Escherichia coli invasion of brain microvascular endothelial cells in vitro and in vivo: Molecular cloning and characterization of invasion gene ibe10

Most cases of neonatal Escherichia coli meningitis develop as a result of hematogenous spread, but it is not clear how circulating E. coli crosses the blood-brain barrier. In an attempt to identify E. coli structures contributing to invasion into the central nervous system (CNS), TnphoA mutagenesis was performed with an invasive CSF isolate of E. coli K1 strain RS218 (O18:K1:H7), and TnphoA mutants were examined for their noninvasive capability in brain microvascular endothelial cells (BMEC). The noninvasive mutants exhibited the invasive ability of < 1% of the parent strain. One of the noninvasive mutants (10A-23) with a single TnphoA insertion and no changes in phenotypic characteristics was found to be significantly less invasive into the CNS in the newborn rat model of hematogenous E. coli meningitis. The TnphoA inserts with flanking sequences were cloned and sequenced. A novel open reading frame (8.2 kDa) was identified. Open reading frame analysis indicated that the 8.2-kDa protein (Ibe10) contained multiple transmembrane domains. ibe10 was cloned into an expression vector, pQE30, and the purified Ibe10 was shown to inhibit invasion of BMEC by strain RS218. These findings indicate that ibe10 is one of the E. coli genes involved in the invasion of BMEC in vitro and in vivo.