Engineered Probiotics Protect against Pseudomonas aeruginosa Post Antibiotic Exposure.

Broad-spectrum antibiotics profoundly disrupt the commensal microbiota and compromise mucosal immunity, creating a vulnerable postantibiotic window that predisposes hosts to opportunistic infections such as Pseudomonas aeruginosa. Strategies for restoring host homeostasis and stimulating mucosal immunity during this period remain limited. Here, we developed a modular and programmable probiotic strategy based on engineered Escherichia coli Nissle 1917 (EcN) that integrates metabolite replenishment with antigen-specific mucosal immunization. The butyrate-overproducing strain restored immune homeostasis after antibiotic exposure, enhanced baseline pulmonary neutrophil levels, and promoted bacterial clearance upon P. aeruginosa challenge. Additionally, synthetic EcN strains were engineered to release outer-membrane vesicles displaying P. aeruginosa antigens (PcrV or OprL), eliciting a robust pathogen-specific mucosal immunity. In antibiotic-exposed mice, metabolite and immunization modules independently reduced the pulmonary bacterial burden and improved survival following P. aeruginosa infection. Combined administration exerted synergistic protection, resulting in higher survival than the butyrate-overproducing strain alone under a lethal P. aeruginosa challenge. Together, this work establishes a versatile synthetic biology framework in which independently engineered probiotic modules can be flexibly combined to regulate microbiota function and deliver targeted immunotherapy, offering a promising alternative to antibiotic-dependent infection control.