J I Keenan1, R A Allardyce. 1. Department of Surgery, Christchurch School of Medicine, New Zealand. jacqui.keenan@chmeds.ac.nz
Abstract
BACKGROUND: Helicobacter pylori shed outer membrane vesicles (OMV) in vitro and in vivo. These OMV, which contain active VacA, provide a potential vehicle for the delivery of H. pylori virulence factors to the gastric mucosa. OBJECTIVE: To assess the influence of environmental iron levels on H. pylori OMV VacA and protease expression in vitro. METHODS: Three well-characterized H. pylori type-strains were grown for 72 h under normal (Brucella broth, 5% fetal calf serum) and iron-limiting (Brucella broth, 5% fetal calf serum, 50 micromol/l 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. RESULTS: 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. CONCLUSIONS: These results suggest that OMV release by H. pylori is influenced by environmental iron levels, and that the qualitative changes that occur in outer membrane composition may contribute to the clinical patterns of H. pylori-associated disease.
BACKGROUND:Helicobacter pylori shed outer membrane vesicles (OMV) in vitro and in vivo. These OMV, which contain active VacA, provide a potential vehicle for the delivery of H. pylori virulence factors to the gastric mucosa. OBJECTIVE: To assess the influence of environmental iron levels on H. pylori OMV VacA and protease expression in vitro. METHODS: Three well-characterized H. pylori type-strains were grown for 72 h under normal (Brucella broth, 5% fetal calf serum) and iron-limiting (Brucella broth, 5% fetal calf serum, 50 micromol/l 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. RESULTS: 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. CONCLUSIONS: These results suggest that OMV release by H. pylori is influenced by environmental iron levels, and that the qualitative changes that occur in outer membrane composition may contribute to the clinical patterns of H. pylori-associated disease.
Authors: Rafael Prados-Rosales; Brian C Weinrick; Daniel G Piqué; William R Jacobs; Arturo Casadevall; G Marcela Rodriguez Journal: J Bacteriol Date: 2014-01-10 Impact factor: 3.490