binding to and invasion of brain microvascular endothelial cells derived from humans and rats of different ages. In contrast, the 65-kDa protein, which could be the internal O-Phospho-L-serine 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. K1 meningitis is the most common infection of the central nervous system in neonates. The O-Phospho-L-serine mortality and morbidity associated with this disease have remained unchanged despite advances in antimicrobial chemotherapy (5, 10, 11, 13, 26). The reasons for the poor outcome has been attributed to limited knowledge of pathogenesis and pathophysiology of the disease. Although most cases of meningitis occur via hematogenous spread, it is not clear what microbial and host factors are responsible for the ability of neurotropic strains of to cross the blood-brain barrier, which is formed by a single layer of brain microvascular endothelial cells (BMEC). The interaction of specific determinants with their corresponding BMEC receptors may dictate the tissue tropism in neonatal meningitis. Non-brain endothelial cells, which have generally been used to study the interaction of that causes meningitis, are not an ideal target cell culture model because they differ considerably from BMEC (1, 12, 28, 34). Thus, we developed an in vitro model of the blood-brain barrier using BMEC derived from humans, cows, and rats (24, 25). Several investigators have made use of cultured mammalian cells to identify the mechanisms of bacterial entry into these cells (2, 3, 9, 19, 20). Many microorganisms utilize integrins on host cells as the receptor molecule for binding to and invasion of eukaryotic cells, e.g., spp. and enteropathogenic O-Phospho-L-serine (2, 3, 27, 29, 31, 32). We have shown that S fimbriae are required for binding to NeuAc2,3-galactose-containing glycoproteins and sulfated glycolipids of BMEC (16, 25). We further Mouse monoclonal to PR showed that S fimbria interacts with a 65-kDa BMEC glycoprotein specifically expressed on brain endothelial cells but not on systemic endothelial cells (15). However, The binding via S fimbriae was not accompanied by invasion in vitro, suggesting that S fimbriae might mediate adherence of to BMEC in vivo. O-Phospho-L-serine After initial adherence mediated by S fimbriae, additional cell surface molecules are thought to contribute to the invasion of bacteria into host cells. Several nonfimbriated determinants have been subsequently identified that contribute to the invasion of BMEC, e.g., OmpA, IbeA, IbeB, and Yijp (7, 8, 17, 30). Except for OmpA, neither the surface localization nor mode of action of other determinants is known. OmpA, a 35-kDa cell surface transmembrane protein with four extracellular loops, is highly conserved on many gram-negative bacteria. We showed that OmpA expression enhances invasion of BMEC by 25- to 50-fold compared strains without OmpA (17). This OmpA-mediated invasion occurs via the interaction of N-terminal loops of OmpA with GlcNAc1,4-GlcNAc epitopes of BMEC surface glycoproteins (18). Receptor analogues, the chitooligomers (GlcNAc1,4-GlcNAc polymers), although at high concentrations, blocked invasion of BMEC both in vitro and in the newborn rat model of hematogenous meningitis, suggesting that the glycoproteins bearing these epitopes are indeed involved in entry into the central nervous system. Furthermore, molecular modeling of GlcNAc1,4-GlcNAc sugar interaction with the canyon formed by the loops 1 and 2 of OmpA showed favorable energy levels and conformations compared to any other area. (D. Datta, N. Vaidehi, W. O-Phospho-L-serine B. Florino, N. V. Prasadarao, and W. A. Goddard III, unpublished results). One of the salient findings of our studies so far has been that S-fimbriae-mediated binding and OmpA-contributed invasion of are specific to BMEC but not to systemic endothelial cells of non-brain origin, e.g., human umbilical vein endothelial cells (HUVEC), human arterial aortic endothelial cells, and human ileac vein endothelial cells (18). Therefore, we speculated that selective invasion of into BMEC compared to systemic endothelial cells might be a result of specific interaction of determinants with corresponding ligands specifically expressed on BMEC. I report here the identification of a 95-kDa human BMEC (HBMEC) surface protein (Ecgp) that binds to OmpA on that was partially purified by lectin affinity chromatography. Partial N-terminal amino acid sequence of Ecgp suggests that it is a gp96-like molecule and its homologue Hsp-90. Inhibition of invasion into HBMEC by affinity-purified Ecgp and the presence of Ecgp on microvessels in.