Helicobacter pylori-Induced TLR9 Activation and Injury Are Associated With the Virulence-Associated Adhesin HopQ.

Helicobacter pylori is the strongest risk factor for gastric adenocarcinoma. The H. pylori cancer-associated cag pathogenicity island (cag-PAI) encodes a type IV secretion system (T4SS), which translocates microbial DNA and activates TLR9; however, most cag-PAI+-infected persons do not develop cancer and cag-PAI-independent regulators of pathogenesis, including strain-specific adhesins, remain understudied. We defined the relationships between H. pylori HopQ adhesin allelic type, gastric injury, and TLR9 activation. Type I hopQ alleles were significantly associated with magnitude of injury, cag-T4SS function, and TLR9 activation. Genetic deletion of hopQ significantly decreased H. pylori-induced TLR9 activation, implicating this adhesin in H. pylori-mediated disease.

Helicobacter pylori antimicrobial resistance and antibiotic consumption in the low-resource Central America setting.

BACKGROUND: Antimicrobial resistance is a global public health problem, particularly in low- and middle-income countries (LMICs), where antibiotics are often obtained without a prescription. H. pylori antimicrobial resistance patterns are informative for patient care and gastric cancer prevention programs, have been shown to correlate with general antimicrobial consumption, and may guide antimicrobial stewardship programs in LMICs. We report H. pylori resistance and antimicrobial utilization patterns for western Honduras, representative of rural Central America. METHODS: In the context of the western Honduras gastric cancer epidemiology initiative, gastric biopsies from 189 patients were studied for culture and resistance patterns. Antimicrobial utilization was investigated for common H. pylori treatment regimens from regional public (7 antimicrobials) and national private (4 antimicrobials) data, analyzed in accordance with WHO anatomical therapeutic chemical defined daily doses (DDD) method and expressed as DDD/1000 inhabitants per day (DID) and per year (DIY). RESULTS: H. pylori was successfully cultured from 116 patients (56% males, mean age: 54), and nearly all strains were cagA+ and vacAs1m1+ positive (99% and 90.4%, respectively). Unexpectedly, high resistance was noted for levofloxacin (20.9%) and amoxicillin (10.7%), while metronidazole (67.9%) and clarithromycin (11.2%) were similar to data from Latin America. Significant associations with age, gender, or histology were not noted, with the exception of levofloxacin (28%, P = 0.01) in those with histology limited to non-atrophic gastritis. Total antimicrobial usage in western Honduras of amoxicillin (17.3 DID) and the quinolones had the highest relative utilizations compared with other representative nations. CONCLUSIONS: We observed significant H. pylori resistance to amoxicillin and levofloxacin in the context of high community antimicrobial utilization. This has implications in Central America for H. pylori treatment guidelines as well as antimicrobial stewardship programs.

Helicobacter pylori-induced cell death is counteracted by NF-κB-mediated transcription of DARPP-32.

OBJECTIVE: DARPP-32 is a frequently amplified and overexpressed gene that promotes several oncogenic functions in gastric cancer. Herein, we investigated the relationship between Helicobacter pylori infection, proinflammatory NF-κB activation and regulation of DARPP-32. DESIGN: The study used in vivo and in vitro experiments. Luciferase reporter, quantitative real-time PCR, immunoblot, chromatin immunoprecipitation (ChIP), cell viability, H. pylori infection, tissue microarrays and immunohistochemical assays were used. RESULTS: Our results indicated that H. pylori infection increased the DARPP-32 mRNA and protein levels in gastric cancer cell lines and gastric mucosa of mice. H. pylori infection increased the activity of NF-κB reporter and p-NF-κB (S536) protein level in vitro and in vivo. To investigate the transcriptional regulation of DARPP-32, we cloned a 3019 bp of the DARPP-32 promoter into the luciferase reporter (pGL3-Luc). Both H. pylori infection and tumour necrosis factor-α treatment induced DARPP-32 reporter activity (p<0.01). Using deletion constructs of DARPP-32 promoter and ChIP assay, we demonstrated that the sequence -996 to -1008 bp containing putative NF-κB-binding sites is the most active region. The induction of DARPP-32 expression by H. pylori infection counteracted H. pylori-induced cell death through activation of serine/threonine-specific protein kinase (AKT), as determined by ATP-Glo and clonogenic survival assays. Immunohistochemistry analysis demonstrated a significant positive correlation between NF-κB and DARPP-32 expression levels in gastric cancer tissues (r2=0.43, p<0.01). CONCLUSIONS: Given the high frequency of DARPP-32 overexpression and its prosurvival oncogenic functions, the induction of DARPP-32 expression following H. pylori infection and activation of NF-κB provides a link between infection, inflammation and gastric tumourigenesis.

TLR9 activation suppresses inflammation in response to Helicobacter pylori infection.

Helicobacter pylori (H. pylori) induces chronic gastritis in humans, and infection can persist for decades. One H. pylori strain-specific constituent that augments disease risk is the cag pathogenicity island. The cag island encodes a type IV secretion system (T4SS) that translocates DNA into host cells. Toll-like receptor 9 (TLR9) is an innate immune receptor that detects hypo-methylated CpG DNA motifs. In this study, we sought to define the role of the H. pylori cag T4SS on TLR9-mediated responses in vivo. H. pylori strain PMSS1 or its cagE- mutant, which fails to assemble a T4SS, were used to infect wild-type or Tlr9-/- C57BL/6 mice. PMSS1-infected Tlr9-/- mice developed significantly higher levels of inflammation, despite similar levels of colonization density, compared with PMSS1-infected wild-type mice. These changes were cag dependent, as both mouse genotypes infected with the cagE- mutant only developed minimal inflammation. Tlr9-/- genotypes did not alter the microbial phenotypes of in vivo-adapted H. pylori strains; therefore, we examined host immunological responses. There were no differences in levels of TH1 or TH2 cytokines in infected mice when stratified by host genotype. However, gastric mucosal levels of IL-17 were significantly increased in infected Tlr9-/- mice compared with infected wild-type mice, and H. pylori infection of IL-17A-/- mice concordantly led to significantly decreased levels of gastritis. Thus loss of Tlr9 selectively augments the intensity of IL-17-driven immune responses to H. pylori in a cag T4SS-dependent manner. These results suggest that H. pylori utilizes the cag T4SS to manipulate the intensity of the host immune response.

Trefoil factor 1 expression suppresses Helicobacter pylori-induced inflammation in gastric carcinogenesis.

BACKGROUND: Infection with Helicobacter pylori, a high-risk factor for gastric cancer, is frequently associated with chronic inflammation through activation of nuclear factor κB (NF-κB). Trefoil factor 1 (TFF1) is a constitutively expressed protein in the stomach that has tumor-suppressor functions and plays a critical role in maintaining mucosal integrity. This study investigated the role of TFF1 in regulating the proinflammatory response to H. pylori infections. METHODS: For in vitro studies, immunofluorescence, luciferase reporter assays, Western blots, and quantitative real-time polymerase chain reaction were performed to investigate the activation of NF-κB and its target genes in response to infections with H. pylori strains J166 and 7.13. In addition, Tff1-knockout (KO) and Tff1-wild-type mice were used for infections with the H. pylori strain called premouse Sydney strain 1. RESULTS: The reconstitution of TFF1 expression in gastric cancer cells significantly suppressed H. pylori-mediated increases in NF-κB-p65 nuclear staining, transcriptional activity, and expression of proinflammatory cytokine genes (tumor necrosis factor α, interleukin 1ß, chemokine [C-X-C motif] ligand 5, and interleukin 4 receptor) that were associated with reductions in the expression and phosphorylation of NF-κB-p65 and IκB kinase α/ß proteins. The in vivo studies using the Tff1-KO mouse model of gastric neoplasia confirmed the in vitro findings. Furthermore, they demonstrated increases in chronic inflammation scores and in the frequency of invasive gastric adenocarcinoma in the Tff1-KO mice infected with H. pylori versus the uninfected Tff1-KO mice. CONCLUSIONS: These findings underscore an important protective role of TFF1 in abrogating H. pylori-mediated inflammation, a crucial hallmark of gastric tumorigenesis. Therefore, loss of TFF1 expression could be an important step in H. pylori-mediated gastric carcinogenesis.

Genetic Manipulation of Helicobacter pylori Virulence Function by Host Carcinogenic Phenotypes.

Helicobacter pylori is the strongest risk factor for gastric adenocarcinoma, yet only a minority of infected persons ever develop this malignancy. One cancer-linked locus is the cag type 4 secretion system (cagT4SS), which translocates an oncoprotein into host cells. A structural component of the cagT4SS is CagY, which becomes rapidly altered during in vivo adaptation in mice and rhesus monkeys, rendering the cagT4SS nonfunctional; however, these models rarely develop gastric cancer. We previously demonstrated that the H. pylori cag+ strain 7.13 rapidly induces gastric cancer in Mongolian gerbils. We now use this model, in conjunction with samples from patients with premalignant lesions, to define the effects of a carcinogenic host environment on the virulence phenotype of H. pylori to understand how only a subset of infected individuals develop cancer. H. pylori cagY sequence differences and cagT4SS function were directly related to the severity of inflammation in human gastric mucosa in either a synchronous or metachronous manner. Serial infections of Mongolian gerbils with H. pylori strain 7.13 identified an oscillating pattern of cagT4SS function. The development of dysplasia or cancer selected for attenuated virulence phenotypes, but robust cagT4SS function could be restored upon infection of new hosts. Changes in the genetic composition of cagY mirrored cagT4SS function, although the mechanisms of cagY alterations differed in human isolates (mutations) versus gerbil isolates (addition/deletion of motifs). These results indicate that host carcinogenic phenotypes modify cagT4SS function via altering cagY, allowing the bacteria to persist and induce carcinogenic consequences in the gastric niche. Cancer Res; 77(9); 2401-12. ©2017 AACR.

Induction of gastric epithelial cell apoptosis by Helicobacter pylori vacuolating cytotoxin.

Chronic gastritis induced by Helicobacter pylori is a strong risk factor for the development of distal gastric adenocarcinoma. A specific host response to H. pylori that may contribute to gastric carcinogenesis is epithelial cell apoptosis. The aim of this study was to investigate the capacity of H. pylori vacuolating toxin (VacA) to induce gastric epithelial cell apoptosis. When cocultured with AGS gastric epithelial cells, H. pylori strain 60190, which expresses a type s1/m1 VacA toxin, induced significantly higher levels of apoptosis than did an isogenic vacA null mutant strain. VacA purified from strain 60190 induced apoptosis in a dose-dependent manner, which required acid activation of the purified toxin and the presence of ammonium chloride. In contrast, apoptosis was not induced after incubation with a chimeric s2/m1 toxin (in which the s1 sequence at the NH(2) terminus of VacA from strain 60190 was replaced with the s2 sequence from the nontoxigenic strain Tx30a) or a VacA mutant protein (VacA Delta 6-27) that lacks a unique strongly hydrophobic region near the VacA NH(2) terminus. Moreover, when an equimolar mixture of purified VacA Delta 6-27 and purified wild-type VacA were added simultaneously to AGS cells, the mutant toxin exhibited a dominant negative effect, completely inhibiting the apoptosis-inducing activity of wild-type VacA. These results indicate that VacA induces gastric epithelial cell apoptosis and suggest that differences in levels of gastric mucosal epithelial apoptosis among H. pylori-infected persons may result from strain-dependent variations in VacA structure.

Draft Genome Sequence of Gerbil-Adapted Carcinogenic Helicobacter pylori Strain 7.13.

We report here the draft genome sequence of Helicobacter pylori strain 7.13, a gerbil-adapted strain that causes gastric cancer in gerbils. Strain 7.13 is derived from clinical strain B128, isolated from a patient with a duodenal ulcer. This study reveals genes associated with the virulence of the strain.

Helicobacter pylori promotes the expression of Krüppel-like factor 5, a mediator of carcinogenesis, in vitro and in vivo.

Helicobacter pylori is the strongest known risk factor for the development of gastric adenocarcinoma. H. pylori expresses a repertoire of virulence factors that increase gastric cancer risk, including the cag pathogenicity island and the vacuolating cytotoxin (VacA). One host element that promotes carcinogenesis within the gastrointestinal tract is Krüppel-like factor 5 (KLF5), a transcription factor that mediates key cellular functions. To define the role of KLF5 within the context of H. pylori-induced inflammation and injury, human gastric epithelial cells were co-cultured with the wild-type cag(+) H. pylori strain 60190. KLF5 expression was significantly upregulated following co-culture with H. pylori, but increased expression was independent of the cag island or VacA. To translate these findings into an in vivo model, C57BL/6 mice were challenged with the wild-type rodent-adapted cag(+) H. pylori strain PMSS1 or a PMSS1 cagE(-) isogenic mutant. Similar to findings in vitro, KLF5 staining was significantly enhanced in gastric epithelium of H. pylori-infected compared to uninfected mice and this was independent of the cag island. Flow cytometry revealed that the majority of KLF5(+) cells also stained positively for the stem cell marker, Lrig1, and KLF5(+)/Lrig1(+) cells were significantly increased in H. pylori-infected versus uninfected tissue. To extend these results into the natural niche of this pathogen, levels of KLF5 expression were assessed in human gastric biopsies isolated from patients with or without premalignant lesions. Levels of KLF5 expression increased in parallel with advancing stages of neoplastic progression, being significantly elevated in gastritis, intestinal metaplasia, and dysplasia compared to normal gastric tissue. These results indicate that H. pylori induces expression of KLF5 in gastric epithelial cells in vitro and in vivo, and that the degree of KLF5 expression parallels the severity of premalignant lesions in human gastric carcinogenesis.

p120 and Kaiso regulate Helicobacter pylori-induced expression of matrix metalloproteinase-7.

Helicobacter pylori is the strongest known risk factor for gastric adenocarcinoma, yet only a fraction of infected persons develop cancer. One H. pylori constituent that augments disease risk is the cytotoxin-associated gene (cag) pathogenicity island, which encodes a secretion system that translocates bacterial effector molecules into host cells. Matrix metalloproteinase (MMP)-7, a member of a family of enzymes with tumor-initiating properties, is overexpressed in premalignant and malignant gastric lesions, and H. pylori cag(+) strains selectively increase MMP-7 protein levels in gastric epithelial cells in vitro and in vivo. We now report that H. pylori-mediated mmp-7 induction is transcriptionally regulated via aberrant activation of p120-catenin (p120), a component of adherens junctions. H. pylori increases mmp-7 mRNA levels in a cag- and p120-dependent manner and induces translocation of p120 to the nucleus in vitro and in a novel ex vivo gastric gland culture system. Nuclear translocation of p120 in response to H. pylori relieves Kaiso-mediated transcriptional repression of mmp-7, which is implicated in tumorigenesis. These results indicate that selective and coordinated induction of mmp-7 expression by H. pylori cag(+) isolates may explain in part the augmentation in gastric cancer risk associated with these strains.

Helicobacter pylori flagellin evades toll-like receptor 5-mediated innate immunity.

Helicobacter pylori colonizes the human stomach for decades unless pharmacologically eradicated. We hypothesized that this flagellated pathogen escapes immune clearance, in part, by avoiding detection by the flagellin receptor Toll-like receptor 5 (TLR5). In contrast to other gram-negative microbes, H. pylori did not release flagellin. Furthermore, recombinant H. pylori flagellin (FlaA) was significantly less potent (1000-fold) than Salmonella typhimurium flagellin in activating TLR5-mediated interleukin (IL)-8 secretion. TLR5 can mediate flagellin-induced IL-8 secretion via p38 mitogen-activated protein kinase signaling; however, compared with potent induction by S. typhimurium flagellin, H. pylori FlaA-dependent p38 activation was substantially attenuated. In addition, disruption of H. pylori flaA decreased motility but had no effect on H. pylori-induced IL-8 secretion, which indicates that H. pylori flagellin plays no role in activating epithelial orchestration of inflammation. We conclude that H. pylori evades TLR5-mediated detection, which may contribute to its long-term persistence in individual hosts.

Helicobacter pylori strain-selective induction of matrix metalloproteinase-7 in vitro and within gastric mucosa.

BACKGROUND AND AIMS: Helicobacter pylori strains that possess the cag pathogenicity island (cag(+)) augment the risk for distal gastric cancer. Matrix metalloproteinase (MMP)-7, an epithelial cell-derived MMP that is induced by bacterial contact, is overexpressed within human gastric adenocarcinoma specimens and enhances tumor formation in rodents. We determined whether H. pylori alters MMP-7 expression and investigated the molecular pathways required for these events. METHODS: MMP-7 was detected in human gastric mucosa by immunohistochemistry and in H. pylori/AGS gastric epithelial cell coculture supernatants by Western analysis. AGS cells were cocultured with wild-type H. pylori, or isogenic cagA(-), cagE(-), or vacA(-) mutants, in the absence or presence of inhibitors of nuclear factor kappaB activation, p38, or extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase. RESULTS: H. pylori cag(+) strains increased MMP-7 expression in AGS cells 5-7-fold, whereas cag(-) isolates had no effect. Inactivation of cagE, but not cagA or vacA, completely attenuated induction of MMP-7, and inhibition of ERK 1/2 decreased MMP-7 production. In vivo, MMP-7 was expressed in gastric epithelial cells in specimens from 80% of cag(+)-colonized persons but in none of the cag(-) or uninfected subjects. CONCLUSIONS: H. pylori cag(+) strains enhance levels of MMP-7 within inflamed mucosa. In vitro, cag(+) isolates selectively induce MMP-7, and this is dependent on activation of ERK 1/2 by specific components within the cag island. Differential induction of MMP-7 by H. pylori cag(+) isolates may explain in part the augmentation in gastric cancer risk associated with these strains.