Research Project
10003340
Alternative therapeutic targets have become a major topic in research with growing concerns over the alarming rate of antibacterial resistance. In contrast, many symbiotic bacteria that aid in human health prevent disease and are often inadvertently targeted with broad-spectrum antimicrobials. New therapeutic targets that selectively treat pathogenic but not symbiotic bacteria are highly valuable to balance the unique role they serve in human health. Bacterial surface polysaccharides are a crucial component to this host-microbe interaction, and underlie both disease and commensalism. These complex glycopolymers are highly unique in composition, even among the same bacterial species, and are not otherwise found in mammalian systems making them excellent therapeutic targets. The difficulty of studying these systems is the inherent complexity associated with glycopolymer composition coupled with the lack of elegant tools to ubiquitously characterize them. This hinders the ability to identify novel targets to inhibit these pathways, or methods to reconstruct glycopolymers for glyco-based vaccines. Robust probes that can be used to track the formation of these complex surface polysaccharides in vitro and in live cells are vital to exploiting them for therapeutic benefit. The use of tagged biomolecules has been invaluable in protein and nucleic acid biochemistry. Tagged sugar residues are the closest equivalent, however these do not preserve the native identity of the glycopolymer. Alternatively, formation of critical surface structures such as exopolysaccharides, teichoic acids, and O-antigens are dependent on the common lipid carrier bactoprenyl phosphate (BP) ? a polyisopreniod carbon chain. Because BP is utilized in each step during polysaccharide assembly, it would provide a means to track the formation of these materials. Installation of foreign tags to long carbon chain BP by chemical synthesis is nontrivial and would entail several iterations of isoprene addition over many lengthy steps.1 Alternatively, research proposed here will focus on the chemoenzymatic preparation of a series of highly fluorescent and click-enabled tags appended to BP. Such probes will greatly expand the range of glycopolymer applications to include low-level detection or facile addition of application-dependent moieties, such as biotinylation for immobilization, or tags to aid in characterization, such as signal enhancing mass spectrometry tags. These types of tagged BP substrates are also well suited to in vivo reconstitution of glycopolymers as they occur in nature. Incorporation of these robust tools in live cell cultures is an excellent method to probe the dynamic assembly of critical surface polysaccharides and will facilitate the development of novel therapeutics to reproduce or disrupt these pathways.
