Interaction of Helicobacter pylori with gastric epithelium

Many Helicobacter pylori organisms colonizing the human stomach are free-floating in the mucous layer covering the gastric epithelium. Only a fraction of these bacteria are found to adhere to the luminal surface of the epithelium either with close contact, apparent fusion or juxtaposition of the bacterial outer membrane with the luminal membrane or with a number of thin filaments radiating from the bacterial surface and joining the two membranes1,2. It has been shown that bacterial adhesion causes rearrangement of the underlying cell cytoskeleton and a number of other morphological and biochemical changes. It is postulated that at the site of adhesion Cag proteins of the bacterial membrane, encoded by genes of the so-called pathogenicity island, may form secretory channels allowing direct delivery of bacterial constituents from the bacterial body into the cell cytoplasm3. Other bacterial products, such as the vacuolating cytotoxin, VacA, are believed to be actively secreted, probably by a type II mechanism. This implies transport of the VacA protoxin across the inner bacterial membrane, via a sec-mediated pathway, followed by transport across the outer membrane via a channel formed by the C-terminus of VacA itself4,6. In support of this concept, we localized VacA immunoreactivity by the immunogold test in the periplasm and outer membrane of intact bacteria, either cultured in vitro or colonizing the gastric mucosa in vivo. In addition, we detected free VacA in the peribacterial space, within culture medium or gastric mucus, a likely result of active toxin secretion. During these investigations outer membrane vesicles (OMVs), 50–250 nm in size, have been found to be released by the bacteria, both in culture and in the gastric lumen, through budding of outer membrane blebs enveloping periplasmic expansions, which later detach from the bacterium to form free-floating vesicles carrying VacA immunoreactivity6. Thus, OMVs were found free in bacterial culture medium, during both growth and stationary phases. They were easily separated from the bacteria by centrifugation, while largely passing through a 0.22-μm cellulose acetate filter, so that they were partly retained in the broth culture filtrates used in tests on gastric MKN28 cell cultures7. In these conditions OMVs were found to adhere in part to the cell membrane at sites of luminal-type differentiation, similar to the behaviour of intact H. pylori organisms. Both free VacA and OMVs contacting the cell membrane can be taken up by the cell, apparently by endocytosis, to be concentrated in cytoplasmic vesicles of an endosomal nature, where they may persist for more than 3 days while retaining VacA biological activity6,7. A similar behaviour of free VacA and VacA-immunoreactive OMVs in respect to colonized gastric epithelium has been observed in gastric biopsies.