Oligomeric and subunit structure of the Helicobacter pylori vacuolating cytotoxin.

Abstract

Abstract. Disease-associated strains of Helicobacter pylori produce a potent toxin that is believed to play a key role in peptic ulcer disease in man. In vitro the toxin causes severe vacuolar degeneration in target cells and has thus been termed VacA (for vacuolating c_.ytotoxin A). Cytotoxic activity is associated with a >600-kD protein consisting of several copies of a 95-kD polypeptide that undergoes specific proteolytic cleavage after release from the bacteria to produce 37-and 58-kD fragments. Quick freeze, deep etch electron microscopy has revealed that the native cytotoxin is formed as regular oligomers with either six-or sevenfold radial symmetry. Within each monomer, two domains can clearly be distinguished, suggesting that the 37-and 58-kD fragments derive from proteolytic cleavage between discrete subunits of the monomer. Analysis of preparations of the toxin that had undergone extensive cleavage into the 37-and 58-kD subunits supports this interpretation and reveals that after cleavage the subunits remain associated in the oligomeric structure. The data suggest a structural similarity with AB-type toxins.

A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain

“…Under nondenaturing conditions at neutral pH, WT VacA assembles into water-soluble flower-shaped oligomeric structures (26)(27)(28)(29)(30)(31) (Fig. 2A); however, when exposed to low pH, VacA disassembles into monomers (Fig.…”

“…WT VacA protein assembles into a heterogeneous assortment of oligomeric structures (26)(27)(28)(29)(30)(31), which is suboptimal for crystal formation. We reasoned that the purified VacA-⌬(346-347)-Str 312 protein might be more suitable for crystallization than WT VacA.…”

“…The 88-kDa VacA protein secreted by H. pylori can assemble into multiple types of water-soluble flower-shaped oligomeric structures, including both double-layered forms (dodecamers and tetradecamers) and single-layered forms (hexamers and heptamers) (26)(27)(28)(29)(30)(31). The single-layered water-soluble oligomers are proposed to be structurally related to VacA membrane channels (17,19,31).…”

Helicobacter pylori toxin VacA induces vacuole formation by acting in the cell cytosol

“…VacA is produced as a 140 kDa precursor, is cleaved at the C-terminal domain and released into the extracellular medium as a 95 kDa mature protein that oligomerizes into heptamers and hexamers (Cover et al, 1994;Phadnis et al, 1994;Schmitt and Haas, 1994;Telford et al, 1994b;Lupetti et al, 1996). VacA shows the unique property of being a protein activated by, and resistant to, pH values as low as 1.5, an acidity that may be reached in the stomach lumen .…”

A New Method to Visualize the Helicobacter pylori-associated Lewisb-binding Adhesin Utilizing SDS-digested Freeze-fracture Replica Labeling

“…Thus far, these aspects have never been demonstrated on an en face view of the H. pylori bacterial envelope. Moreover, from a methodological point of view, there have been few publications concerning freeze-fracture of H. pylori bacteria (Noach et al 1994;Lupetti et al 1996;Cover et al 1997;Lanzavecchia et al 1998): None of these concerns the visualization of specific adhesins or outer membrane receptors.…”