The research proposed in the present application is part of a long-term goal to understand the molecular events controlling the synthesis and transport of the polysialic acid capsule of Escherichia coli K1. The capsule is an essential virulence determinant allowing E. coli to evade non-specific host defense mechanisms. Strains that produce the K1 capsule account for 80% of E. coli neonatal meningitis, and this disease continues to portend a poor prognosis in infected neonates. Given the poor prospect of developing vaccines based on purified K1 polysaccharide, the investigator proposes that an understanding of the key reactions involved in the synthesis and export of the capsule is needed to provide novel rationales for intervention in the disease process. The K1 polysaccharide is synthesized in the cytoplasm and transported through two lipid bilayers by a complex and poorly understood process. The 17-kb kps gene cluster encoding the proteins necessary for capsule expression includes two genes, kpsM and kpsT, that are required for polymer transport across the cytoplasmic membrane. KpsM is a hydrophobic integral inner membrane protein, while KpsT is a peripheral inner membrane protein that binds ATP. These proteins belong to the ATP- binding cassette (ABC) superfamily of transport proteins. To study the role of KpsT in polymer transport, the investigator has isolated and characterized a dominant negative mutation of plasmid-encoded kpsT. These studies allowed the investigator to develop a working model for polysaccharide export in E. coli. The essential feature of the model is a cycle of ATP-driven insertion and deinsertion of KpsT into the cytoplasmic membrane governed by its association with ATP, polysaccharide and KpsM. The proposed studies therefore focus on the KpsMT exporter and are broken down into four specific questions that test this export model. (1) Does purified KpsT bind and hydrolyze ATP? Inclusion bodies from cells overexpressing KpsT will be solubilized with urea and the protein purified by dye-ligand chromatography. The investigator will also use limited proteolysis and tryptophan fluorescence to determine if ATP- binding to purified KpsT induces a conformational change in the protein. (2) Is KpsT exposed to the periplasmic face of the cytoplasmic membrane during the translocation process? These studies will include protease and biotinylation accessibility in oriented membrane vesicles. (3) Is KpsT attached to polymer during the transport process? In these studies, the investigator proposes to purify polysialic acid from cells using an immunoaffinity column of specifically purified horse antiserum, and polyclonal anti-KpsT antibody will be used to detect KpsT attached to polysaccharide. (4) What is the composition of the KpsMT transporter complex? Co-immunoprecipitation and chemical cross-linking techniques will be used for these studies. The investigator believes that the studies proposed in this application should result in considerable progress not only in the understanding of macromolecular export in medically important bacteria, but also in the mechanism by which the ABC superfamily of membrane transporters functions.
