Phage-resistance alters Lipid A reactogenicity: a new strategy for LPS-based conjugate vaccines against Salmonella Rissen

<i>Salmonella enterica</i> serovar Rissen (<i>S.</i> Rissen) is an emerging causative agent of foodborne diseases. The current emergence of antibiotic resistance makes necessary alternative therapeutic strategies. In this study, we investigated the potential of a phage-resistant strain of <i>S.</i> Rissen (R^R) as a tool for developing an effective lipopolysaccharide (LPS)-based vaccine. The LPS O-antigen is known to play critical roles in protective immunity against <i>Salmonella</i>. However, the high toxicity of the LPS lipid A moiety limits its use in vaccines. Here, we demonstrated that the acquisition of bacteriophage resistance by <i>S.</i> Rissen leads to structural modifications in the LPS structure. Using NMR and mass spectrometry, we characterized the LPS from phage-resistant strains as a smooth variant bearing under-acylated Lipid A portions (penta- and tetra-acylated forms). We then combined RT-qPCR and NMR-based metabolomics to explore the effects of phage resistance and LPS modification on bacterial fitness and virulence. Finally, we conducted <i>in vivo</i> studies to determine whether lysogeny-induced remodeling of LPS affects the host immune response. Results revealed that the under-acylated variant of LPS from <i>R^R</i> attenuates the inflammatory response in BALB/c mice, while eliciting a specific antibody response that protects against <i>S.</i> Rissen (R^W) infection. In conclusion, our findings suggest that phage resistance, through lipid A modification, may offer a novel strategy for reducing LPS toxicity, highlighting its potential as a promising biological approach for developing LPS-based vaccines against <i>Salmonella</i> infections.