Abstract

Based on the structural knowledge of TLR5 surface and using blind docking platforms, peptides derived from a truncated HMGB1 acidic tail from Salmo salar was designed as TLR5 agonistic. Additionally, a template peptide with the native N-terminal of the acidic tail sequence as a reference was included (SsOri). Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. The best peptides, termed 6WK and 5LWK, were selected for chemical synthesis and experimental functional assay. The agonist activity by immunoblotting and immunocytochemistry was determined following the NF-kappa Bp65 phosphorylation (p-NF-kappa Bp65) and the nuclear translocation of the NF-kappa Bp65 subunit from the cytosol, respectively. HeLa cells stably expressing a S. salar TLR5 chimeric form (TLR5c7) showed increased p-NF-kappa Bp65 levels regarding extracts from flagellin-treated cells. No statistically significant differences (p > 0.05) were found in the detected p-NF-kappa Bp65 levels between cellular extracts treated with peptides or flagellin by one-way ANOVA. The image analysis of NF-kappa Bp65 immunolabeled cells obtained by confocal microscopy showed increased nuclear NF-kappa Bp65 co-localization in cells both 5LWK and flagellin stimulated, while 6WK and SsOri showed less effect on p65 nuclear translocation (p < 0.05). Also, an increased transcript expression profile of proinflammatory cytokines such as TNF alpha, IL-1 beta, and IL-8 in HKL cells isolated from Salmo salar was evidenced in 5LWK -stimulated by RT-PCR analysis. Overall, the result indicates the usefulness of novel peptides as a potential immunostimulant in S. salar.