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Infection and Immunity, August 2002, p. 4556-4563, Vol. 70, No. 8
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.8.4556-4563.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of Escherichia coli Outer Membrane Protein A Receptor on Human Brain Microvascular Endothelial Cells

Nemani V. Prasadarao*

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

Neonatal Escherichia coli meningitis continues to be a diagnostic and treatment challenge despite the availability of active antibiotics. Our earlier studies have shown that outer membrane protein A (OmpA) is one of the major factors responsible for Escherichia coli traversal across the blood-brain barrier that constitutes a lining of brain microvascular endothelial cells (BMEC). In this study we showed that OmpA binds to a 95-kDa human BMEC (HBMEC) glycoprotein (Ecgp) for E. coli invasion. Ecgp was partially purified by wheat germ agglutinin and Maackia amurensis lectin (MAL) affinity chromatography. The MAL affinity-purified HBMEC proteins bound to OmpA+ E. coli but not to OmpA- E. coli. In addition, the deglycosylated MAL-bound proteins still interact with OmpA+ E. coli, indicating the role of protein backbone in mediating the OmpA binding to HBMEC. Interestingly, the MAL affinity-bound fraction showed one more protein, a 65-kDa protein that bound to OmpA+ E. coli in addition to Ecgp. Further, the 65-kDa protein was shown to be a cleavage product of Ecgp. Immunocytochemistry of HBMEC infected with OmpA+ E. coli by using anti-Ecgp antibody suggests that Ecgp clusters at the E. coli entry site. Anti-Ecgp antibody also reacted to microvascular endothelium on human brain tissue sections, indicating the biological relevance of Ecgp in E. coli meningitis. Partial N-terminal amino acid sequence of Ecgp suggested that it has 87% sequence homology to gp96, an endoplasmic reticulum-resident molecular chaperone that is often expressed on the cell surface. In contrast, the 65-kDa protein, which could be the internal portion of Ecgp, showed 70% sequence homology to an S-fimbria-binding sialoglycoprotein reported earlier. These results suggest that OmpA interacts with Ecgp via the carbohydrate epitope, as well as with the protein portion for invading HBMEC.

* Mailing address: Division of Infectious Diseases, MS 51, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027. Phone: (323) 669-5465. Fax: (323) 660-2661. E-mail: pnemani{at}chla.usc.edu.

Editor: E. I. Tuomanen

Infection and Immunity, August 2002, p. 4556-4563, Vol. 70, No. 8
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.8.4556-4563.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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