Identification and characterization of a novel Ibe10 binding protein that contributes to Escherichia coli invasion of brain microvascular endothelialcells

The molecular basis of Escherichia coli traversal of the blood-brain barrie r in the development of E. coli meningitis is not well understood. We have previously shown that a novel Ibe10 protein found in cerebrospinal fluid is olates of E. coli is necessary for invasion of the brain microvascular endo thelial cells (BMEC) that constitute the blood-brain barrier both in vitro and in a newborn rat model of hematogenous meningitis. Here we identified a novel Ibe10 binding molecule/receptor (Ibe10R) on both bovine BMEC (HBMEC) and human BMEC (HBMEC) that is responsible for invasion by E. coli. Ibe10R , an approximately 55-kDa protein, was purified from BBMEC by Ibe10-Ni-Seph arose affinity chromatography. Bovine Ibe10R, as well as polyclonal antibod ies to Ibe10R, blocked E. call invasion of BBMEC very effectively. The N-te rminal amino acid sequence of Ibe10R showed 75% homology to serum albumin. However, the amino acid sequence of an Ibe10R fragment generated by limited enzymatic digestion did not reveal homology to any other proteins, suggest ing that Ibe10R represents a novel albumin-like protein. Immunocytochemical analysis of BBMEC using anti-Ibe10R antibody suggested that only a subset of cultured BBMEC express Ibe10R on their surface. Enrichment of Ibe10R-pos itive BBMEC by fluorescence-activated cell sorting with antl-Ibe10R antibod y resulted in enhanced invasion by E. coli, The anti-Ibe10R antibody raised against bovine Ibe10R also blocked E. coli invasion of HBMEC very effectiv ely. Interestingly, anti-Ibe10R antibody affinity chromatography of HBMEC m embrane proteins revealed a smaller protein with an approximate molecular m ass of 45 kDa. These results suggest that the Ibe10 of E. coli interacts wi th a novel BMEC surface protein, Ibe10R, for invasion of both BBMEC and HBM EC.