Neonatal Escherichia coil meningitis continues to be a diagnostic and treatment challenge despite the availability of active antibiotics. Investigations on the understanding of the pathogenesis and pathophysiology are needed to develop novel strategies to prevent the high morbidity and mortality associated with this disease. OmpA of E. coil is one of the major factors responsible for E. coil traversal across the blood-brain barrier that constitutes a lining of brain microvascular endothelial cells (BMEC). Ecgp, a novel blood-brain barrier specific glycoprotein interacts with OmpA to induce actin rearrangement for subsequent penetration into the central nervous system, has been identified and characterized. Recombinant Ecgp significantly blocked the E. coli traversal across the blood-brain barrier indicating the biological relevance of OmpA-Ecgp interaction in the development of this disease. Ecgp itself is phosphorylated to interact with two BMEC cytoplasmic signaling proteins, FAK and Vav2. in response to E coli infection. The identification of ligand binding and signaling domains of Ecgp will help elucidate the molecular mechanisms of OmpA-Ecgp interaction and its importance in the pathogenesis and pathophysiology of E. coli meningitis. Hypothesis: OmpA-Ecgp interaction is a key step that signals BMEC for E. coil traversal across the blood-brain barrier to cause meningitis. The following are the specific aims to test this hypothesis: 1. Characterize the antigenic structure of Ecgp using monoclonal antibodies against Ecgp that block E. coli invasion of HBMEC both in vitro and in the newborn rat model of hematogenous meningitis. 2. Identify the minimum structures of the Ecgp extracellular domains that interact with OmpA responsible for E. coli invasion of HBMEC by molecular modeling and site directed mutagenesis of Ecgp. 3. Identify the intracellular motifs of Ecgp that interact with the HBMEC cytoplasmic proteins, FAK and Vav2.
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