Structure/activity evaluation of the bacterial membrane constituents involved in the immune innate system

Glycidic cell membrane constituents are a class of carbohydrate-based molecules that include Lipopolysaccharides (LPSs), Capsular polysaccharides (CPSs) and Peptidoglycan (PGN). Surrounding the external surface of cell membrane, these molecules are strongly involved in different biological mechanisms such as adhesion, colonization and infection of host organisms. Thus, the knowledge of oligosaccharidic moiety is fundamental to understand the bacterial infection mechanisms . This PhD project, during these last three years, involved performing the analysis of the structure of Lipopolysaccharides (LPSs) and Capsular polysaccharides (CPSs) produced by different bacterial strains and the preparation of new glycomolecules with potential biological activity. In this paragraph are summarized the themes developed in the four chapters that follow. This PhD project can be divided under four different main topics: 1. Use of lipooligosaccharide to prepare liposomes; 2. Analysis of the glycidic constituents of cell membrane from bacteria isolated from mice intestinal crypt; 3. Characterization of CPSs produced by five clinical isolates of Acinetobacter baumannii. 4. Preparation of the building block for the synthesis of new glycoconjugates with potential activity against HIV-1 The first thematic was developed in collaboration with Prof. L. Paduano’s team. The principal aim was to use oligosaccharides from bacteria to functionalize Ru(II)-liposome in order to increase their life-time in the body and to promote, eventually, a target specific delivery . Lipooligosaccharide from Rhizobium rubiT was selected because this bacterium produces a LOS without inflammatory activity on human cells and its outer core displays the human Lewis B domain . Different liposomes with different percentage of LOS were obtained and characterized by DLS and Cryo-TEM analyses. The second thematic was developed in collaboration with Prof. Sansonetti of Pasteur Institute of Paris that have isolated a panel of different bacteria from the intestinal crypt of healthy mice. He introduced the concept of Crypt-Specific Core Microbiota (CSCM): a panel of different bacteria capable to colonize murine colinic crypt . The existence of CSCM opens many questions, and the main point regards the role of bacterial CPSs and LPSs in the host immune system evasion of these bacteria. In this context the structure of the O-antigen from LPS produced by Stenotrophomonas maltophilia and Delftia acidovorans and the structure of LOS produced by Acinetobacter parvus were resolved. With regard to the third thematic, this work was conducted in collaboration with Dr. Johanna Kenyon at the University of Sydney who works on the elucidation of genes responsible for LPS and CPS biosynthesis in Acinetobacters species relevant in nosocomial infections. The final purpose of this project is understanding the mechanisms involved in triggering inflammation by bacteria and escaping the host immune system, together with the idea to understand the biosynthetic pathway of CPS. So far the structure of CPSs produced by different strains of Acinetobacter baumannii were resolved on the basis of chemical and spectroscopical analysis. The last part of this thesis was focused on the preparation of building blocks for the synthesis of new aggregates with biological activity against HIV-1. Recently a monoclonal antibody (2G12) was isolated that recognized mannose oligosaccharide repeating units present on the capsid of surface of HIV-1 virus and inhibits its proliferation . Unfortunately, this antibody is originated only after the HIV infection. In this context, this project was carried out involving the preparation of new glycoconjugates presenting a mannose a-(1→2) oligosaccharide moiety anchored on a chitosan oligosaccharide via click conjugation. According to the strategy chosen, the azide carrier is a low molecular weight derivative of chitosan, while the alkyne derivative was prepared starting from yeast mannan. Chemical procedures for preparation, purification and characterization of different chitoazidooligosaccharides and mannose alkyn- oligosaccharides having different length and different grade of functionalization were optimized.