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Infection and Immunity, March 1999, p. 1131-1138, Vol. 67, No. 3
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Identification and Characterization of a Novel Ibe10 Binding Protein That Contributes to Escherichia coli Invasion of Brain Microvascular Endothelial Cells

Nemani V. Prasadarao,* Carol A. Wass, Sheng-He Huang, and Kwang Sik Kim

Division of Infectious Diseases, Childrens Hospital Los Angeles, and University of Southern California School of Medicine, Los Angeles, California 90027

Received 18 September 1998/Returned for modification 29 October 1998/Accepted 1 December 1998

The molecular basis of Escherichia coli traversal of the blood-brain barrier in the development of E. coli meningitis is not well understood. We have previously shown that a novel Ibe10 protein found in cerebrospinal fluid isolates of E. coli is necessary for invasion of the brain microvascular endothelial 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-Sepharose affinity chromatography. Bovine Ibe10R, as well as polyclonal antibodies to Ibe10R, blocked E. coli invasion of BBMEC very effectively. The N-terminal 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, suggesting 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-positive BBMEC by fluorescence-activated cell sorting with anti-Ibe10R antibody resulted in enhanced invasion by E. coli. The anti-Ibe10R antibody raised against bovine Ibe10R also blocked E. coli invasion of HBMEC very effectively. Interestingly, anti-Ibe10R antibody affinity chromatography of HBMEC membrane proteins revealed a smaller protein with an approximate molecular mass of 45 kDa. These results suggest that the Ibe10 of E. coli interacts with a novel BMEC surface protein, Ibe10R, for invasion of both BBMEC and HBMEC.

* Corresponding author. Mailing address: Division of Infectious Diseases, #51, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027. Phone: (323) 669-5622. Fax: (323) 660-2661. E-mail: nemani{at}hsc.usc.edu.

Infection and Immunity, March 1999, p. 1131-1138, Vol. 67, No. 3
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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