The gram-negative bacterium E. coli K1 is the leading cause of meningitis during the neonatal period. The mortality and long-term morbidity rates associated with E. coli meningitis have remained relatively unchanged despite the availability of active antimicrobial agents. The disease is fatal in 5% to 40% of infected neonates. Incomplete understanding of the pathogenesis and pathophysiology of this infection has hampered attempts to develop novel therapeutic approaches for this deadly disease. A characteristic pathophysiological feature of E. coli K1 meningitis is the disruption of the blood-brain barrier (BBB), and is mainly attributed for the neurological sequelae of this disease. However, the mechanisms by which E. coli K1 disrupts the BBB are poorly understood. We have demonstrated that E. coli K1 interacts with GlcNAd, 4GlcNAc epitopes and the protein backbone of a novel brain microvascular endothelial cell (BMEC) glycoprotein receptor (Ecgp) to invade BMEC, an in vitro model of the BBB. In addition, OmpA-Ecgp mediated invasion of E. coli also increases the permeability of the BBB both in vitro and in the animal model. Anti-Ecgp antibodies exhibited significant inhibitory effect on E. coli invasion and on the permeability of BMEC. We have also grossly defined the structural motifs of Ecgp required for interaction with OmpA by mutational analysis. We further demonstrated that E. coli K1 invasion via Ecgp induces several signaling cascades for invasion as well as to increase the permeability of the BBB. Therefore, we hypothesize that binding of OmpA to Ecgp is essential to initiate signaling events that induce E. coli invasion and increased permeability of the BBB. The project includes three specific aims: 1. To further characterize the domains of Ecgp that interact with OmpA for their ability to mediate E. coli invasion of HBMEC as well as increased BBB permeability. 2. To determine whether the interaction of the C-terminal domains of Ecgp with caveolin-1 initiates the internalization of E. coli and alter the BBB permeability via eNOS activation. 3. To examine whether blocking of OmpA-Ecgp interaction by peptides and non-peptide molecules prevent the E. coli invasion and associated BBB permeability. A better understanding of Ecgp interaction with OmpA will help elucidate the molecular mechanisms involved in the development of E. coli meningitis. Screening of small molecule antagonists to Ecgp function will lay the foundation for the development of new therapeutic strategies to prevent this deadly disease.
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