The proposed research is intended to elucidate the mechanism of translocation of the E. coli K1 capsular polysaccharide from its site of synthesis on the cytoplasmic membrane to the bacterial cell surface. This work is a continuation of our analyses of the cloned K1 antigen genes and will focus on the product of the kpsD gene, a 60-kDa periplasmic protein required for the translocation process. The proposed research is aimed at providing information on the role of the kpsD protein in polymer transport and identifying important functional domains of the protein. We also intend to further define the contribution of K1 transport of other genes (kpsB, C, E, and F) located in the 11.6-kb transport region. The research we propose is part of our long-term goal to elucidate the molecular and genetic events controlling the synthesis and assembly of the K1 capsular polysaccharide. K1 producing E. coli remain the most common gram negative organism causing sepsis and meningitis in the neonate. The K1 capsule is an essential virulence determinant. Unfortunately , purified K1 is a poor immunogen in humans, perhaps a consequence of immune tolerance due to cross reactive brain components. An alternative to anticapsular immunity may be needed to render these organisms vulnerable to host defenses and an understanding of the key reactions involved in synthesis, assembly, and export of the K1 polysaccharide is needed.
The specific aims of this proposal and methods for achieving them include: 1) purification of the kpsD protein by immunoaffinity chromatography; 2) functional analyses of KpsD; these will include chemical and physical characterization of polysaccharide accumulating in kpsD mutants, KpsD- affinity chromatography to detect specific protein-protein interactions, equilibrium dialysis to determine KpsD-oligosaccharide interactions, and ultrastructural localization of KpsD by immunoelectronmicroscopy; 3) identification of important functional domains of the kpsD protein by site- directed mutagenesis; 4) analysis of the contribution to K1 transport of kpsB, C, E, and F by Tn1000 mutagenesis, and construction of site-directed insertion mutations. The subcellular location and membrane topology of proteins encoded by these genes will be determined by biochemical techniques and TnphoA fusion technology.
