Group B streptococci (GBS) are the most common cause of bacterial sepsis and meningitIs in the newborn infant. Although an extensive literature exists describing the immunology and epidemiology of GBS infections in neonates, very little information exists on the specific mechanisms this organism uses to induce disease. We had previously demonstrated, in an in utero model of neonatal sepsis induced by GBS in subhuman primates, that this organism is capable of invading alveolar respiratory epithelial cells. Based on this data, we developed in vitro assays for investigating epithelial cell invasion by GBS. During the first two years of the funded proposal, we demonstrated that GBS were capable of invading respiratory epithelial cells and identified some of the important bacterial and cellular processes necessary for invasion. During the current year, we have investigated the ability of GBS to traverse an intact polar epithelial monolayer in vitro. In addition, we have begun to generate isogenic invasion mutants by transposon mutagenesis in order to define the molecular pathogenesis of cellular invasion by this organism. To extend our previous observations, we propose to continue to use transposon mutagenesis to derive isogenic mutant strains of GBS with altered abilities to invade epithelial cells in vitro. Mutants which invade poorly relative to the parent strain, remain as adherent to epithelial cells and are otherwise unchanged in their phenotype, will be analyzed further. The genes important for invasion determinants, identified by transposon mutagenesis or genomic cloning into B. subtilis, will be analyzed by nucleotide sequencing, and the gene products identified using standardized gene expression assays. The ability of the wild type invasion genes to complement these mutants will be determined by using cloning vectors and transformation procedures we have recently adapted for GBS. The virulence of well characterized invasion mutants will be tested for their ability to induce GBS infections in neonatal rat GBS infection models compared to the parent strain, confirming the relevance of our in vitro findings. Effects of the mutations on GBS surface composition or secretion of extracellular products, the ability of surface extracts of GBS to augment invasion, and the induction of proteins from GBS during the intracellular state, will be investigated in pilot experiments for future studies. We anticipate these studies should begin to identify the bacterial traits important in the early steps in the pathogenesis of neonatal infections caused by group B streptococci.
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