Screening of bacterial molecules with antagonistic or adjuvant activity
Creators: name: Fregolino, Eleonora
Publisher: name: Università degli Studi di Napoli Federico II
Year: 2010

description: The Lipopolysaccharides (LPSs) are the major constituents of the outer membrane of Gram-negative bacteria and the interest in their chemical characterization is motivated by the close correlation existing between the structure and the biological activity of these molecules. In particular, the glycolipid portion of the LPS, the Lipid A, is the real endotoxic principle since it triggers the innate immune system of infected organism. The intrinsic structural variability of the Lipid A is on the basis of its different biological activity, ranging from the activation to the inhibition of an inflammatory process, and for this reason a Lipid A is classified as agonist or antagonist. The ability of Lipid A to stimulate the innate immune system that in turn activates the adaptive immunity, makes this molecule a useful vaccine adjuvant.  
The present thesis deals with the structural study of Lipopolysaccharides and Capsular polysaccharides (CPSs) from different Gram-negative bacteria: 
The Lipooligosaccharide (LOS) structure produced by Rhyzobium radiobacter Rv3 was determined through spectroscopic studies. It shows the occurrence of a mannose oligosaccharide analogue to the D1 arm of the gp120 oligosaccharide shield, recognized by the broadly neutralizing human anti-HIV antibody 2G12. This discovery opened the attractive way to the use of this bacterium for potential HIV vaccine development: a new neoglycoprotein was realized, possessing as antigen portion the modified LOS linked, through a spacer, to the commercial Bovine Serum Albumin (BSA).   
In order to find a Lipid A with a potential antagonistic activity, structural features of this molecule, produced by the non-pathogen Cupriavidus necator  DSM 13523, were explored. It produces mainly a symmetric hexa-acyl Lipid A made up of a diphosphorylated disaccharide glucosamine backbone, peculiarly substituted only by fourteen carbons chains. Different Lipid As from non-pathogenic bacteria as Rhodobacter capsulatus, Rhodobacter spheroids and Chromobacter violaceum were described as antagonists; in particular, the Lipid A from Chromobacter violaceum possessed an hexa-acyl symmetric structure, similarly to that found for Cupriavidus necator. These assumptions increase the interest to the investigation of the biological activity of Cupriavidus necator Lipid A. The O-polysaccharide repeating unit produced by Cupriavidus necator DSM 13523 was also disclosed through spectroscopic analyses and it consisted only of deoxy sugars: four rhamnose units and one 4-deoxy-β-D-arabinohexose unit. 
The complete structural characterization of the Lipooligosaccharide produced by a clinical isolate of Acinetobater baumannii strain SMAL was determined to provide valuable information on the mechanism activity adopted by this emerging pathogen. The Core oligosaccharide is formed of twelve sugar residues, organized in a highly branched inner Core moiety. It was found to be similar to that of Acinetobacter baumannii ATCC 19606, although the LOS from the strain SMAL presented an enhanced zwitterionic character. The features presented by this structure, might reflect the adaptation of the bacterium to the host environment; in particular, the presence of cationic amino sugars decrease the net negative charge of the Core region and thus might shield the bacterial membrane from the effect of host defence agents, like the cationic antimicrobial peptides. Regarding to the Lipid A structure, mass spectrometry experiments revealed that it is composed of a heterogeneous blend of molecules differing for type and degree of acylation. The proportion among the differently acylated species changed in response to the growth conditions. 
 Capsular Polysaccharides of two clinical isolates of A. baumannii strains SMAL and MG1 were isolated and their structure was elucidated. The A. baumannii MG1 CPS consisted of a linear aminopolysaccharide and it is quite similar to the O-Chain of the LPS from A.baumannii strain 24. The repeating unit of the CPS produced by strain SMAL comprised a branched pentasaccharide backbone, which was previously described as the O-antigen of another Acinetobacter baumannii species (strain ATCC 17961), while here it occurs as a capsule. These results highline that care must be taken in the development of these diagnostic devices, because similar if not identical polysaccharides might be constituents of the Lipopolysaccharides O-antigen or of the bacterial envelope capsule, as reported for A. baumannii SMAL and MG1. Furthermore, this study may contribute significantly to the establishment of serotyping scheme for this bacterium. 
The study conducted on the LPS produced by the enterohaemorrhagic E.coli O157:H-, subject of a collaboration, demonstrated that it is was not affected by the phenomenon of the phase variation but also that it produced two cyclic forms of the Enterobacterial common antigen: the tetrameric ECACYC and the pentameric ECBCYC. The presence of ECACYC for a pathogenic strain of E.coli together with the pentameric form, ECBCYC, represents a new datum and further studies will be focused on the study of biological functions of these molecules and eventually on their implication as a virulence factor. 
The last part of the thesis is the result of a collaboration with the  Novartis Vaccines Diagnostic of Siena concerning the analyses of the polysaccharide components on the Outer Membrane Vesicles (OMVs) of Neisseria meningitidis group B (MenB). In particular, one of the target was focused on the isolation and structural characterization of the Lipooligosaccharide in order to define the LOS immunotypes of the strain used for vaccine production. The Lipid A family of the strain was also structurally defined through MALDI mass spectrometry and the penta-acyl Lipid A resulted the predominant specie. Finally, the content of LOS and CPS outside vesicles was determined, as well.