Group B Streptococcus (GBS) colonizes mucosal surfaces of the human gastrointestinal and gynecological tracts and causes disease in a wide range of patients. Invasive illness occurs after organisms cross an epithelial boundary and enter deeper tissues. The broad scientific goal of this project is to better characterize the molecular events occurring during interaction of alpha-like proteins (Alps) on the surface of GBS with epithelial cells. Alpha C protein (ACP) is the prototype of this family of highly conserved Gram-positive bacterial surface proteins sharing similar N-terminal regions and long tandem repeating regions. Prior work shows ACP binds to cervical epithelial cells, mediates GBS entry into these cells, and contributes to GBS movement across a multi-layer configuration of these cells. The ACP N-terminal region is particularly implicated in these events. Both full ACP and the N-terminal region induce protective antibody responses in a mouse model of infection. Preliminary studies suggest that exposure of epithelial cells to free ACP or to live GBS expressing surface ACP results in actin-dependent accumulation of intracellular ACP. Additional preliminary work indicates Alp3 protein, with 78% identity to ACP in the N terminal region, shares similar functions to ACP. These data support a hypothesis that Alps trigger actin-dependent internalization of GBS through interaction of an Alp N-terminal domain with an epithelial receptor.
The specific aims of this proposal are 1. to identify the molecular events allowing accumulation of cell-associated Alps over time by assessing the impact of interference with specific Rho GTPase-mediated actin rearrangements, 2. to map important ACP epitopes for epithelial cell interaction by producing altered versions of the ACP N-terminal region and comparing their functions to those of native ACP, and 3. to assess the efficacy of the ACP variants as vaccines or as pharmacologic agents in preventing invasive disease in a mouse model of GBS infection. The training goal of this project is to prepare the applicant for a career as a physician/scientist performing independent investigation in the field of cellular microbiology/bacterial pathogenesis, by providing didactic education and the opportunity to develop laboratory expertise in this field. ? ?
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