Richard M. Peek

Clinical and pathological importance of heterogeneity in vacA, the vacuolating cytotoxin gene of Helicobacter pylori

BACKGROUND & AIMS vacA encodes the vacuolating cytotoxin of Helicobacter pylori and exhibits marked variation in signal sequence and midgene coding regions. The implications for gastroduodenal pathology are unknown. The aim of this study was to define the association of vacA genotype with gastric inflammation and injury, in vitro cytotoxin activity, and peptic ulceration. METHODS Sixty-one consecutive dyspeptic patients underwent endoscopy and gastric biopsy. The biopsy specimens were processed for H. pylori culture, and 52 specimens were also processed for histology. H. pylori vacA was typed by polymerase chain reaction and colony hybridization. Cytotoxin activity was assessed by a HeLa cell vacuolation assay. RESULTS vacA signal sequence type s1a strains were associated with greater antral mucosal neutrophil and lymphocyte infiltration than s1b or s2 strains (P < 0.05). vacA midregion type m1 strains were associated with greater gastric epithelial damage than m2 strains (P < 0.05). Both midregion and signal sequence were associated with cytotoxin activity in vitro. Duodenal ulcer disease occurred in 89% of 18 patients with s1a strains vs. 29% of 14 with s1b strains (P < 0.01), 20% of 10 with s2 strains (P < 0.001), and 16% of 19 uninfected patients (P < 0.001). CONCLUSIONS H. pylori strains of vacA signal sequence type s1a are associated with enhanced gastric inflammation and duodenal ulceration. vacA s2 strains are associated with less inflammation and lower ulcer prevalence.

Helicobacter pylori strain-specific differences in genetic content, identified by microarray, influence host inflammatory responses

Helicobacter pylori enhances the risk for ulcer disease and gastric cancer, yet only a minority of H. pylori-colonized individuals develop disease. We examined the ability of two H. pylori isolates to induce differential host responses in vivo or in vitro, and then used an H. pylori whole genome microarray to identify bacterial determinants related to pathogenesis. Gastric ulcer strain B128 induced more severe gastritis, proliferation, and apoptosis in gerbil mucosa than did duodenal ulcer strain G1.1, and gastric ulceration and atrophy occurred only in B128+ gerbils. In vitro, gerbil-passaged B128 derivatives significantly increased IL-8 secretion and apoptosis compared with G1.1 strains. DNA hybridization to the microarray identified several strain-specific differences in gene composition including a large deletion of the cag pathogenicity island in strain G1.1. Partial and complete disruption of the cag island in strain B128 attenuated induction of IL-8 in vitro and significantly decreased gastric inflammation in vivo. These results indicate that the ability of H. pylori to regulate epithelial cell responses related to inflammation depends on the presence of an intact cag pathogenicity island. Use of an H pylori whole genome microarray is an effective method to identify differences in gene content between H. pylori strains that induce distinct pathological outcomes in a rodent model of H. pylori infection.

Colon-specific delivery of a probiotic-derived soluble protein ameliorates intestinal inflammation in mice through an EGFR-dependent mechanism

Probiotic bacteria can potentially have beneficial effects on the clinical course of several intestinal disorders, but our understanding of probiotic action is limited. We have identified a probiotic bacteria-derived soluble protein, p40, from Lactobacillus rhamnosus GG (LGG), which prevents cytokine-induced apoptosis in intestinal epithelial cells. In the current study, we analyzed the mechanisms by which p40 regulates cellular responses in intestinal epithelial cells and p40s effects on experimental colitis using mouse models. We show that the recombinant p40 protein activated EGFR, leading to Akt activation. Activation of EGFR by p40 was required for inhibition of cytokine-induced apoptosis in intestinal epithelial cells in vitro and ex vivo. Furthermore, we developed a pectin/zein hydrogel bead system to specifically deliver p40 to the mouse colon, which activated EGFR in colon epithelial cells. Administration of p40-containing beads reduced intestinal epithelial apoptosis and disruption of barrier function in the colon epithelium in an EGFR-dependent manner, thereby preventing and treating DSS-induced intestinal injury and acute colitis. Furthermore, p40 activation of EGFR was required for ameliorating colon epithelial cell apoptosis and chronic inflammation in oxazolone-induced colitis. These data define what we believe to be a previously unrecognized mechanism of probiotic-derived soluble proteins in protecting the intestine from injury and inflammation.

Iron deficiency accelerates Helicobacter pylori–induced carcinogenesis in rodents and humans

Gastric adenocarcinoma is strongly associated with Helicobacter pylori infection; however, most infected persons never develop this malignancy. H. pylori strains harboring the cag pathogenicity island (cag+), which encodes CagA and a type IV secretion system (T4SS), induce more severe disease outcomes. H. pylori infection is also associated with iron deficiency, which similarly augments gastric cancer risk. To define the influence of iron deficiency on microbial virulence in gastric carcinogenesis, Mongolian gerbils were maintained on iron-depleted diets and infected with an oncogenic H. pylori cag+ strain. Iron depletion accelerated the development of H. pylori-induced premalignant and malignant lesions in a cagA-dependent manner. H. pylori strains harvested from iron-depleted gerbils or grown under iron-limiting conditions exhibited enhanced virulence and induction of inflammatory factors. Further, in a human population at high risk for gastric cancer, H. pylori strains isolated from patients with the lowest ferritin levels induced more robust proinflammatory responses compared with strains isolated from patients with the highest ferritin levels, irrespective of histologic status. These data demonstrate that iron deficiency enhances H. pylori virulence and represents a measurable biomarker to identify populations of infected persons at high risk for gastric cancer.

Loss of TFF1 is associated with activation of NF-κB-mediated inflammation and gastric neoplasia in mice and humans.

Trefoil factor 1 (TFF1) is a tumor suppressor gene that encodes a peptide belonging to the trefoil factor family of protease-resistant peptides. Although TFF1 expression is frequently lost in gastric carcinomas, the tumorigenic pathways this affects have not been determined. Here we show that Tff1-knockout mice exhibit age-dependent carcinogenic histological changes in the pyloric antrum of the gastric mucosa, progressing from gastritis to hyperplasia, low-grade dysplasia, high-grade dysplasia, and ultimately malignant adenocarcinoma. The histology and molecular signatures of gastric lesions in the Tff1-knockout mice were consistent with an inflammatory phenotype. In vivo, ex-vivo, and in vitro studies showed that TFF1 expression suppressed TNF-α-mediated NF-κB activation through the TNF receptor 1 (TNFR1)/IκB kinase (IKK) pathway. Consistent with these mouse data, human gastric tissue samples displayed a progressive decrease in TFF1 expression and an increase in NF-κB activation along the multi-step carcinogenesis cascade. Collectively, these results provide evidence that loss of TFF1 leads to activation of IKK complex-regulated NF-κB transcription factors and is an important event in shaping the NF-κB-mediated inflammatory response during the progression to gastric tumorigenesis.

Delineation of a Carcinogenic Helicobacter pylori Proteome

Helicobacter pylori is the strongest known risk factor for gastric adenocarcinoma, yet only a fraction of infected persons ever develop cancer. The extensive genetic diversity inherent to this pathogen has precluded comprehensive analyses of constituents that mediate carcinogenesis. We previously reported that in vivo adaptation of a non-carcinogenic H. pylori strain endowed the output derivative with the ability to induce adenocarcinoma, providing a unique opportunity to identify proteins selectively expressed by an oncogenic H. pylori strain. Using a global proteomics DIGE/MS approach, a novel missense mutation of the flagellar protein FlaA was identified that affects structure and function of this virulence-related organelle. Among 25 additional differentially abundant proteins, this approach also identified new proteins previously unassociated with gastric cancer, generating a profile of H. pylori proteins to use in vaccine development and for screening persons infected with strains most likely to induce severe disease.

EGFR regulates macrophage activation and function in bacterial infection

EGFR signaling regulates macrophage function, but its role in bacterial infection has not been investigated. Here, we assessed the role of macrophage EGFR signaling during infection with Helicobacter pylori, a bacterial pathogen that causes persistent inflammation and gastric cancer. EGFR was phosphorylated in murine and human macrophages during H. pylori infection. In human gastric tissues, elevated levels of phosphorylated EGFR were observed throughout the histologic cascade from gastritis to carcinoma. Deleting Egfr in myeloid cells attenuated gastritis and increased H. pylori burden in infected mice. EGFR deficiency also led to a global defect in macrophage activation that was associated with decreased cytokine, chemokine, and NO production. We observed similar alterations in macrophage activation and disease phenotype in the Citrobacter rodentium model of murine infectious colitis. Mechanistically, EGFR signaling activated NF-κB and MAPK1/3 pathways to induce cytokine production and macrophage activation. Although deletion of Egfr had no effect on DC function, EGFR-deficient macrophages displayed impaired Th1 and Th17 adaptive immune responses to H. pylori, which contributed to decreased chronic inflammation in infected mice. Together, these results indicate that EGFR signaling is central to macrophage function in response to enteric bacterial pathogens and is a potential therapeutic target for infection-induced inflammation and associated carcinogenesis.

Spermine oxidase, a polyamine catabolic enzyme that links Helicobacter pylori CagA and gastric cancer risk

We have recently reported that Helicobacter pylori strains expressing the virulence factor cytotoxin-associated gene A (CagA) stimulate increased levels of spermine oxidase (SMO) in gastric epithelial cells, while cagA– strains did not. SMO catabolizes the polyamine spermine and produces H2O2 that results in both apoptosis and DNA damage. Exogenous overexpression of CagA confirmed these findings, and knockdown or inhibition of SMO blocked CagA-mediated apoptosis and DNA damage. The strong association of SMO, apoptosis, and DNA damage was also demonstrated in humans infected with cagA+, but not cagA– strains. In infected gerbils and mice, DNA damage was CagA-dependent and only present in epithelial cells that expressed SMO. We also discovered SMOhigh gastric epithelial cells from infected animals with dysplasia that are resistant to apoptosis despite high levels of DNA damage. Inhibition of polyamine synthesis or SMO could abrogate the development of this cell population that may represent precursors for neoplastic transformation.

Diet, Helicobacter pylori strain-specific infection, and gastric cancer risk among Chinese men.

Evidence for the association of diet and gastric cancer is equivocal, and the majority of previous studies have not evaluated the interaction of diet and infection with Helicobacter pylori, the leading risk factor for gastric cancer. We examined these associations among 226 cases and 451 controls nested within a prospective cohort. Dietary intakes were calculated from validated food frequency questionnaires. Blood levels of 15 antibodies to Helicobacter pylori proteins were assessed using multiplex serology. Odds ratios (ORs) were calculated using logistic regression. Among individuals infected with high-risk Helicobacter pylori (sero-positivity to 5–6 virulent H. pylori proteins), increasing intake of red meat, heme iron, and sodium increased risk (comparing highest tertile to lowest: ORs [95% confidence interval {CI}]: 1.85 [1.01–3.40]; 1.95 [1.06–3.57]; and 1.76 [0.91–3.43], respectively) while increasing intake of fruit decreased gastric cancer risk (comparing highest tertile of intake to lowest: OR [95% CI]: 0.52 [0.28–0.94]). No associations of diet with risk were found among individuals infected with low-risk H. pylori (P for interaction for red meat and sodium: 0.02 and 0.01, respectively). In this population with over 90% prevalence of CagA-positive H. pylori infection, categorizing individuals using H. pylori multiplex serology may identify individuals for whom a diet intervention may be effective.

The gastric microbiome, its interaction with Helicobacter pylori , and its potential role in the progression to stomach cancer

Gastric adenocarcinoma is the third leading cause of cancer-related death worldwide, accounting for more than 720,000 deaths annually [1]. The strongest known risk factor for this devastating disease is infection with Helicobacter pylori, which drives the development of premalignant lesions (such as gastric atrophy, intestinal metaplasia, and dysplasia) that can lead to gastric cancer (Fig 1). However, although H. pylori is the most common bacterial infection worldwide and colonizes greater than 50% of the global population, only 1%–3% of infected individuals ever develop gastric cancer. Open in a separate window Fig 1 Alterations in the gastric microbiota following Helicobacter pylori infection and gastric disease progression. (A) Schematic representation of the predominant phyla of the gastric microbiota based on H. pylori infection status. H. pylori-negative individuals harbor a microbiota that is more complex and highly diverse compared to H. pylori-positive individuals. (B) Schematic representation of the predominant genera at different stages within the gastric carcinogenesis cascade. Following infection with H. pylori, Proteobacteria and specifically H. pylori dominate the gastric microbiota. This leads to the development of chronic gastritis. In the later stages of the disease, ranging from intestinal metaplasia to gastric adenocarcinoma, a number of genera are enriched. These include Escherichia-Shigella and Burkholderia within the Proteobacteria phylum; Lactobacillus, Lachnospiraceae, Streptococcus, and Veillonella within the Firmicutes phylum; and Prevotella within the Bacteroidetes phylum.