Uptake ofHelicobacter pyloriOuter Membrane Vesicles by Gastric Epithelial Cells

Abstract

Helicobacter pylori bacteria colonize the human stomach where they stimulate a persistent inflammatory response. H. pylori is considered noninvasive; however, lipopolysaccharide (LPS)-enriched outer membrane vesicles (OMV), continuously shed from the surface of this bacterium, are observed within gastric epithelial cells. The mechanism of vesicle uptake is poorly understood, and this study was undertaken to examine the roles of bacterial VacA cytotoxin and LPS in OMV binding and cholesterol and clathrin-mediated endocytosis in vesicle uptake by gastric epithelial cells. OMV association was examined using a fluorescent membrane dye to label OMV, and a comparison was made between the associations of vesicles from a VacA ؉ strain and OMV from a VacA ؊ isogenic mutant strain. Within 20 min, essentially all associated OMV were intracellular, and vesicle binding appeared to be facilitated by the presence of VacA cytotoxin. Uptake of vesicles from the VacA ؉ strain was inhibited by H. pylori LPS (58% inhibition with 50 g/ml LPS), while uptake of OMV from the VacA ؊ mutant strain was less affected (25% inhibition with 50 g/ml LPS). Vesicle uptake did not require cholesterol. However, uptake of OMV from the VacA ؊ mutant strain was inhibited by a reduction in clathrinmediated endocytosis (42% with 15 g/ml chlorpromazine), while uptake of OMV from the VacA ؉ strain was less affected (25% inhibition with 15 g/ml chlorpromazine). We conclude that VacA toxin enhances the association of H. pylori OMV with cells and that the presence of the toxin may allow vesicles to exploit more than one pathway of internalization.Infection with the gastric pathogen Helicobacter pylori results in chronic gastritis (13) and is associated with increased risk for the development of peptic ulcer disease (35), gastric carcinoma (41, 57), and gastric lymphoma (5, 60). H. pylori persistence, in an environment where peristalsis and sloughing of cells are continually occurring, is mediated by a variety of adhesins present on the bacterial surface (14,21,36,40). However, despite the ability to adhere to the gastric epithelium, the majority of organisms remain unattached to surface cells (32), leading to speculation that lipopolysaccharide (LPS)-enriched outer membrane vesicles (OMV) shed by these bacteria (15,19,26) contribute to H. pylori pathogenesis via the persistent delivery of bacterial virulence factors (including the vacuolating cytotoxin VacA) and antigens to the gastric mucosa (26,27). Observations that H. pylori OMV modulate gastric epithelial cell proliferation (22), induce apoptosis (3), stimulate secretion of the proinflammatory cytokine interleukin-8 (22), increase micronucleus formation (8), and are at the luminal surface (15, 26) and within cells of the gastric epithelium (15) support this hypothesis.OMV shedding by Gram-negative bacteria is well described in the literature (reviewed by Kuehn and Kesty [33]), yet little is known of the mechanisms of vesicle adherence to and internalization within mammalian host cells. The adherence of...

Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles Are Internalized in Human Host Cells and Trigger NOD1- and NOD2-Dependent NF-κB Activation

“…Internalization of OMVs in human cells, which may also allow further intracellular trafficking of the vesicles, is in accordance with several recent studies on OMVs from various bacterial species, including the human pathogens B. abortus, E. coli, H. pylori, P. aeruginosa, P. gingivalis, and V. cholerae (23)(24)(25)(26)(27)(28) and also nonvirulent E. coli K-12 strains (63). Colocalization of A. actinomycetemcomitans OMVs with the ER is consistent with findings with vesicles from enterotoxigenic E. coli and P. aeruginosa (24,26).…”

“…The inhibitor monensin (Sigma-Aldrich) was used at a final concentration of 2 M or 10 M. To estimate the effect of these inhibitors on cell viability, a neutral red uptake assay was carried out as described previously (51). Similar to the procedure in other studies (27,52), to minimize detrimental effects on the cells, we assessed the effect of the inhibitors mainly by pretreating the target cells for 30 min. The cells were then washed twice, and the medium was replaced prior to the incubation with OMVs.…”

“…It was not known if A. actinomycetemcomitans OMVs can be internalized into the interior of nonphagocytic human cells via endocytic pathways, analogously to vesicles derived from other species, such as Brucella abortus, Escherichia coli, Helicobacter pylori, Pseudomonas aeruginosa, Porphyromonas gingivalis, and Vibrio cholerae (23)(24)(25)(26)(27)(28). In addition to protein delivery, internalization of OMVs represents an important mechanism to expose the host cells to pathogen-associated molecular patterns (PAMPs), e.g., peptidoglycan (PGN) fragments and LPS, which are associated with intact OMVs and/or with fragments from ruptured vesicles within the cell and are recognized by intracellular host pattern recognition receptors (PRRs).…”

Exploring the immunomodulatory potential of microbial-associated molecular patterns derived from the enteric bacterial microbiota

“…Enterotoxigenic E. coli MVs preferentially package heat-labile toxin in their luminal space, protecting it from extracellular enzymic activity and deliver it directly to the cytoplasm of target cells (Kesty et al, 2004). A similar system was also seen in Helicobacter pylori, with its MVs encapsulating and transporting its major virulence factor, H. pylori vacuolating toxin (Parker et al, 2010). The immunomodulatory potential of MVs was recognized when MVs isolated from H. pylori were shown to elicit IL-8 release in human gastric epithelial cells (Ismail et al, 2003).…”

In vitro characterization of multivalent adhesion molecule 7-based inhibition of multidrug-resistant bacteria isolated from wounded military personnel

“…33, 34 We have tested all isolates for the presence of hemolysins using blood agar plating assays ( Table 1), but failed to detect any hemolytic activity in any of the isolates. However, this does not fully exclude the presence of toxins with other functions and this option will be more thoroughly explored in future studies.…”