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.