Cloning and expression of the Escherichia coli K1 outer membrane protein a receptor, a gp96 homologue

Nemani V. Prasadarao, Pramod K. Srivastava, Rajyalakshmi S. Rudrabhatla, Kwang Sik Kim, Sheng He Huang, Sunil K. Sukumaran

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1680-1688
Number of pages9
JournalInfection and immunity
Volume71
Issue number4
DOIs
StatePublished - Apr 1 2003

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

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