Identification of Escherichia coli outer membrane protein A receptor on human brain microvascular endothelial cells.
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.
Division of Infectious Diseases, Childrens Hospital Los Angeles, and Keck School of Medicine, University of Southern California, Los Angeles 90027, USA. pnemani@chla.usc.edu
Nemani V
NV
Prasadarao
D000595:Amino Acid Sequence; D000906:Antibodies / Q000378:metabolism; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001921:Brain / Q000098:blood supply*; D002196:Capillaries / Q000378:metabolism; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010835:Phytohemagglutinins / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D001425:Bacterial Outer Membrane Proteins; C464645:GPATCH1 protein, human; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010835:Phytohemagglutinins; D011956:Receptors, Cell Surface; C100156:leukoagglutinins, plants; C078167:OMPA outer membrane proteins