García, PatriciaRodríguez-Coello, AriannaGarcía-Pose, AndreaFernández-López, María del CarmenMuras, AndreaMoscoso, MiriamBeceiro Casas, AlejandroBou, Germán2025-07-072025-07-072025-06-19García P, Rodríguez-Coello A, García-Pose A, Fernández-López MDC, Muras A, Moscoso M, Beceiro A, Bou G. Engineering and evaluation of a live-attenuated vaccine candidate with enhanced Type 1 fimbriae expression to optimize protection against Salmonella Typhimurium. Vaccines (Basel). 2025 Jun 19;13(6):659.2076-393Xhttps://hdl.handle.net/2183/45479[Abstract] Background:Salmonella 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. Methods: PtetA-driven expression of the fim operon was achieved by λ-Red mutagenesis. fimA 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 PtetA::fim. 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. Results: IRTA ΔΔΔ PtetA::fim showed robust fimA 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 Salmonella, and enhanced the ability of fecal extracts to inhibit the adherence of wild-type strains. Upon challenge (IRTA wild-type/20220258), IRTA ΔΔΔ PtetA::fim 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. Conclusions: Inducible expression of type 1 fimbriae enhances mucosal immunity and protection, supporting their inclusion in next-generation Salmonella vaccines. Future work should assess cross-protection and optimize FimH-mediated targeting for mucosal delivery.engAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/FimHSalmonella TyphimuriumFecal IgAIntestinal infection modelLive auxotrophic vaccinesMucosal vaccineType 1 fimbriaejournal articleopen access10.3390/vaccines13060659