Functional complementation reveals the importance of intermolecular monomer interactions for Helicobacter pylori VacA vacuolating activity.
The Helicobacter pylori vacuolating cytotoxin (VacA) induces degenerative vacuolation of sensitive mammalian cell lines. Although evidence is accumulating that VacA enters cells and functions from an intracellular site of action, the biochemical mechanism by which VacA mediates cellular vacuolation has not been established. In this study, we used functional complementation and biochemical approaches to probe the structure of VacA. VacA consists of two discrete fragments, p37 and p58, that are both required for vacuolating activity. Using a transient transfection system, we expressed genetically modified forms of VacA and identified mutations in either p37 or p58 that inactivated the toxin. VacA with an inactivating single-residue substitution in the p37 domain [VacA (P9A)] functionally complemented a second mutant form of VacA with an inactivating two-residue deletion in the p58 domain [VacA Delta(346-347)]. VacA (P9A) and VacA Delta(346-347) also co-immunoprecipitated from vacuolated monolayers, supporting the hypothesis that these two inactive mutants associate directly to function in trans. p37 and p58 interact directly when expressed as separate fragments within HeLa cells, suggesting that p37-p58 inter-actions facilitate VacA monomer associations. Collectively, these results support a model in which the active form of VacA requires assembly into a complex of two or more monomers to elaborate toxin function.
Ye
Dan
D
Department of Biology and Biochemistry, University of Houston, 369 Science and Research Building II, Houston, TX 77204-5001, USA.
Blanke
Steven R
SR
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005816:Genetic Complementation Test; D006367:HeLa Cells; D016480:Helicobacter pylori / Q000235:genetics / Q000472:pathogenicity*; D006801:Humans; D009154:Mutation; D013329:Structure-Activity Relationship; D014162:Transfection; D014617:Vacuoles / Q000382:microbiology*
D001426:Bacterial Proteins; C074689:VacA protein, Helicobacter pylori