title: Random mutagenesis of Helicobacter pylori vacA to identify amino acids essential for vacuolating cytotoxic activity.
abstract: VacA is a secreted toxin that plays a role in Helicobacter pylori colonization of the stomach and may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. In this study, we analyzed a library of plasmids expressing randomly mutated forms of recombinant VacA and identified 10 mutant VacA proteins that lacked vacuolating cytotoxic activity when added to HeLa cells. The mutations included six single amino acid substitutions within an amino-terminal hydrophobic region and four substitutions outside the amino-terminal hydrophobic region. All 10 mutations mapped within the p33 domain of VacA. By introducing mutations into the H. pylori chromosomal vacA gene, we showed that secreted mutant toxins containing V21L, S25L, G121R, or S246L mutations bound to cells and were internalized but had defects in vacuolating activity. In planar lipid bilayer and membrane depolarization assays, VacA proteins containing V21L and S25L mutations were defective in formation of anion-selective membrane channels, whereas proteins containing G121R or S246L mutations retained channel-forming capacity. These are the first point mutations outside the amino-terminal hydrophobic region that are known to abrogate vacuolating toxin activity. In addition, these are the first examples of mutant VacA proteins that have defects in vacuolating activity despite exhibiting channel activities similar to those of wild-type VacA.
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, and Veterans Affairs Tennessee Valley Healthcare System, Nashville, TN 37232, USA.
authors.forename: Mark S
authors.initials: MS
authors.lastname: McClain
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Czajkowsky
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Torres
authors.forename: Gabor
authors.initials: G
authors.lastname: Szabo
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Shao
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Cover
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity*; D003603:Cytotoxins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000633:toxicity*; D019281:Dimerization; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D006367:HeLa Cells; D016480:Helicobacter pylori / Q000235:genetics* / Q000472:pathogenicity*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D016296:Mutagenesis; D012694:Serine / Q000737:chemistry / Q000235:genetics; D014617:Vacuoles / Q000382:microbiology / Q000473:pathology*; D014633:Valine / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003603:Cytotoxins; C074689:VacA protein, Helicobacter pylori; D012694:Serine; D014633:Valine; D005998:Glycine