Antibodies to the capsular polysaccharide effectively protect newborn infants against infection with group B Streptococcus (GBS), the leading cause of life-threatening bacterial infection in neonates. However, at least five antigenically distinct capsular types of GBS are important causes of infection and immunity is type-specific. Studies on the genetics and biochemistry of capsule synthesis in type III GBS have implicated several putative genes spanning a 16 kb region of the chromosome. However, specific functions have been assigned to only two capsule synthesis genes. The goal of this proposal is to determine the function of the many other genes involved in GBS capsule expression. It is proposed to characterize the capsule gene region of the GBS chromosome for types Ia and Ib GBS, serotypes whose polysaccharide structures contain identical sugars as the type III GBS capsule but unique glycosidic linkages. Cloning the capsule gene regions of types Ia and Ib GBS will permit detailed comparison of the architecture of the capsule loci and the sequences of individual genes in order to identify conserved genes encoding proteins common to all serotypes as well as unique genes encoding serotype-specific glycosyltransferases. The function of each gene will be studied by constructing isogenic mutants and determining the structure of the resultant capsular polysaccharides. Glycosyltransferases will be investigated by expressing individual recombinant enzymes in E. coli then identifying their glycolipid products in an in vitro assay system. The structural basis for the specificity of glycosyltransferases will be determined using site-specific mutagen&sis to identify domains of the protein responsible for glycosidic bond specificity. An in vitro capsule biosynthesis assay will be used to characterize glycolipid intermediates and the function of gene products involved in polysaccharide polymerization and export. These studies will advance understanding of the molecular basis for emergence of new serotypes of GBS and may suggest new targets for pharmacologic therapy.
