Differential Adjuvant Activity by Flagellins from Escherichia coli, Salmonella enterica Serotype Typhimurium, and Pseudomonas aeruginosa

(1) <b>Background</b>: The adjuvant properties of flagellin from various bacterial species have been extensively studied; however, a systematic comparison of the immunoadjuvant effects of flagellins from different bacterial species is lacking. This study aims to analyze the amino acid sequences and structural features of flagellins from <i>Escherichia coli</i> (FliC<i>_E.C</i>), <i>Salmonella enterica</i> serotype Typhimurium (FliC<i>_S.T</i>), and <i>Pseudomonas aeruginosa</i> (FliC<i>_P.A</i>), and to evaluate their adjuvant activities in terms of Toll-like receptor 5 (TLR5) activation, antibody production, and cytokine responses in a murine model. (2) <b>Methods</b>: Bioinformatics analysis was conducted to compare the amino acid sequences and structural domains (D0, D1, D2, and D3) of flagellins from the three bacterial species. PyMol atomic models were used to confirm structural differences. Toll-like receptor 5 (TLR5) activation assays were performed to measure IL-8 and TNF-α production in vitro. The IgG antibody titers against the model antigen FaeG and cytokine responses, including IL-4 and TNF-α secretion were evaluated in a murine model. (3) <b>Results</b>: Bioinformatics analysis revealed that the D0 and D1 domains are highly conserved, whereas the D2 and D3 domains exhibit significant variability across the three species. Structural analysis via PyMol confirmed these differences, particularly in the D2 and D3 domains. TLR5 activation assays showed that FliC<i>_S.T</i> and FliC<i>_P.A</i> induced higher levels of IL-8 and TNF-α production compared to FliC<i>_E.C</i>, indicating species-specific variations in TLR5 activation. In the murine model, FliC<i>_S.T</i> as an adjuvant produced higher antibody titers against FaeG and increased IL-4 secretion in splenocytes compared to FliC<i>_E.C</i> and FliC<i>_P.A</i>. FliC<i>_P.A</i> induced higher TNF-α expression than FliC<i>_S.T</i> and FliC<i>_E.C</i>, suggesting FliC<i>_S.T</i> and FliC<i>_P.A</i> are more effective at inducing T-cell responses. (4) <b>Conclusions</b>: This study highlights the potential of FliC<i>_S.T</i> and FliC<i>_P.A</i> as potent vaccine adjuvants. The results provide insights into the structure-function relationships of these flagellins and support their application in enhancing immune responses against diverse pathogens.