. Many of these are based on highly conserved molecules in microorganisms, known as pathogen-associated molecular patterns (PAMPs), which are recognized by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), present in both leukocytes and epithelial cells. In rainbow trout, two isoforms of TLR5 have been identified: the membrane-bound TLR5M and the soluble TLR5S, both capable of binding flagellin. Activation of TLR5M induces the expression of TLR5S, thereby amplifying the immune response through the expression of pro-inflammatory cytokines and chemokines. Based on the interaction model between Salmonella typhimurium flagellin and mammalian TLR5, synthetic peptides derived from the D1 domain of Vibrio anguillarum flagellin were designed, as well as recombinant proteins corresponding to the complete molecule and to the N-terminal portion of the D1 domain. Activation of model immune cells was evaluated through the overexpression of TLR5S in rainbow trout liver cells and in macrophages from gilthead seabream and rainbow trout, analyzing the expression of genes associated with the innate immune response. The results demonstrated that peptide S18 and the recombinant proteins induced the overexpression of cytokines IL-1β and TNF-α, as well as the chemokine IL-8 in vitro—key pro-inflammatory genes involved in the regulation of the innate immune response in fish. In conclusion, the molecules generated in this thesis, both synthetic and recombinant, exhibit immunomodulatory potential comparable to the effect of PAMPs on fish leukocytes. Future in vivo studies could confirm their potential application in modulating a stronger and more robust immune response in mass-cultured fish.