Engineering low-endotoxin lipid A in a double auxotroph Pseudomonas aeruginosa to develop safer whole-cell vaccines

Introduction Pseudomonas aeruginosa is a highly adaptable Gram-negative bacterium causing severe respiratory infections, particularly in vulnerable populations. The rise of antimicrobial resistance highlights the urgent need for effective vaccines. We previously developed a live-attenuated vaccine candidate, PAO1 Δ murI Δ alr Δ dadX , a genetically stable double auxotrophic strain that exhibited dose-dependent reactogenicity upon intranasal administration in mice, likely due to lipid A component of lipopolysaccharide (LPS). Methods To reduce LPS-associated toxicity while preserving immunogenicity, we engineered novel strains by inactivating genes involved in lipid A biosynthesis ( htrB1 , htrB2 ) or modification ( pagP , pagL ). Lipid A structural modifications were confirmed by MALDI-TOF MS. Mutants were evaluated for Toll-like receptor 4 (TLR4) activation, virulence attenuation, and their ability to induce immune responses and protection in an acute pneumonia model. Results All engineered strains displayed modified lipid A structures. Mutations in htrB1 and htrB2 reduced TLR4 activation and significantly attenuated virulence following intraperitoneal challenge in mice. Inactivation of pagL resulted in minimal attenuation, whereas pagP inactivation led to marked attenuation without altering TLR4 activation. In the acute pneumonia model, all mutants elicited robust systemic and mucosal immune responses, and conferred strong protection, despite transient weight loss following intranasal administration. Discussion Targeted lipid A modification represents an effective strategy to reduce in vitro reactogenicity while preserving immunogenicity in live-attenuated P. aeruginosa vaccine candidates. Although further optimization may minimize residual in vivo effects, these findings support the potential of engineered strains as vaccine candidates for preventing respiratory infections caused by P. aeruginosa .