The principal investigator has established an infant rat model of experimental hematogenous meningitis, which mimics human E. coli meningitis (i.e., hematogenous infection of the meninges without the need for adjuvant or direct inoculation of bacteria into cerebrospinal fluid). They have established an in vitro model of blood-brain barrier using brain microvascular endothelial cells (BMEC). Using both in vitro and in vivo systems and the BMEC model, they have shown that successful traversal of E. coli across the blood-brain barrier may be a complex process involving separate steps of E. coli-BMEC interactions, i.e., binding to BMEC and invasion of BMEC. They have shown that S fimbriae are the major E. coli structures contributing to binding to BMEC. They therefore hypothesize that the role of S fimbriae is to have a more intimate contact for circulating E. coli to BMEC to withstand blood flow, which may be required for subsequent crossing of the blood-brain barrier, but there is no information to support this hypothesis. The overall aim of the proposal is to study the role of binding via S fimbriae in the pathogenesis of E. coli meningitis, both in vitro (using BMEC in culture) and in vivo (using an experimental animal model of hematogenous E. coli meningitis that closely mimics the pathogenesis of human E. coli meningitis).
The specific aims are: (1)to continue to construct isogenic S fimbriae operon deletion (delta sfaII) mutant of invasive E. coli K1 strain RS218 by chromosomal gene replacement (allelic exchange); (2)to examine the ability of S fimbriae negative (delta sfaII) mutant to bind and invade brain microvascular endothelial cells (BMEC) in vitro and in vivo; (3)to determine whether it is possible to restore the ability of S fimbriae negative (delta sfaII) mutant to bind and invade BMEC by complementation with the cloned sfaII gene cluster; (4)to assess which specific subunits of S fimbriae are responsible for binding to BMEC by constructing single gene deletion mutants (delta sfaIIS, delta sfaIIA, delta sfaIIG, delta sfaIIH); (5)and to identify and characterize BMEC glycoprotein(s) interactive with S fimbriae.
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