Expression and secretion of wild-type and mutant VacA proteins. H. pylori wild- type strain 60190, strains expressing mutant forms of VacA, and a vacA null mutant strain (VM018)  were grown in broth culture. Broth cultures were normalized by optical density (OD 600 nm) and then pellets (A) and unconcentrated broth culture supernatants (C) were analyzed by immunoblot assay using polyclonal anti-VacA serum #958. Samples were also immunoblotted with a control antiserum against H. pylori heat shock protein (HspB). The intensity of immunoreactive VacA bands was quantified by densitometry (panels B and D). Wild-type VacA and each of the mutant proteins were expressed and proteolytically processed to yield ~85-88 kDa proteins that were secreted into the broth culture supernatant. Western blots depict representative results from one of three independent experiments; histograms represent results pooled from three independent experiments. Results represent the mean ± SD. *, p < 0.05 compared to wild-type VacA, as determined by Student's t-test.