Cloning and expression of the Escherichia coli K1 outer membrane protein A receptor, a gp96 homologue.
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.
Prasadarao
Nemani V
NV
Division of Infectious Diseases, MS #51, Children's Hospital Los Angeles and Keck School of Medicine, University of Southern California, 4650 Sunset Boulevard, Los Angeles, CA 90027, USA. pnemani@chla.usc.edu
Srivastava
Pramod K
PK
Rudrabhatla
Rajyalakshmi S
RS
Kim
Kwang Sik
KS
Huang
Sheng-he
SH
Sukumaran
Sunil K
SK
D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001921:Brain / Q000098:blood supply; D016466:CHO Cells; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000166:cytology / Q000382:microbiology; D004926:Escherichia coli / Q000378:metabolism / Q000472:pathogenicity*; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D000951:Antigens, Neoplasm; D001425:Bacterial Outer Membrane Proteins; C464645:GPATCH1 protein, human; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; C052646:sarcoma glycoprotein gp96 rejection antigens; C078167:OMPA outer membrane proteins