title: Biophysical Characterization and Activity of Lymphostatin, a Multifunctional Virulence Factor of Attaching and Effacing Escherichia coli.
abstract: Attaching and effacing Escherichia coli cause diarrhea and typically produce lymphostatin (LifA), an inhibitor of mitogen-activated proliferation of lymphocytes and pro-inflammatory cytokine synthesis. A near-identical factor (Efa1) has been reported to mediate adherence of E. coli to epithelial cells. An amino-terminal region of LifA shares homology with the catalytic domain of the large clostridial toxins, which are retaining glycosyltransferases with a DXD motif involved in binding of a metal ion. Understanding the mode(s) of action of lymphostatin has been constrained by difficulties obtaining a stably transformed plasmid expression clone. We constructed a tightly inducible clone of enteropathogenic E. coli O127:H6 lifA for affinity purification of lymphostatin. The purified protein inhibited mitogen-activated proliferation of bovine T lymphocytes in the femtomolar range. It is a monomer in solution and the molecular envelope was determined using both transmission electron microscopy and small-angle x-ray scattering. Domain architecture was further studied by limited proteolysis. The largest proteolytic fragment containing the putative glycosyltransferase domain was tested in isolation for activity against T cells, and was not sufficient for activity. Tryptophan fluorescence studies indicated thatlymphostatin binds uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) but not UDP-glucose (UDP-Glc). Substitution of the predicted DXD glycosyltransferase motif with alanine residues abolished UDP-GlcNAc binding and lymphostatin activity, although other biophysical properties were unchanged. The data indicate that lymphostatin has UDP-sugar binding potential that is critical for activity, and is a major leap toward identifying the nature and consequences of modifications of host cell factors.
authors.affiliation: From the Roslin Institute, Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, United Kingdom.
authors.forename: Robin L
authors.initials: RL
authors.lastname: Cassady-Cain
authors.affiliation: the Centre for Translational and Chemical Biology (CTCB), University of Edinburgh, Michael Swann Building, King's Buildings, Edinburgh EH9 3BF, United Kingdom, and.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Blackburn
authors.affiliation: the Department of Microbiology & Infection Control, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark.
authors.forename: Husam
authors.initials: H
authors.lastname: Alsarraf
authors.affiliation: the Department of Microbiology & Infection Control, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark.
authors.forename: Emil
authors.initials: E
authors.lastname: Dedic
authors.affiliation: From the Roslin Institute, Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, United Kingdom.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Bease
authors.affiliation: the Centre for Translational and Chemical Biology (CTCB), University of Edinburgh, Michael Swann Building, King's Buildings, Edinburgh EH9 3BF, United Kingdom, and.
authors.forename: Bettina
authors.initials: B
authors.lastname: Böttcher
authors.affiliation: the Department of Microbiology & Infection Control, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark.
authors.forename: René
authors.initials: R
authors.lastname: Jørgensen
authors.affiliation: the Centre for Translational and Chemical Biology (CTCB), University of Edinburgh, Michael Swann Building, King's Buildings, Edinburgh EH9 3BF, United Kingdom, and.
authors.forename: Martin
authors.initials: M
authors.lastname: Wear
authors.affiliation: From the Roslin Institute, Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, United Kingdom,. Electronic address: Mark.Stevens@roslin.ed.ac.uk.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Stevens
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000276:immunology*; D002417:Cattle; D002418:Cattle Diseases / Q000276:immunology* / Q000382:microbiology; D054308:Enteropathogenic Escherichia coli / Q000737:chemistry / Q000276:immunology*; D004927:Escherichia coli Infections / Q000276:immunology / Q000382:microbiology / Q000662:veterinary*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000276:immunology*; D016695:Glycosyltransferases / Q000737:chemistry / Q000276:immunology; D006801:Humans; D008213:Lymphocyte Activation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D016415:Sequence Alignment; D013601:T-Lymphocytes / Q000276:immunology / Q000382:microbiology*; D037521:Virulence Factors / Q000276:immunology; D014961:X-Ray Diffraction
chemical_list: D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D037521:Virulence Factors; C406587:lymphostatin protein, E coli; D016695:Glycosyltransferases
keywords: Escherichia coli (E. coli); LifA; T-cell; cell proliferation; circular dichroism (CD); electron microscopy (EM); glycosyltransferase; lymphostatin; small-angle X-ray scattering (SAXS); structural biology