Validation of a Novel Immunoline Assay for Patient Stratification according to Virulence of the Infecting Helicobacter pylori Strain and Eradication Status.

Helicobacter pylori infection shows a worldwide prevalence of around 50%. However, only a minority of infected individuals develop clinical symptoms or diseases. The presence of H. pylori virulence factors, such as CagA and VacA, has been associated with disease development, but assessment of virulence factor presence requires gastric biopsies. Here, we evaluate the H. pylori recomLine test for risk stratification of infected patients by comparing the test score and immune recognition of type I or type II strains defined by the virulence factors CagA, VacA, GroEL, UreA, HcpC, and gGT with patient's disease status according to histology. Moreover, the immune responses of eradicated individuals from two different populations were analysed. Their immune response frequencies and intensities against all antigens except CagA declined below the detection limit. CagA was particularly long lasting in both independent populations. An isolated CagA band often represents past eradication with a likelihood of 88.7%. In addition, a high recomLine score was significantly associated with high-grade gastritis, atrophy, intestinal metaplasia, and gastric cancer. Thus, the recomLine is a sensitive and specific noninvasive test for detecting serum responses against H. pylori in actively infected and eradicated individuals. Moreover, it allows stratifying patients according to their disease state.

Comparison of enzymatic properties and small molecule inhibition of γ-glutamyltranspeptidases from pathogenic and commensal bacteria.

Helicobacter pylori infects the stomach of 50% of the population worldwide, thus causing chronic gastritis. Although this infection can be cured by antibiotic treatment, therapeutic options are increasingly limited due to the development of resistances. The γ-glutamyl-transpeptidase (gGT) of H. pylori (HpgGT) is a virulence factor important for colonization and contributes to bacterial immune evasion. Therefore, this enzyme is a potential target for developing new anti-infectives. As species specificity of such compounds is required in order to avoid off-target or adverse effects, comparative analysis of the gGTs from different organisms is a prerequisite for drug development. To allow detailed biochemical and enzymatic characterization, recombinant gGTs from five different bacteria as well as Homo sapiens were characterized and compared. Investigation of the enzymatic activity, the binding modes of known inhibitors to the catalytic center, and a high resolution X-ray structure of the HpgGT provided a starting point for the identification of new inhibitory substances targeting HpgGT. Inhibitors with Ki values in the nm to mm range were identified and their binding modes were analyzed by mass spectrometry. The results of this study provide a basis for the development of species-specific lead compounds with anti-infective potential by effectively inhibiting HpgGT.

Helicobacter bilis gamma-glutamyltranspeptidase enhances inflammatory stress response via oxidative stress in colon epithelial cells.

Helicobacter bilis (H. bilis) infection is associated with cases of inflammatory bowel Disease, thyphlocolitis, hepatitis and cholecystitis. However, little is known about the bacterial virulence determinants or the molecular mechanisms involved. Recently, H. bilis γ-glutamyltranspeptidase (HBgGT) was shown to be a virulence factor decreasing host cell viability. Bacterial gGTs play a key role in synthesis and degradation of glutathione and enables the bacteria to utilize extracellular glutamine and glutathione as sources of glutamate. gGT-mediated loss of cell viability has so far been linked to DNA damage via oxidative stress, but the signaling cascades involved herein have not been described. In this study, we identified enhanced ROS production induced by HBgGT as a central factor involved in the activation of the oxidative stress response cascades, which finally activate CREB, AP-1 and NF-κB in H. bilis infected colon cancer cells. IL-8, an important pro-inflammatory chemokine that is a common downstream target of these transcription factors, was up-regulated upon H. bilis infection in an HBgGT dependent manner. Moreover, the induction of these signaling responses and inflammatory cytokine production in host cells could be linked to HBgGT-mediated glutamine deprivation. This study implicates for the first time HBgGT as an important regulator of signaling cascades regulating inflammation in H. bilis infected host epithelial cells that could be responsible for induction of inflammatory disorders by the bacterium.

A novel line immunoassay based on recombinant virulence factors enables highly specific and sensitive serologic diagnosis of Helicobacter pylori infection.

Helicobacter pylori colonizes half of the world's population, and infection can lead to ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Serology is the only test applicable for large-scale, population-based screening, but current tests are hampered by a lack of sensitivity and/or specificity. Also, no serologic test allows the differentiation of type I and type II strains, which is important for predicting the clinical outcome. H. pylori virulence factors have been associated with disease, but direct assessment of virulence factors requires invasive methods to obtain gastric biopsy specimens. Our work aimed at the development of a highly sensitive and specific, noninvasive serologic test to detect immune responses to important H. pylori virulence factors. This line immunoassay system (recomLine) is based on recombinant proteins. For this assay, six highly immunogenic virulence factors (CagA, VacA, GroEL, gGT, HcpC, and UreA) were expressed in Escherichia coli, purified, and immobilized to nitrocellulose membranes to detect serological immune responses in patient's sera. For the validation of the line assay, a cohort of 500 patients was screened, of which 290 (58.0%) were H. pylori negative and 210 (42.0%) were positive by histology. The assay showed sensitivity and specificity of 97.6% and 96.2%, respectively, compared to histology. In direct comparison to lysate blotting and enzyme-linked immunosorbent assay (ELISA), the recomLine assay had increased discriminatory power. For the assessment of individual risk for gastrointestinal disease, the test must be validated in a larger and defined patient cohort. Taking the data together, the recomLine assay provides a valuable tool for the diagnosis of H. pylori infection.

Evidence for conserved function of γ-glutamyltranspeptidase in Helicobacter genus.

The confounding consequences of Helicobacter bilis infection in experimental mice populations are well recognized, but the role of this bacterium in human diseases is less known. Limited data are available on virulence determinants of this species. In Helicobacter pylori, γ-glutamyltranspeptidase (γGT) contributes to the colonization of the gastric mucosa and to the pathogenesis of peptic ulcer. The role of γGT in H. bilis infections remains unknown. The annotated genome sequence of H. bilis revealed two putative ggt genes and our aim was to characterize these H. bilis γGT paralogues. We performed a phylogenetic analysis to understand the evolution of Helicobacter γGTs and to predict functional activities of these two genes. In addition, both copies of H. bilis γGTs were expressed as recombinant proteins and their biochemical characteristics were analysed. Functional complementation of Esherichia coli deficient in γGT activity and deletion of γGT in H. bilis were performed. Finally, the inhibitory effect of T-cell and gastric cell proliferation by H. bilis γGT was assessed. Our results indicated that one gene is responsible for γGT activity, while the other showed no γGT activity due to lack of autoprocessing. Although both H. bilis and H. pylori γGTs exhibited a similar affinity to L-Glutamine and γ-Glutamyl-p-nitroanilide, the H. bilis γGT was significantly less active. Nevertheless, H. bilis γGT inhibited T-cell proliferation at a similar level to that observed for H. pylori. Finally, we showed a similar suppressive influence of both H. bilis and H. pylori γGTs on AGS cell proliferation mediated by an apoptosis-independent mechanism. Our data suggest a conserved function of γGT in the Helicobacter genus. Since γGT is present only in a few enterohepatic Helicobacter species, its expression appears not to be essential for colonization of the lower gastrointestinal tract, but it could provide metabolic advantages in colonization capability of different niches.

Helicobacter pylori induces miR-155 in T cells in a cAMP-Foxp3-dependent manner.

Amongst the most severe clinical outcomes of life-long infections with Helicobacter pylori is the development of peptic ulcers and gastric adenocarcinoma--diseases often associated with an increase of regulatory T cells. Understanding H. pylori-driven regulation of T cells is therefore of crucial clinical importance. Several studies have defined mammalian microRNAs as key regulators of the immune system and of carcinogenic processes. Hence, we aimed here to identify H. pylori-regulated miRNAs, mainly in human T cells. MicroRNA profiling of non-infected and infected human T cells revealed H. pylori infection triggers miR-155 expression in vitro and in vivo. By using single and double H. pylori mutants and the corresponding purified enzymes, the bacterial vacuolating toxin A (VacA) and gamma-glutamyl transpeptidase (GGT) plus lipopolysaccharide (LPS) tested positive for their ability to regulate miR-155 and Foxp3 expression in human lymphocytes; the latter being considered as the master regulator and marker of regulatory T cells. RNAi-mediated knockdown (KD) of the Foxp3 transcription factor in T cells abolished miR-155 expression. Using adenylate cyclase inhibitors, the miR-155 induction cascade was shown to be dependent on the second messenger cyclic adenosine monophosphate (cAMP). Furthermore, we found that miR-155 directly targets the protein kinase A inhibitor alpha (PKIalpha) mRNA in its 3'UTR, indicative of a positive feedback mechanism on the cAMP pathway. Taken together, our study describes, in the context of an H. pylori infection, a direct link between Foxp3 and miR-155 in human T cells and highlights the significance of cAMP in this miR-155 induction cascade.

ITF-2 is disrupted via allelic loss of chromosome 18q21, and ITF-2B expression is lost at the adenoma-carcinoma transition.

BACKGROUND & AIMS: The ubiquitously expressed basic helix-loop-helix transcription factor ITF-2B has an important role in differentiation processes, and its transcription is regulated by beta-catenin. The ITF-2 gene is located in the chromosomal region 18q21; allelic loss of this locus occurs in 70% of colorectal cancers. We analyzed the expression, regulation, and function of ITF-2B in colorectal carcinogenesis. METHODS: The loss-of-heterozygosity (LOH) status of 18q21 and expression of ITF-2B were studied in colorectal carcinomas using polymerase chain reaction-based methods and immunohistochemistry. The biologic effects of ITF-2B were studied in colorectal cancer cells. Reporter gene assays and chromatin immunoprecipitation were utilized to analyze effects of ITF-2B on gene transcription. RESULTS: ITF-2B is strongly expressed in colon adenomas but frequently down-regulated in carcinomas because of LOH at 18q21. ITF-2B induces cell cycle arrest and regulates the expression of p21(Cip1) via newly identified E-boxes in the CDKN1A gene, independently of p53. Loss of ITF-2B expression correlates with loss of p21(Cip1) expression in primary colon carcinomas. CONCLUSIONS: Accumulation of mutations and allelic losses are driving forces of colorectal carcinogenesis. ITF-2B, which is up-regulated during early colorectal carcinogenesis because of loss of adenomatous polyposis coli, is a target for LOH on chromosome 18q, along with deleted in colorectal carcinoma and Smad4. This finding, along with the fact that ITF-2B is a regulator of the key cell cycle inhibitor p21(Cip1), indicates that ITF-2B is a tumor suppressor that has an important function at the adenoma to carcinoma transition.