FIG. 5.

Inhibition of wild-type VacA cytotoxic activity by VacA Δ346-347. Preparations of acid-activated wild-type VacA (WT; 15 μg/ml) were incubated with 15 μg of acid-activated VacA Δ6-27/ml, 8 μg of acid-activated VacA Δ346-347/ml, or an equivalent volume of an acidified preparation from a vacA-null mutant strain (vacA::km) as described in Materials and Methods, and the mixtures were then added to the medium overlying HeLa cells for 1 h at 37°C. The toxins were removed, and fresh medium containing 5 mM ammonium chloride was added to HeLa cells for 5 h at 37°C. The vacuolating activity was quantified by using a neutral red uptake assay. The results represent the mean ± the SD from triplicate samples. *, P ≤ 0.05 as determined by using ANOVA, followed by Dunnett's post hoc test compared to WT.