2C and 2D). Analysis of the culture supernatants by ELISA yielded similar results (data not shown). Thus, all eight of the mutant proteins were expressed and underwent proteolytic processing similar to that of wild-type VacA, but there was substantial variation among the mutant proteins in the levels of

expression and secretion. Figure 2 Expression and secretion of wild-type and mutant VacA proteins. H. pylori wild- type GDC-0941 purchase strain 60190, strains expressing mutant forms of VacA, and a vacA null mutant strain (VM018) [36] 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 click here 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 CHIR 99021 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. Susceptibility of VacA mutant proteins

to proteolytic cleavage by trypsin Previous studies have shown that the wild-type 88 kDa VacA passenger domain is secreted and released into the extracellular space and that 88 kDa proteins also remain localized on the surface of H. pylori [40]. To investigate whether the mutant VacA proteins were able to localize on the bacterial surface similar to wild-type VacA, the wild-type and mutant H. pylori strains were harvested from blood agar plates and treated with trypsin as described in Methods. Trypsin

is expected to proteolytically cleave proteins on the surface Palmatine of the bacteria, but not intracellular proteins [7]. Each of the ~85 kDa mutant proteins was cleaved by trypsin (Fig. 3A), which provided evidence that these mutant VacA proteins are transported across the inner and outer membranes and localize on the surface of the bacteria. Figure 3 Susceptibility of VacA proteins to proteolytic cleavage by trypsin. A) Intact H. pylori strains [wild-type strain 60190, strains expressing mutant forms of VacA, and a vacA null mutant strain (VM018)] were suspended in PBS and incubated in the presence (+) or absence (-) of trypsin as described in Methods. After centrifugation, bacterial pellets were analyzed by immunoblot analysis using polyclonal anti-VacA serum #958. (B) H. pylori strains were sonicated as described in Methods. After centrifugation, the soluble fractions were analyzed further. The total protein concentration of each sample was approximately 7.