title: Purification and characterization of the vacuolating toxin from Helicobacter pylori.
abstract: A vacuolating toxin was purified to homogeneity from broth culture supernatant of the human gastric bacterium, Helicobacter pylori. The procedure for isolating the toxin included ammonium sulfate precipitation, hydrophobic interactive chromatography, size exclusion chromatography, and anion exchange chromatography, which together resulted in a greater than 5000-fold purification of toxin activity. The molecular mass of the purified, denatured toxin was 87,000 +/- 320 daltons, and the native toxin was an aggregate with a molecular mass greater than or equal to 972,000 daltons. The amino-terminal sequence of the purified toxin was partially homologous with internal sequences of numerous transport or ion channel proteins. Antiserum raised against the M(r) = 87,000 protein neutralized toxin activity, whereas preimmune serum did not. When reacted with specific antiserum to the M(r) = 87,000 protein in an enzyme-linked immunosorbent (ELISA) assay, culture supernatants from eight tox+ H. pylori strains produced significantly higher optical density readings than eight tox- supernatants (0.614 +/- 0.11 versus 0.046 +/- 0.01, p less than 0.0001). Sera from H. pylori-infected humans recognized the purified M(r) = 87,000 protein significantly better by ELISA than sera from uninfected persons (0.424 +/- 0.06 versus 0.182 +/- 0.02, p = 0.0009). Finally, ELISA recognition of the purified M(r) = 87,000 protein by human sera was significantly associated with toxin-neutralizing activity (p = 0.019, r = 0.518).
authors.affiliation: Infectious Disease Section, Department of Veterans Affairs Medical Center, Nashville, Tennessee 37212.
authors.forename: T L
authors.initials: TL
authors.lastname: Cover
authors.forename: M J
authors.initials: MJ
authors.lastname: Blaser
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006367:HeLa Cells; D016480:Helicobacter pylori / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; C074689:VacA protein, Helicobacter pylori