Identification and Characterization of an Escherichia coli Invasion Gene Locus, ibeB , Required for Penetration of Brain Microvascular Endothelial Cells

Abstract

ABSTRACT Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis, but the mechanism by which E. coli K1 crosses the blood-brain barrier is incompletely understood. We have previously described the cloning and molecular characterization of a determinant, ibeA (also called ibe10 ), from the chromosome of an invasive cerebrospinal fluid isolate of E. coli K1 strain RS218 (O18:K1:H7). Here we report the identification of another chromosomal locus, ibeB , which allows RS218 to invade brain microvascular endothelial cells (BMEC). The noninvasive Tn phoA mutant 7A-33 exhibited <1% the invasive ability of the parent strain in vitro in BMEC and was significantly less invasive in the central nervous system in the newborn rat model of hematogenous E. coli meningitis than the parent strain. The Tn phoA insert with flanking sequences was cloned and sequenced. A 1,383-nucleotide open reading frame (ORF) encoding a 50-kDa protein was identified and termed ibeB . This ORF was found to be 97% identical to a gene encoding a 50-kDa hypothetical protein (p77211) and located in the 13-min region of the E. coli K-12 genome. However, no homology was observed between ibeB and other known invasion genes when DNA and protein databases in GenBank were searched. Like the Tn phoA insertion mutant 7A-33, an isogenic ibeB deletion mutant (IB7D5) was unable to invade BMEC. A 7.0-kb locus containing ibeB was isolated from a LambdaGEM-12 genomic library of E. coli RS218 and subcloned into a pBluescript KS vector (pKS7-7B). pKS7-7B was capable of completely restoring the BMEC invasion of the noninvasive Tn phoA mutant 7A-33 and the ibeB deletion mutant IB7D5 to the level of the parent strain. More importantly, the ibeB deletion mutant IB7D5 was fully complemented by pFN476 carrying the ibeB ORF (pFN7C), indicating that ibeB is required for E. coli K1 invasion of BMEC. Taken together, these findings indicate that several E. coli determinants, including ibeA and ibeB , contribute to crossing of the blood-brain barrier.