Jacqueline I. Keenan

Helicobacter pylori outer membrane vesicles modulate proliferation and interleukin-8 production by gastric epithelial cells.

ABSTRACT Helicobacter pylori infection, which is always associated with gastritis, can progress to ulceration or malignancy. The diversity in clinical outcomes is partly attributed to the expression of virulence factors and adhesins by H. pylori. However, H. pylori may not have to adhere to the epithelium to cause gastritis. We hypothesize that outer membrane vesicles (OMV), which are constantly shed from the surface of H. pylori, play a role as independent activators of host cell responses. In this study, we found that low doses of OMV from cag PAI+ toxigenic and cag PAI− nontoxigenic strains increased proliferation of AGS gastric epithelial cells. At higher doses, we detected growth arrest, increased toxicity, and interleukin-8 (IL-8) production. The only strain differences detected were vacuolation with the toxigenic strain and higher levels of IL-8 production with OMV from the cag PAI− nontoxigenic strain. In summary, we suggest that constitutively shed OMV play a role in promoting the low-grade gastritis associated with H. pylori infection.

Immune Response to an 18-Kilodalton Outer Membrane Antigen Identifies Lipoprotein 20 as a Helicobacter pylori Vaccine Candidate

ABSTRACT Experiments were performed using the standardized murine model ofHelicobacter pylori infection to determine the immunogenicity of H. pylori outer membrane vesicles in immune protection. These vesicles, which are naturally shed from the surface of the bacterium, induce a protective response when administered intragastrically to mice in the presence of cholera holotoxin, despite the absence of the urease enzyme and associated Hsp54 chaperonin. Immunoblotting identified a specific serum immunoglobulin G (IgG) response to an 18-kDa outer membrane protein in a significant number of immunized animals. This commonly expressed, immunodominant protein was subsequently identified as lipoprotein 20 (Lpp20). Hybridoma backpacks secreting an IgG1 subclass monoclonal antibody to Lpp20 were generated in H. pylori-infected mice and were found to significantly reduce bacterial numbers, providing evidence that this surface-exposed antigen is a true vaccine candidate and not merely an antigenic marker for successful, protective immunization.

Uptake of Helicobacter pylori Outer 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 μg/ml LPS), while uptake of OMV from the VacA− mutant strain was less affected (25% inhibition with 50 μg/ml LPS). Vesicle uptake did not require cholesterol. However, uptake of OMV from the VacA− mutant strain was inhibited by a reduction in clathrin-mediated endocytosis (42% with 15 μg/ml chlorpromazine), while uptake of OMV from the VacA+ strain was less affected (25% inhibition with 15 μg/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.

Oxidant-mediated phosphatidylserine exposure and macrophage uptake of activated neutrophils: possible impairment in chronic granulomatous disease

The removal of neutrophils from inflammatory sites is essential for the resolution of inflammation. Surface changes, including phosphatidylserine exposure, label neutrophils for phagocytosis by macrophages. Here, we demonstrate that externalization of phosphatidylserine and uptake by monocyte‐derived macrophages occurred in human neutrophils ingesting Staphylococcus aureus. Both processes were dependent on oxidant production from the neutrophil NADPH oxidase. There was no requirement for myeloperoxidase, and H2O2 was identified as the most likely trigger for PS exposure. We hypothesize that clearance of stimulated neutrophils would be delayed in chronic granulomatous disease (CGD) neutrophils, which lack a functional NADPH oxidase. To explore this possibility, heat‐killed S. aureus were injected into the peritoneum of CGD and normal mice. Elevated neutrophil numbers were observed in the inflammatory exudate of the CGD animals, consistent with impaired recognition and clearance.

The action of bismuth against Helicobacter pylori mimics but is not caused by intracellular iron deprivation.

ABSTRACT Helicobacter pylori is highly susceptible to bismuth, a heavy metal with antimicrobial activity linked to its effect on bacterial iron uptake. Three strains of H. pylori were analyzed for indicators of iron limitation following exposure to the MIC of colloidal bismuth subcitrate (MICCBS). Similar morphologic and outer membrane changes were observed following growth in iron-limiting medium and at the MICCBS that inhibited the growth of all three strains. These changes, which were also observed for iron-limited bacteria, were alleviated by the addition of iron to the cultures. H. pylori ATP levels, reduced in iron-limiting medium, were below the limits of detection in two of the three strains following exposure to bismuth. The addition of iron partially restored bacterial ATP levels in these two strains, although not to normal concentrations. In contrast, exposure of the same strains to the MICCBS failed to deplete intracellular levels of iron, which were significantly reduced by culturing in iron-limiting medium. Thus, the antimicrobial effect of bismuth and of iron limitation on H. pylori may be similar. However, the respective mechanisms of intracellular action would appear to be mediated by different pathways within the cell.

Outer membrane vesicles enhance the carcinogenic potential of Helicobacter pylori

Chronic Helicobacter pylori infection is associated with an increased risk of gastric carcinogenesis. These non-invasive bacteria colonize the gastric mucosa and constitutively shed small outer membrane vesicles (OMV). In this study, we investigated the direct effect of H.pylori OMV on cellular events associated with carcinogenesis. We observed increased micronuclei formation in AGS human gastric epithelial cells treated with OMV isolated from a toxigenic H.pylori strain (60190). This effect was absent in OMV from strain 60190v:1 that has a mutant vacA, indicating VacA-dependent micronuclei formation. VacA induces intracellular vacuolation, and reduced acridine orange staining indicated disruption in the integrity of these vacuoles. This was accompanied by an alteration in iron metabolism and glutathione (GSH) loss, suggesting a role for oxidative stress in genomic damage. Increasing intracellular GSH levels with a GSH ester abrogated the VacA-mediated increase in micronuclei formation. In conclusion, OMV-mediated delivery of VacA to the gastric epithelium may constitute a new mechanism for H.pylori-induced gastric carcinogenesis.

Lewis Epitopes on Outer Membrane Vesicles of Relevance to Helicobacter pylori Pathogenesis

Background.  Helicobacter pylori extrudes protein‐ and lipopolysaccharide‐enriched outer membrane vesicles from its cell surface which have been postulated to act to deliver virulence factors to the host. Lewis antigen expression by lipopolysaccharide of H. pylori cells has been implicated in a number of pathogenic roles. The aim of this study was to further characterize the expression of lipopolysaccharide on the surface of these outer membrane vesicles and, in particular, expression of Lewis antigens and their association with antibody production in the host.

Differences in immunogenicity and protection in mice and guinea pigs following intranasal immunization with Helicobacter pylori outer membrane antigens

Mice and guinea pigs were intranasally immunized with either recombinant lipoprotein 20 or Helicobacter pylori outer membrane vesicles (OMV). Cholera toxin was used as mucosal adjuvant. In mice, both vaccines elicited systemic and local IgG responses, which correlated with significantly lower levels of H. pylori colonization. In contrast, only OMV proved immunogenic in guinea pigs, with the development of both systemic and local immune responses. These antibodies did not, however, correlate with protection in these animals, which suggests that vaccine formulation is as important as choice of antigen in the development of an H. pylori vaccine.

Composition and function of Helicobacter pylori outer membrane vesicles.

The gastric pathogen Helicobacter pylori sheds outer membrane vesicles (OMV) that possess many of the surface elements of the bacterium. Here we review current knowledge on the composition of H. pylori OMV and discuss evidence for their potential roles in bacterial survival and pathogenesis.

Iron influences the expression of Helicobacter pylori outer membrane vesicle-associated virulence factors.

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 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. 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.