Synthesis and immunochemistry of lipopolysaccharide epitopes containing 4-Amino-4-deoxy-L-arabinose

Gram-negative bacteria have various efficient defense mechanisms against the immune system of their respective host at their disposal. The outer membrane of the bacterial cell wall exerts a protective function and is characterized by the presence of numerous negatively charged substituents such as sugar acids and –phosphates. The barrier function of the cell membrane, however, is counteracted by positively charged proteins (cationic antimicobrial peptides) provided by the innate immune system. Conversely, by decorating their membrane with positively charged aminosugars, bacteria become resistant. Whereas the genetic and biosynthetic basis of this resistance mechanism is being intensively studied, much less is known on the molecular details and antigenic properties of these additional sugar modifications. Moreover, the incorporation of these molecules may serve as potential signals for the further elongation of the sugar chains and for the attachment of fatty acids onto membrane components. An example is seen in bacteria causing bubonic plague, where the degree of aminosugar substitution seems to be correlated with decreasing growth temperature as a means of microbial adaption to the insect host. These modifications are of importance in phytopathogenic Burkholderia which is used as a biological fungicide and for soil decontamination, but which is of increasing clinical importance as a human pathogen in cystic fibrosis leading via dysfunction of the respiratory tract to the letal cepacia syndrome. Burkholderia strains are often multiresistant and may even survive within macrophages. In the framework of the project complex structural units of these membrane domains will be synthesized and linked to bovine serum albumin to generate immunoreagents. The target compounds comprise the aminosugar as well as additional sugar acid and phosphate substituents, to investigate their steric and chemical interactions. Within well-established international cooperations the synthetic antigens will serve to characterize existing antibodies and to generate novel antibodies which will be employed as tools to study the biosynthesis and further modifcations of the membrane. In this context, the biosynthesis of a stable sugar acid will be addressed, which most likely is being generated by a novel enzymatic activity. Finally, the compounds will be used as ligands for crystal studies with antibody fragments in order to investigate the influence of the aminosugar substitution on the recognition or masking of exposed sugar acid residues.