Identification and characterization of an Escherichia coli invasion gene locus, ibeB, required for penetration of brain microvascular endothelial cells

Escherichia coli K1 is the most common gram-negative organism causing neona tal meningitis, but the mechanism by which E. coli K1 crosses the blood-bra in barrier is incompletely understood. We have previously described the clo ning and molecular characterization of a determinant, ibeA (also called ibe 10), from the chromosome of an invasive cerebrospinal fluid isolate of E. c oli K1 strain RS218 (O18:K1:H7). Here we report the identification of anoth er chromosomal locus, ibeB, which allows RS218 to invade brain microvascula r endothelial cells (BR-IEC), The noninvasive TnphoA mutant 7A-33 exhibited <1% the invasive ability of the parent strain in vitro in BMEC and was sig nificantly less invasive in the central nervous system in the newborn rat m odel of hematogenous E, coli meningitis than the parent strain. The TnphoA insert,vith Ranking sequences was cloned and sequenced. A 1,383-nucleotide open reading frame (ORF) encoding a 50-kDa protein was identified and terme d 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, co li K-12 genome, However, no homology was observed between ibeB and other kn own invasion genes when DNA and protein databases in GenBank were searched, Like the TnphoA insertion mutant 7A-33, an isogenic ibeB deletion mutant ( TS7D5) was unable to invade BMEC, A 7.0-kb locus containing ibeB was isolat ed 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 restor ing the BMEC invasion of the noninvasive TnphoA mutant 7A-33 and the ibeB d eletion mutant IB7D5 to the level of the parent strain, More importantly, t he 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 dete rminants, including ibeA and ibeB, contribute to crossing of the blood-brai n barrier.