Free-soluble and outer membrane vesicle-associated VacA from Helicobacter pylori: Two forms of release, a different activity

This paper is devoted to the memory of Dr. G. Menestrina, leading biophysicist and toxinologist, untimely died in a motorcycle accident in July 2004.
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Abstract

Helicobacter pylori releases VacA both as free-soluble and as outer membrane vesicle (OMV)-associated toxin. In this study, we investigated the amount of VacA released in each of the two forms and the role of each form in VacA-induced cell vacuolation in vitro. We found that: (1) free-soluble toxin accounted for about 75% of released VacA, while the remaining 25% was OMV-associated; (2) although OMV-associated VacA caused a statistically significant vacuolation, virtually all the vacuolating activity of a H. pylori broth culture filtrate was due to free-soluble VacA. While it is widely accepted that OMVs may represent an important vehicle for delivering virulence factors to the gastric mucosa, our results suggest that OMV-associated VacA could play a pathobiological role different from that of free-soluble toxin. This conclusion fits with mounting evidence that VacA exerts a large pattern of pathobiological effects among which cell vacuolation might not be the main one.

Section snippets

Materials and methods

Bacterial strains, broth culture filtrate preparation, and OMV purification. Two well-characterized VacA-producing H. pylori strains (both with a s1a/m1 vacA genotype) were used: 60190 (ATCC 49503) and CCUG 17874 (from Culture Collection University of Göteborg, Göteborg, Sweden). VacA-containing broth culture filtrate (VacA+ BCF) was produced as described by Ricci et al. [19], [20]. Briefly, bacteria were grown in Brucella broth (Difco, Detroit, MI) supplemented with 1% Vitox (Oxoid,

Results and discussion

Mounting evidence suggests an important role for OMVs in the pathogenesis of bacterial diseases. OMVs are released by several Gram-negative bacteria, including Neisseria meningitidis, Haemophilus influenzae, Borrelia burgdorferi, Pseudomonas aeruginosa, Campylobacter jejuni, and enterotoxigenic Escherichia coli[16], [31], [32], [33]. OMVs, consisting of outer membrane lipids and a subset of outer membrane proteins and soluble periplasmic components, have been proposed to be vehicles for

Acknowledgments

This research was supported in part by grants from the Italian Ministry of University and Research (PRIN 2004 to V.R. and U.V.), from the Italian Ministry of Health to IRCCS Policlinico S. Matteo, and from the Pavia University.

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