title: Properties of haemolysin E (HlyE) from a pathogenic Escherichia coli avian isolate and studies of HlyE export.
abstract: Haemolysin E (HlyE) is a novel pore-forming toxin first identified in Escherichia coli K-12. Analysis of the 3-D structure of HlyE led to the proposal that a unique hydrophobic beta-hairpin structure (the beta-tongue, residues 177-203) interacts with the lipid bilayer in target membranes. In seeming contradiction to this, the hlyE sequence from a pathogenic E. coli strain (JM4660) that lacks all other haemolysins has been reported to encode an Arg residue at position 188 that was difficult to reconcile with the proposed role of the beta-tongue. Here it is shown that the JM4660 hlyE sequence encodes Gly, not Arg, at position 188 and that substitution of Gly188 by Arg in E. coli K-12 HlyE abolishes activity, emphasizing the importance of the head domain in HlyE function. Nevertheless, 76 other amino acid substitutions were confirmed compared to the HlyE protein of E. coli K-12. The JM4660 HlyE protein was dimeric, suggesting a mechanism for improving toxin solubility, and it lysed red blood cells from many species by forming 36-41 A diameter pores. However, the haemolytic phenotype of JM4660 was found to be unstable due to defects in HlyE export, indicating that export of active HlyE is not an intrinsic property of the protein but requires additional components. TnphoA mutagenesis of hlyE shows that secretion from the cytoplasm to the periplasm does not require the carboxyl-terminal region of HlyE. Finally, disruption of genes associated with cell envelope function, including tatC, impairs HlyE export, indicating that outer membrane integrity is important for effective HlyE secretion.
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Neil R
authors.initials: NR
authors.lastname: Wyborn
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Angela
authors.initials: A
authors.lastname: Clark
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Ruth E
authors.initials: RE
authors.lastname: Roberts
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Jamieson
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Svetomir
authors.initials: S
authors.lastname: Tzokov
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Per A
authors.initials: PA
authors.lastname: Bullough
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Stillman
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Artymiuk
authors.affiliation: Center for Vaccine Development, Division of Infectious Diseases and Tropical Pediatrics, University of Maryland School of Medicine, 685 W. Baltimore St, Baltimore, MD 21201, USA.
authors.forename: James E
authors.initials: JE
authors.lastname: Galen
authors.affiliation: Center for Vaccine Development, Division of Infectious Diseases and Tropical Pediatrics, University of Maryland School of Medicine, 685 W. Baltimore St, Baltimore, MD 21201, USA.
authors.forename: Licheng
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Center for Vaccine Development, Division of Infectious Diseases and Tropical Pediatrics, University of Maryland School of Medicine, 685 W. Baltimore St, Baltimore, MD 21201, USA.
authors.forename: Myron M
authors.initials: MM
authors.lastname: Levine
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Green
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002645:Chickens / Q000382:microbiology*; D004251:DNA Transposable Elements; D004912:Erythrocytes; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity*; D004927:Escherichia coli Infections / Q000382:microbiology / Q000662:veterinary; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006168:Guinea Pigs; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D011201:Poultry Diseases / Q000382:microbiology*; D017510:Protein Folding; D011817:Rabbits
chemical_list: D001427:Bacterial Toxins; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins