title: Identification of minimum carbohydrate moiety in N-glycosylation sites of brain endothelial cell glycoprotein 96 for interaction with Escherichia coli K1 outer membrane protein A.
abstract: Bacterial meningitis is a serious central nervous system infection and Escherichia coli K1 (E. coli K1) is one of the leading etiological agents that cause meningitis in neonates. Outer membrane protein A (OmpA) of E. coli K1 is a major virulence factor in the pathogenesis of meningitis, and interacts with human brain microvascular endothelial cells (HBMEC) to cross the blood-brain barrier. Using site-directed mutagenesis, we demonstrate that two N-glycosylation sites (NG1 and NG2) in the extracellular domain of OmpA receptor, Ecgp96 are critical for bacterial binding to HBMEC. E. coli K1 invasion assays using CHO-Lec1 cells that express truncated N-glycans, and sequential digestion of HBMEC surface N-glycans using specific glycosidases showed that GlcNAc1-4GlcNAc epitopes are sufficient for OmpA interaction with HBMEC. Lack of NG1 and NG2 sites in Ecgp96 inhibits E. coli K1 OmpA induced F-actin polymerization, phosphorylation of protein kinase C-α, and disruption of transendothelial electrical resistance required for efficient invasion of E. coli K1 in HBMEC. Furthermore, the microvessels of cortex and hippocampus of the brain sections of E. coli K1 infected mice showed increased expression of glycosylated Ecgp96. Therefore, the interface of OmpA and GlcNAc1-4GlcNAc epitope interaction would be a target for preventative strategies against E. coli K1 meningitis.
authors.affiliation: Division of Infectious Diseases and Department of Pediatrics, Children's Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027, USA.
authors.forename: Subramanian
authors.initials: S
authors.lastname: Krishnan
authors.affiliation: Division of Infectious Diseases and Department of Pediatrics, Children's Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027, USA; Department of Surgery, Children's Hospital Los Angeles and University of Southern California, 4650 Sunset Blvd., Los Angeles, CA 90027, USA. Electronic address: pnemani@chla.usc.edu.
authors.forename: Nemani V
authors.initials: NV
authors.lastname: Prasadarao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D001921:Brain / Q000166:cytology* / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D042783:Endothelial Cells / Q000737:chemistry / Q000378:metabolism* / Q000382:microbiology; D004926:Escherichia coli / Q000378:metabolism* / Q000472:pathogenicity; D054884:Host-Pathogen Interactions; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009154:Mutation; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D008562:Membrane Glycoproteins; D011134:Polysaccharides; C078167:OMPA outer membrane proteins
keywords: Brain endothelium; Escherichia coli K1; Glycoprotein; Hsp90; Invasion; Meningitis