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

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: P_tetA-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 P_tetA::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 P_tetA::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 P_tetA::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.

Keywords: FimH; Salmonella Typhimurium; fecal IgA; intestinal infection model; live auxotrophic vaccines; mucosal vaccine; type 1 fimbriae.

Figure 1

Construction and verification of S. Typhimurium IRTA ΔΔΔ P_tetA::fim auxotrophic derivative with inducible expression of type 1 fimbriae. (a) PCR verification of aph(3′)-IIIa tetR P_tetA cassette integration in the native fimA promoter region. The amplicon sizes are 535 bp (IRTA ΔΔΔ) and 2663 bp (IRTA ΔΔΔ P_tetA::fim). Lane M, GeneRuler 1 Kb Plus DNA Ladder. A detailed schematic representation of the mutagenesis protocol is depicted in Figure S1. (b) Expression levels of the fimA gene, determined by qRT-PCR (mean ± SD; n = 3 biological replicates) in IRTA GN-3728 and its auxotrophic derivatives obtained under different culture conditions, and normalized to the rpoD reference gene. Primers and UPL probes used are listed in Table S1. Student’s t-test (Welch’s correction): * p < 0.05, *** p < 0.005, **** p < 0.0001, relative to IRTA GN-3728, and ^# p < 0.05, ^### p < 0.005 between the indicated groups. (c) TEM micrographs of fimbriation patterns. Scale bar: 0.5 µm. Static, 3rd serial passage without agitation; ATc, 100 ng/mL.

Figure 2

Characterization of S. Typhimurium IRTA ΔΔΔ P_tetA::fim auxotrophic derivative. (a) In vitro binding to immobilized BSA-mannose measured as absorbance (mean ± SD) following crystal violet staining. (b) Percentage of bacterial adherence to HT-29 cells (mean ± SD). **** p < 0.0001, Student’s t-test (Welch’s correction). n.s., not significant. D-mannose: 4-Aminophenil α-D-mannopyranoside.

Figure 3

Comparison of humoral immune responses induced by S. Typhimurium auxotrophic derivatives. (a,b) Log₁₀ 1/Endpoint titer of fecal IgA and serum IgG, IgG1 and IgG2a antibodies produced by BALB/c mice on day 47 post-vaccination. ELISA plates were coated with (a) IRTA GN-3728 or (b) IRTA ΔΔΔ P_tetA::fim. Antibody responses were measured in mice immunized with three doses of 10⁹ CFU of IRTA ΔΔΔ or IRTA ΔΔΔ P_tetA::fim, as well as in control (saline-treated) mice. Each experimental group consisted of 6–9 mice. (c) Percentage adherence of the indicated S. Typhimurium strains to HT-29 cells (mean ± SD) after pre-incubation with fecal extracts from control mice injected with saline (open circles) or mice immunized with IRTA ΔΔΔ (gray squares) or IRTA ΔΔΔ P_tetA::fim (black triangles). Data are expressed relative to adhesion after pre-incubation with fecal extracts from control mice (100% reference value). Student’s t-test with Welch’s correction: * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001, relative to control mice, or ^# p < 0.05, ^## p < 0.005 between vaccinated groups. n.s., Not significant.

Figure 4

Comparison of protective efficacy mediated by S. Typhimurium auxotrophic derivatives. Bacterial load (Log₁₀ CFU/g) in organs recovered from immunized and control (administered saline) mice, after infection with (a) IRTA GN-3728 (4 × 10⁶ CFU) and (b) 20220258 (2 × 10⁵ CFU). Mann–Whitney U test: * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001, relative to control mice, or ^# p < 0.05, ^## p < 0.005 between vaccinated groups. n.s., Not significant.