Aminoarabinose LPS epitopes

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.

Numerous Gram-negative pathogenic bacteria exert efficient defense mechanisms against the immune system of their respective host organisms. Acidic and phosphate-substituted carbohydrates located in the outer membrane of the bacterial cell wall play a major role in bacterial resistance. In particular, positively charged amino sugars (Aminoarabinose) in the bacterial cell membrane counteract the impact of positively charged peptides. Within the project, an efficient synthesis of aminoarabinose has been developed in a multigram scale. Subsequently aminoarabinose was coupled to the sugar acids deoxyoctulosonic acid (Kdo) and octulosonic acid (Ko) corresponding to part structures of the cell wall components of Burkholderia and Proteus strains. Major accomplishments have been achieved by coupling of these carbohydrate ligands to proteins. Thus, reactive end groups were introduced into the carbohydrate units which could be selectively conjugated to bovine serum albumin. The glycoconjugates were then used for the immunization of mice and rabbits and the resulting sera were tested for reactivity with several bacterial lipopolysaccharides. The compounds were highly immunogenic and the sera displayed reactivity against accessible aminoarabinose units in the bacterial cell membrane. These antibody specificities may thus be used for diagnosis of Burkholderia and Proteus infections. Additional conjugates allowing for differentiation between Burkholderia and Proteus are being tested. The neoglycoconjugates will further be exploited in a recently approved international follow-up project in the context of lung diseases such as cystic fibrosis. By contrast, aminoarabinose-phosphate linked to the lipid A part in the membrane was not bound by the antibodies. In order to conclusively assess the antigenic properties of the distal aminoarabinose-phosphate entity, glycoconjugates containing the phosphate-linked aminoarabinose have been prepared and will be used for immunization. The synthesized sugar acids have been cocrystallized with Kdo-specific antibody fragments. Several crystal structures have been solved and the binding affinities have been determined using surface plasmon resonance spectroscopy. It could be shown that additional hydrogen bonding within the binding site did not lead to enhanced binding affinities, whereas hydrophobic modification of the carbohydrate ligands resulted in substantial increase both in specificity and binding affinity, respectively. These results are thus of general interest in the study of antibody-carbohydrate interactions.