title: Cloning and molecular analysis of the Isi1 (rfaF) gene of Neisseria meningitidis which encodes a heptosyl-2-transferase involved in LPS biosynthesis: evaluation of surface exposed carbohydrates in LPS mediated toxicity for human endothelial cells.
abstract: Neisseria meningitidis, but not Haemophilus influenzae, damage cultured human endothelial cells. We have undertaken a study to generate genetically and structurally defined lipopolysaccharide (LPS) mutant strains of meningococci for functional studies to assess the role of surface exposed oligosaccharides in imparting specificity of toxic damage to human endothelial cells. The Isi1 gene, which had been shown to be involved in LPS biosynthesis of Neisseria gonorrhoeae, was amplified by PCR and cloned. Nucleotide sequence analysis confirmed the identity of the clone and revealed homology with Isi1 of N. gonorrhoeae and the rfaF gene of Salmonella typhimurium which encodes a heptosyl-2-transferase involved in LPS biosynthesis. The identity of the cloned Isi1 gene, as a functional rfaF homologue, was confirmed by the complementation of a S. typhimurium rfaF mutant using a P22 phage sensitivity test. An Isi1 mutant meningococcal strain was constructed, and structural analysis of the mutant LPS molecule revealed a single heptose in the core structure, consistent with a heptosyl-2-transferase deficient mutant. In order to investigate the relative cytotoxicities of meningococci expressing native and altered LPS, wild type, Isi1, and galE strains were compared in cytotoxicity assays using human umbilical vein endothelial cells (Huvecs) in culture. Analysis using Huvecs derived from several individuals (cords) showed that the three phenotypes were almost equally cytotoxic. Removal of the terminal portion (galE mutant) or the majority (Isi mutant) of the oligosaccharide did not effect LPS-mediated cytopathic damage to Huvecs in a culture suggesting that the oligosaccharide portion did not play a major role in cytotoxicity.
authors.affiliation: Molecular Infectious Diseases Group, John Radcliffe Hospital, Headington, Oxford, U.K.
authors.forename: M P
authors.initials: MP
authors.lastname: Jennings
authors.forename: M
authors.initials: M
authors.lastname: Bisercic
authors.forename: K L
authors.initials: KL
authors.lastname: Dunn
authors.forename: M
authors.initials: M
authors.lastname: Virji
authors.forename: A
authors.initials: A
authors.lastname: Martin
authors.forename: K E
authors.initials: KE
authors.lastname: Wilks
authors.forename: J C
authors.initials: JC
authors.lastname: Richards
authors.forename: E R
authors.initials: ER
authors.lastname: Moxon
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D005798:Genes, Bacterial / Q000235:genetics*; D005816:Genetic Complementation Test; D016695:Glycosyltransferases / Q000235:genetics*; D006801:Humans; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000201:enzymology / Q000235:genetics*; D009844:Oligosaccharides / Q000032:analysis / Q000633:toxicity; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014471:Umbilical Veins
chemical_list: D008070:Lipopolysaccharides; D009844:Oligosaccharides; D016695:Glycosyltransferases; C086735:heptosyltransferase