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Infection and Immunity, April 2003, p. 1680-1688, Vol. 71, No. 4
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.4.1680-1688.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Cloning and Expression of the Escherichia coli K1 Outer Membrane Protein A Receptor, a gp96 Homologue

Nemani V. Prasadarao,1* Pramod K. Srivastava,2 Rajyalakshmi S. Rudrabhatla,1 Kwang Sik Kim,3 Sheng-he Huang,1 and Sunil K. Sukumaran1

Division of Infectious Diseases, Children's Hospital Los Angeles and Keck School of Medicine, University of Southern California, Los Angeles, California 90027,1 Department of Immunology and Infectious Diseases, University of Connecticut Health Center, Farmington, Connecticut,2 Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland3

Received 16 October 2002/ Returned for modification 12 December 2002/ Accepted 23 December 2002

Escherichia coli is one of the most common gram-negative bacteria that cause meningitis in neonates. Our previous studies have shown that outer membrane protein A (OmpA) of E. coli interacts with a 95-kDa human brain microvascular endothelial cell (HBMEC) glycoprotein, Ecgp, for invasion. Here, we report the identification of a gene that encodes Ecgp by screening of an HBMEC cDNA expression library as well as by 5' rapid amplification of cDNA ends. The sequence of the Ecgp gene shows that it is highly similar to gp96, a tumor rejection antigen-1, and contains an endoplasmic reticulum retention signal, KDEL. Overexpression of either Ecgp or gp96 in both HBMECs and CHO cells increases E. coli binding and invasion. We further show that Ecgp gene-transfected HBMECs express Ecgp on the cell surface despite the presence of the KDEL motif. Northern blot analysis of total RNA from various eukaryotic cells indicates that Ecgp is significantly expressed in HBMECs. Recombinant His-tagged Ecgp blocked E. coli invasion efficiently by binding directly to the bacteria. These results suggest that OmpA of E. coli K1 interacts with a gp96-like molecule on HBMECs for invasion.

* Corresponding author. Mailing address: Division of Infectious Diseases, MS #51, Children's 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: J. N. Weiser

Infection and Immunity, April 2003, p. 1680-1688, Vol. 71, No. 4
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.4.1680-1688.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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