Bacteria produce an astonishingly diverse array of carbohydrate-based macromolecules that serve important physiological roles. The lipopolysaccharide or LPS, for example, is a complex glycoconjugate attached to the outer membranes of Gram-negative bacteria. Conceptually, the LPS can be thought of in terms of three regions: the lipid A component, the core oligosaccharide, and the O-antigen. It is the O-antigen that displays the most variation from species to species and that, in addition to the lipid A moiety, plays a role in virulence. Likewise, the capsular polysaccharides, which surround both pathogenic Gram-positive and Gram- negative bacteria, serve as the first lines of defense against the host immune system. These high molecular weight polysaccharides function by camouflaging cell surface components that would normally elicit the immune response. Often the sugars in the capsular polysaccharides are modified by the attachment of a variety of moieties including an O-methyl phosphoramidate group, which has been shown to be involved in host invasion and bacteriophage recognition. In addition, some capsular polysaccharides contain nonulosonic acids, nine-carbon based monosaccharides that have been implicated in virulence. The intellectual goals of this MIRA award are threefold: (1) to expand upon our current knowledge of the structures and activities of the enzymes involved in the biosynthesis of O-antigen sugars, (2) to provide a molecular framework for understanding the biosynthesis of the O-methyl phosphoramidate group in Campylobacter jejuni, and (3) to explore the structures and functions of the enzymes involved in the biosyntheses of nonulosonic acids from Acinetobacter baumannii, an organism that has been placed into the ?Critical? category by the World Health Organization for the development of new antibiotics. Techniques to be utilized include X-ray crystallography, site-directed mutagenesis, and kinetic analyses. Importantly, preliminary data for many of the proposed investigations are already available. Our studies have and will continue to inform research into bacterial pathogenicity. Given that the LPS and capsular polysaccharides play critical roles in bacterial virulence, the enzymes to be investigated may ultimately serve as targets for antimicrobial drug design.
The goals of this application are to investigate the enzymes involved in the production of unusual dideoxysugars found on the lipopolysaccharides of pathogenic Gram-negative bacteria, to study the enzymes required for the biosynthesis of the O-methyl phosphoramidate modification observed in bacterial capsular polysaccharides, and to examine the enzymes needed for the construction of the nonulosonic acids observed in bacterial surface polysaccharides. The components of this application are linked together in that both unusual sugars and modifications thereof play key roles in bacterial invasion and serum resistance.