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.
Prasadarao
Nemani V
NV
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
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