Engineering and Evaluation of a Live-Attenuated Vaccine Candidate with Enhanced Type 1 Fimbriae Expression to Optimize Protection Against Salmonella Typhimurium

<b>Background:</b><i>Salmonella</i> Typhimurium is a major zoonotic pathogen, in which type 1 fimbriae play a crucial role in intestinal colonization and immune modulation. This study aimed to improve the protective immunity of a previously developed growth-deficient strain-a double auxotroph for D-glutamate and D-alanine-by engineering the inducible expression of type 1 fimbriae. <b>Methods</b>: P<i>_tetA</i>-driven expression of the <i>fim</i> operon was achieved by λ-Red mutagenesis. <i>fimA</i> expression was quantified by qRT-PCR, and fimbriation visualized by transmission electron microscopy. Adhesive properties were evaluated through FimH sequence analysis, yeast agglutination, mannose-binding/inhibition assays, and HT-29 cell adherence. BALB/c mice were immunized orogastrically with IRTA ΔΔΔ or IRTA ΔΔΔ P<i>_tetA</i>::<i>fim</i>. Safety and immunogenicity were assessed by clinical monitoring, bacterial load, fecal shedding, ELISA tests, and adhesion/blocking assays using fecal extracts. Protection was evaluated after challenging with wild-type and heterologous strains. <b>Results:</b> IRTA ΔΔΔ P<i>_tetA</i>::<i>fim</i> showed robust <i>fimA</i> expression, dense fimbrial coverage, a marked mannose-sensitive adhesive phenotype and enhanced HT-29 attachment. Fimbrial overexpression did not alter intestinal colonization or translocation to mesenteric lymph nodes (mLNs). Immunization elicited a mixed IgG1/IgG2a, significantly increased IgA and IgG against type 1 fimbriae-expressing <i>Salmonella</i>, and enhanced the ability of fecal extracts to inhibit the adherence of wild-type strains. Upon challenge (IRTA wild-type/20220258), IRTA ΔΔΔ P<i>_tetA</i>::<i>fim</i> reduced infection burden in the cecum (-1.46/1.47-log), large intestine (-1.35/2.17-log), mLNs (-1.32/0.98-log) and systemic organs more effectively than IRTA ΔΔΔ. <b>Conclusions</b>: Inducible expression of type 1 fimbriae enhances mucosal immunity and protection, supporting their inclusion in next-generation <i>Salmonella</i> vaccines. Future work should assess cross-protection and optimize FimH-mediated targeting for mucosal delivery.