A Helicobacter pylori vacuolating toxin mutant that fails to oligomerize has a dominant negative phenotype.
Most Helicobacter pylori strains secrete a toxin (VacA) that causes massive vacuolization of target cells and which is a major virulence factor of H. pylori. The VacA amino-terminal region is required for the induction of vacuolization. The aim of the present study was a deeper understanding of the critical role of the N-terminal regions that are protected from proteolysis when VacA interacts with artificial membranes. Using a counterselection system, we constructed an H. pylori strain, SPM 326-Delta49-57, that produces a mutant toxin with a deletion of eight amino acids in one of these protected regions. VacA Delta49-57 was correctly secreted by H. pylori but failed to oligomerize and did not have any detectable vacuolating cytotoxic activity. However, the mutant toxin was internalized normally and stained the perinuclear region of HeLa cells. Moreover, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. This loss of activity was correlated with the disappearance of oligomers in electron microscopy. These findings indicate that the deletion in VacA Delta49-57 disrupts the intermolecular interactions required for the oligomerization of the toxin.
IRIS, Chiron Srl, Via Fiorentina, 1, 53100 Siena, Italy.
Christophe
C
Genisset
Cesira L
CL
Galeotti
Pietro
P
Lupetti
David
D
Mercati
David A G
DA
Skibinski
Silvia
S
Barone
Roberto
R
Battistutta
Marina
M
de Bernard
John L
JL
Telford
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D016480:Helicobacter pylori / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008854:Microscopy, Electron; D009154:Mutation; D010641:Phenotype; D020798:Two-Hybrid System Techniques; D014617:Vacuoles / Q000378:metabolism*
D001426:Bacterial Proteins; C074689:VacA protein, Helicobacter pylori