(294e) Development of Anti-Capsular Polysaccharide Aptamers for High-Throughput Screening and Serotyping

Bacterial pathogens are often encapsulated by a viscous layer of polysaccharide known as the capsule, which serves as the first layer of protection against a diverse array of environmental pressures. The mucous-like layer formed by the capsular polysaccharide retains water to prevent desiccation and can even hinder opsonization and subsequent phagocytosis by host immune systems. A unique class of bacteria biosynthesize capsules that are identical to polysaccharides also produced in mammalian tissues and are thus non-immunogenic, further contributing to evasion of host immune response. Pathogens bearing these capsular polysaccharides are implicated in such human diseases as urinary tract infections, strep throat, and bacterial meningitis. Coincidentally these capsular polysaccharides are metabolic engineeering targets as they can also serve as precursors in the production of medically relevant glycosaminoglycans, or highly acidic linear polysaccharides such as heparin and chondroitin sulfate. The utility of a rapid and selective assay quantifying and differentiating between capsular polysaccharides is twofold. Engineered strains could be screened in a high-throughput manner and selected for increased capsular polysaccharide production levels. Furthermore, a molecular screen selective for specific polysaccharides could be utilized in disease diagnosis and in strain serotyping. As antibodies cannot be generated against these non-immunogenic polysaccharides due to their natural presence in host animals, we have instead opted to select DNA aptamers from an oligomer library against capsular polysaccharides purified from three distinct bacterial sources. Heparosan from Escherichia coli K5, chondroitin from E. coli K4, and hyaluronan from Pasteurella multocida were purified, as confirmed by NMR, from fermentation supernatant using anion exchange chromatography. Linkage of capsular polysaccharides to amine-functionalized magnetic microparticles by reductive amination with sodium cyanoborohydride was verified by using a method recently developed in our lab. Specifically, bead-immobilized capsular polysaccharides were digested into disaccharides with their cognate polysaccharide lyases, followed by 2-aminoacridone (AMAC) labeling and quantification in a single, highly sensitive reverse phase ultra-performance liquid chromatography-mass spectrometry (RP-UPLC-MS) experiment. Molecular beacons based on selected aptamers will be further developed for fluorometric high-throughput screens to evaluate the capsular polysaccharide production capacities of metabolically engineered microbes.