Structure of potential LPS agonist and antagonists from different bacterial sources

Lipopolysaccharides (LPS) represent one of the most important glycoconjugates found on the outer membrane (OM) of Gram-negative bacteria cell wall, covering around 75% of its surface. They are crucial for bacterial survival, contributing significantly to the integrity and stability of the OM and protecting the bacterium from the external milieu stress factors. These amphiphilic molecules are divided into three genetically, biologically and chemically distinct domains – a hydrophobic glycolipid portion called lipid A, a repeating glycan termed O-polysaccharide and a core oligosaccharide connecting the two domains. More interestingly, the LPS are classed as PAMPs (Pathogen Associated Molecular Patterns) since they are able to trigger hosts innate immune responses. The key event in the signalling is the recognition of LPS by the TLR4/MD-2 receptorial complex, triggering the activation of immune defences, and stimulating the production of inflammatory cytokines. If the TLR4/MD-2 driven activation of the innate immune response is beneficial to combat the infection, its over-stimulation leads to sepsis and finally life threating septic shock. Nonetheless, modifications in the LPS structure, and more precisely in the lipid A region, affect its immunostimulant properties, including reduction of TLR4/MD-2 activation and even inhibition of signalling caused by agonistic molecules; hence, the search of LPS possessing inhibitory activity is a high importance and interest topic. The PhD thesis is focused on structural or/and immunological characterization of LPSs derived from several human non-pathogenic, Gram-negative bacteria, including acetic acid bacterium, Acetobacter pasteurianus CIP103108; marine sponge symbiont, Endozoicomonas sp. HEX311 and marine bacterium Phaeobacter gallaeciensis BS107 SA/WT. The structural investigations, performed applying various steps of chemical analysis, NMR and mass spectrometry permitted to determinate several novel structures with interesting immunological properties. Moreover, the behaviour of A. pasteurianus and Bradyrhizobium BTAi-1 Δshc LPS upon addition of the MD-2 protein was observed by DOSY-NMR and Cryo-TEM. Finally, the constitution of exopolysaccharide, produced by an ethanologenic bacterium, Zymomonas mobilis was unveiled and its protective role was assessed applying PFG-NMR and DLS measurements.