García Patricia, Rodríguez-Coello Arianna, García-Pose Andrea, Fernández-López María Del Carmen, Muras Andrea, Moscoso Miriam, Beceiro Alejandro, Bou Germán
Servicio de Microbiología, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, 15006 A Coruña, Spain.
Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, 28029 Madrid, Spain.
Vaccines (Basel). 2025 Jun 19;13(6):659. doi: 10.3390/vaccines13060659.
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. : P-driven expression of the operon was achieved by λ-Red mutagenesis. 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::. 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. IRTA ΔΔΔ P:: showed robust 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 , and enhanced the ability of fecal extracts to inhibit the adherence of wild-type strains. Upon challenge (IRTA wild-type/20220258), IRTA ΔΔΔ P:: 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. : Inducible expression of type 1 fimbriae enhances mucosal immunity and protection, supporting their inclusion in next-generation vaccines. Future work should assess cross-protection and optimize FimH-mediated targeting for mucosal delivery.
鼠伤寒沙门氏菌是一种主要的人畜共患病原体，其中1型菌毛在肠道定植和免疫调节中起关键作用。本研究旨在通过对1型菌毛的诱导表达进行工程改造，提高先前开发的生长缺陷型菌株（D-谷氨酸和D-丙氨酸双营养缺陷型）的保护性免疫力。通过λ-Red诱变实现了由P驱动的操纵子表达。通过qRT-PCR对表达进行定量，并通过透射电子显微镜观察菌毛形成情况。通过FimH序列分析、酵母凝集、甘露糖结合/抑制试验以及HT-29细胞黏附来评估黏附特性。用IRTA ΔΔΔ或IRTA ΔΔΔ P::经口胃途径免疫BALB/c小鼠。通过临床监测、细菌载量、粪便排出、ELISA试验以及使用粪便提取物的黏附/阻断试验来评估安全性和免疫原性。在用野生型和异源菌株攻击后评估保护作用。IRTA ΔΔΔ P::表现出强大的表达、密集的菌毛覆盖、明显的甘露糖敏感黏附表型以及增强的HT-29黏附。菌毛过表达未改变肠道定植或向肠系膜淋巴结（mLNs）的转移。免疫引发了混合的IgG1/IgG2a，显著增加了针对表达1型菌毛的IgA和IgG，并增强了粪便提取物抑制野生型菌株黏附的能力。在攻击后（IRTA野生型/20220258），IRTA ΔΔΔ P::比IRTA ΔΔΔ更有效地降低了盲肠（-1.46/1.47对数）、大肠（-1.35/2.17对数）、mLNs（-1.32/0.98对数）和全身器官中的感染负担。1型菌毛的诱导表达增强了黏膜免疫和保护作用，支持将其纳入下一代疫苗。未来的工作应评估交叉保护作用，并优化FimH介导的靶向用于黏膜递送。
Zotero 插件