The mortality and morbidity associated with neonatal bacterial meningitis have remained significant despite advances in antimicrobial chemotherapy and supportive care. Inadequate knowledge of the pathogenesis and pathophysiology has contributed to this high mortality and morbidity. E. coli is the most common gram-negative organism that causes meningitis during the neonatal period. Most cases of E. coli meningitis in newborns develop as a result of hematogenous spread, but it is not clear how circulating E. coli cross the blood-brain barrier. We have established an infant rat model of experimental hematogenous meningitis which mimics human E. coli meningitis (e.g. hematogenous infection of meninges). We have also established an in vitro model of the blood-brain barrier with brain microvascular endothelial cells (BMEC). Using these in vitro and in vivo systems, we have shown that invasion of BMEC is a requirement for E. coli K1 crossing of the blood-brain barrier in vivo. During the previous funding period, we have shown that several E. coli K1 determinants contribute to invasion of BMEC in vitro and crossing of the blood-brain barrier in vivo (i.e., OmpA, IbeA, IbeB, IbeC, CNF1). We also showed that E. coli K1 invasion of endothelial cells is specific to BMEC, and no such invasion was observed for endothelial cells of non-brain origin. We have so far shown that some of the E. coli proteins (e.g., OmpA, IbeA) interact with specific receptors present on BMEC, not on systemic vascular endothelial cells. In addition, we showed that actin cytoskeleton rearrangements are involved in E. coli K1 invasion of BMEC, as shown by invasive E. coli K1-associated F-actin condensation and blockade of invasion by cytochalasin D. Based on the resources and findings derived from the past funding period, we would like to examine the following specific aims. 1. To continue to identify and characterize microbial determinants contributing to E. coli K1 invasion of BMEC in vitro and in vivo 2. To examine the mechanisms involved in E. coli K1 invasion of BMEC, including structure-function analysis of E. coli proteins, and identification and characterization of BMEC receptors. 3. To determine host cell signal transduction pathways involved in E. coli K1 invasion of BMEC, including focal adhesion kinase (FAK), Rho and phosphatidylinositol (PI)3-kinase. Further understanding and characterization of these E. coli K1-BMEC interactions should allow us to develop novel strategies to prevent this serious infection.
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