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Helicobacter pylorished outer membrane vesicles (OMV)in vitroandin vivo.These OMV, which contain active VacA, provide a potential vehicle for the delivery ofH. pylorivirulence factors to the gastric mucosa.To assess the influence of environmental iron levels onH. pyloriOMV VacA and protease expressionin vitro.Three well-characterizedH. pyloritype-strains were grown for 72 h under normal (Brucella broth, 5% fetal calf serum) and iron-limiting (Brucella broth, 5% fetal calf serum, 50 μmol/I deferoxamine) conditions. Following harvesting by differential centrifugation, the ratio of whole cells to OMV was determined. OMV VacA levels in response to iron availability were determined by ELISA and immunolabelling of washed bacteria. Protease activity was detected by zymography of OMV in the presence and absence of enzyme inhibitors and activators. HEp-2 cells were used to assay for OMV-associated cytopathogenic toxins.Decreased iron availability, which limited bacterial growth but not OMV release, also influenced the expression of OMV-associated virulence factors. VacA levels were reduced, whereas two new proteolytic enzymes were expressed on these OMV. When an iron salt was added to counteract the effect of the deferoxamine, VacA levels were restored in the outer membrane and the proteolytic activity disappeared.These results suggest that OMV release byH. pyloriis influenced by environmental iron levels, and that the qualitative changes that occur in outer membrane composition may contribute to the clinical patterns ofH. pylori-associated disease.