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 infection in neonates, very little information exists on the specific mechanisms this organism uses to induce disease. We have developed an in utero model of neonatal sepsis, induced by GBS, in subhuman primates - M. nemestrina or pigtail macaques. These infants display all of the stigmata of human infants infected by GBS in utero. Lung ultrastructural studies of infant primates with GBS pneumonia have shown that GBS are capable of invading alveolar epithelial cells. Based on this data, we have developed in vitro assays of epithelial cell invasion by GBS. This proposal seeks to define the characteristics of entry by GBS into epithelial cells. The investigation will first characterize the bacterial and cellular factors required by GBS for invasion of primarily respiratory epithelial cell lines. Secondly, we will investigate the ability of GBS to enter and traverse (trancytosis) from the apical to the basoloateral surface of a polar monolayer of epithelial cells. These experiments should provide insight into the mechanisms GBS use to penetrate and traverse across eukaryotic cells. We propose to identify the putative virulence factors produced by GBS that are responsible for the invasion phenotype. Transposon mutants in production of capsule and hemolysin have been made previously and will be screened for their ability to invade epithelial cells. Transposon mutagenesis will be used to derive new isogenic mutant strains of B|GBS which are unable to enter epithelial cells in vitro. The virulence of invasion mutants will be tested for their ability to induce GBS infections, first in a neonatal rat sepsis model, and subsequently, specific invasion mutants will be tested in utero in the GBS neonatal primate sepsis model. Those mutants incapable of invasion and subsequently shown to be avirulent in animal models will be further characterized using molecular biologic techniques. The genes important for invasion determinants, identified by transposon mutagenesis, will be cloned and the gene products identified using standardized gene expression assays. These studies should begin to identify using standardized gene expression assays. 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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