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1.
Gut ; 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31857433

RESUMO

OBJECTIVE: Gastrointestinal microbiota may be involved in Helicobacter pylori-associated gastric cancer development. The aim of this study was to explore the possible microbial mechanisms in gastric carcinogenesis and potential dysbiosis arising from H. pylori infection. DESIGN: Deep sequencing of the microbial 16S ribosomal RNA gene was used to investigate alterations in paired gastric biopsies and stool samples in 58 subjects with successful and 57 subjects with failed anti-H. pylori treatment, relative to 49 H . pylori negative subjects. RESULTS: In H. pylori positive subjects, richness and Shannon indexes increased significantly (both p<0.001) after successful eradication and showed no difference to those of negative subjects (p=0.493 for richness and p=0.420 for Shannon index). Differential taxa analysis identified 18 significantly altered gastric genera after eradication. The combination of these genera into a Microbial Dysbiosis Index revealed that the dysbiotic microbiota in H. pylori positive mucosa was associated with advanced gastric lesions (chronic atrophic gastritis and intestinal metaplasia/dysplasia) and could be reversed by eradication. Strong coexcluding interactions between Helicobacter and Fusobacterium, Neisseria, Prevotella, Veillonella, Rothia were found only in advanced gastric lesion patients, and were absent in normal/superficial gastritis group. Changes in faecal microbiota included increased Bifidobacterium after successful H. pylori eradication and more upregulated drug-resistant functional orthologs after failed treatment. CONCLUSION: H. pylori infection contributes significantly to gastric microbial dysbiosis that may be involved in carcinogenesis. Successful H. pylori eradication potentially restores gastric microbiota to a similar status as found in uninfected individuals, and shows beneficial effects on gut microbiota.

2.
Sci Rep ; 9(1): 17401, 2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31758014

RESUMO

Vaccination is the most effective method to prevent infectious diseases. However, approaches to identify novel vaccine candidates are commonly laborious and protracted. While surface proteins are suitable vaccine candidates and can elicit antibacterial antibody responses, systematic approaches to define surfomes from gram-negatives have rarely been successful. Here we developed a combined discovery-driven mass spectrometry and computational strategy to identify bacterial vaccine candidates and validate their immunogenicity using a highly prevalent gram-negative pathogen, Helicobacter pylori, as a model organism. We efficiently isolated surface antigens by enzymatic cleavage, with a design of experiment based strategy to experimentally dissect cell surface-exposed from cytosolic proteins. From a total of 1,153 quantified bacterial proteins, we thereby identified 72 surface exposed antigens and further prioritized candidates by computational homology inference within and across species. We next tested candidate-specific immune responses. All candidates were recognized in sera from infected patients, and readily induced antibody responses after vaccination of mice. The candidate jhp_0775 induced specific B and T cell responses and significantly reduced colonization levels in mouse therapeutic vaccination studies. In infected humans, we further show that jhp_0775 is immunogenic and activates IFNγ secretion from peripheral CD4+ and CD8+ T cells. Our strategy provides a generic preclinical screening, selection and validation process for novel vaccine candidates against gram-negative bacteria, which could be employed to other gram-negative pathogens.

3.
J Immunol ; 203(8): 2183-2193, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31511355

RESUMO

Helicobacter pylori colonizes the stomach of around 50% of humans. This chronic infection can lead to gastric pathologic conditions such as gastric ulcers and gastric adenocarcinomas. The strong inflammatory response elicited by H. pylori is characterized by the induction of the expression of several cytokines. Among those, IL-18 is found highly upregulated in infected individuals, and its expression correlates with the severity of gastric inflammation. IL-18 is produced as inactive proform and has to be cleaved by the multiprotein complex inflammasome to be active. In immune cells, the NLRC4 inflammasome, which is activated by flagellin or bacterial secretion systems, was shown to be dispensable for H. pylori-induced inflammasome activation. However, apart from immune cells, gastric epithelial cells can also produce IL-18. In this study, we analyzed the role of the NLRC4 inflammasome during H. pylori infection. Our results indicate that NLRC4 and a functional type IV secretion system are crucial for the production of IL-18 from human and murine gastric epithelial cells. In vivo, Nlrc4-/- mice failed to produce gastric IL-18 upon H. pylori infection. Compared with wild type mice, Nlrc4-/- mice controlled H. pylori better without showing strong inflammation. Moreover, H. pylori-induced IL-18 inhibits ß-defensin 1 expression in a NF-κB-dependent manner, resulting in higher bacterial colonization. At the same time, inflammasome activation enhances neutrophil infiltration, resulting in inflammation. Thus, NLRC4 inflammasome activation and subsequent IL-18 production favors bacterial persistence by inhibiting antimicrobial peptide production and, at the same time, contributes to gastric inflammation.

4.
Cell Rep ; 28(1): 231-244.e5, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269443

RESUMO

Helicobacter pylori chronically colonizes the stomach and is strongly associated with gastric cancer. Its concomitant occurrence with helminths such as schistosomes has been linked to reduced cancer incidence, presumably due to suppression of H. pylori-associated pro-inflammatory responses. However, experimental evidence in support of such a causal link or the mutual interaction of both pathogens is lacking. We investigated the effects of co-infection during the different immune phases of S. mansoni infection. Surprisingly, co-infected mice had increased H. pylori gastric colonization during the interferon gamma (IFNγ) phase of schistosome infection but reduced infiltration of T cells in the stomach due to misdirection of antigen-experienced CXCR3+ T cells to the liver. Unexpectedly, H. pylori co-infection resulted in partial protection from schistosome-induced liver damage. Here, we demonstrate that an increase in fibrosis-protective IL-13Ra2 is associated with H. pylori infection. Thus, our study strongly points to an immunological interaction of anatomically isolated pathogens, eventually resulting in altered disease pathology.

5.
Sci Rep ; 9(1): 7030, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31065023

RESUMO

Helicobacter pylori infection induces a number of pro-inflammatory signaling pathways contributing to gastric inflammation and carcinogenesis. Among those, NF-κB signaling plays a pivotal role during infection and malignant transformation of the gastric epithelium. However, deficiency of the adaptor molecule myeloid differentiation primary response 88 (MyD88), which signals through NF-κB, led to an accelerated development of gastric pathology upon H. felis infection, but the mechanisms leading to this phenotype remained elusive. Non-canonical NF-κB signaling was shown to aggravate H. pylori-induced gastric inflammation via activation of the lymphotoxin ß receptor (LTßR). In the present study, we explored whether the exacerbated pathology observed in MyD88-deficient (Myd88-/-) mice was associated with aberrant activation of non-canonical NF-κB. Our results indicate that, in the absence of MyD88, H. felis infection enhances the activation of non-canonical NF-κB that is associated with increase in Cxcl9 and Icam1 gene expression and CD3+ lymphocyte recruitment. In addition, activation of signal transducer and activator of transcription 3 (STAT3) signaling was higher in Myd88-/- compared to wild type (WT) mice, indicating a link between MyD88 deficiency and STAT3 activation in response to H. felis infection. Thereby, MyD88 deficiency results in accelerated and aggravated gastric pathology induced by Helicobacter through activation of non-canonical NF-κB.

6.
Cancers (Basel) ; 11(3)2019 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-30884828

RESUMO

The E3 ubiquitin ligase ring finger protein 43 (RNF43) is frequently mutated in gastric tumors and loss of RNF43 expression was suggested to be one of the key events during the transition from adenoma to gastric carcinoma. Functional studies on RNF43 have shown that it acts as a tumor suppressor by negatively regulating Wnt signaling. Interestingly, we observed that RNF43H292R/H295R mice bearing two point mutations in the ring domain displayed thickening of the mucosa at early age but did not develop neoplasia. In this study, we infected these mice for 6 months with Helicobacter pylori, which has been described as one of the major risk factors for gastric cancer. Mice bearing mutant RNF43H292R/H295R showed higher gastritis scores upon H. pylori infection compared to wild-type mice, accompanied by increased lymphocyte infiltration and Ifng levels. Furthermore, infected Rnf43 mutant mice developed atrophy, hyperplasia and MUC2 expressing metaplasia and displayed higher levels of the gastric stem cell marker CD44 and canonical NF-κB signaling. In summary, our results show that transactivating mutations in the tumor suppressor Rnf43 can worsen H. pylori induced pathology.

7.
Carcinogenesis ; 40(4): 551-559, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-30380024

RESUMO

Ring finger protein 43 (RNF43) is an E3 ubiquitin ligase that has been described to be frequently mutated in gastrointestinal cancers. RNF43 downregulation was associated with distant metastasis, TNM stage and poorer survival in patients with gastric and colorectal cancers. Functional analysis has shown that overexpressed RNF43 negatively regulates Wnt signalling by ubiquitinating Frizzled receptors and targeting them for degradation and by sequestering T-cell factor 4 (TCF4) to the nuclear membrane, thereby inhibiting Wnt-mediated transcription. In the stomach, RNF43 overexpression was shown to impair stem-like properties and to be negatively correlated with expression of Wnt-target genes. In this study, we show that RNF43 knockdown enhances the tumourigenic potential of gastric and colorectal cancer cell lines in vitro and in vivo. Thus, loss of RNF43 leads to increased proliferation and anchorage-independent growth as well as increased invasive capacity. In a xenograft model, RNF43 depletion enhanced tumour growth. Furthermore, we established two mouse models in which mutations in the RING domain of RNF43 were introduced. In the intestine and colon, loss of Rnf43 did not induce changes in epithelial architecture or proliferation. In contrast, in the stomach, thickening of the mucosa, hyperplasia and cellular atypia were observed in these mice. Notably, this was independent of elevated Wnt signalling. Together, our results show that RNF43 plays a tumour suppressive role in gastric and colorectal cancer cells and that the loss of its function alters gastric tissue homeostasis in vivo.

8.
Artigo em Inglês | MEDLINE | ID: mdl-29971220

RESUMO

Eradication of Helicobacter pylori has been found to be effective for gastric cancer prevention, but uncertainties remain about the possible adverse consequences such as the potential microbial dysbiosis. In our study, we investigated the association between gut microbiota and H. pylori-related gastric lesions in 47 subjects by deep sequencing of microbial 16S ribosomal RNA (rRNA) gene in fecal samples. The dominant phyla in fecal samples were Bacteroidetes, Firmicutes, and Proteobacteria with average relative abundances of 54.77, 31.37 and 12.91%, respectively. Microbial diversity analysis showed that observed species and Shannon index were increased in subjects with past or current H. pylori infection compared with negative subjects. As for the differential bacteria, the average relative abundance of Bacteroidetes was found to significantly decrease from H. pylori negative (66.16%) to past infection group (33.01%, p = 0.007), as well as from normal (76.49%) to gastritis (56.04%) and metaplasia subjects (46.83%, p = 0.027). For Firmicutes and Proteobacteria, the average relative abundances showed elevated trends in the past H. pylori infection group (47.11, 20.53%) compared to negative group (23.44, 9.05%, p = 0.068 and 0.246, respectively), and similar increased trends were also found from normal (18.23, 5.05%) to gastritis (35.31, 7.23%, p = 0.016 and 0.294, respectively) or metaplasia subjects (32.33, 20.07%, both p < 0.05). These findings suggest that the alterations of fecal microbiota, especially the dominant phyla of Bacteroidetes, Firmicutes and Proteobacteria, may be involved in the process of H. pylori-related gastric lesion progression and provide hints for future evaluation of microbial changes after H. pylori eradication.


Assuntos
DNA Bacteriano/genética , Microbioma Gastrointestinal/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Neoplasias Gástricas/microbiologia , Adulto , Idoso , Disbiose/microbiologia , Disbiose/patologia , Fezes/microbiologia , Feminino , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Gastrite/microbiologia , Gastrite/patologia , Infecções por Helicobacter/diagnóstico , Humanos , Masculino , Metaplasia/microbiologia , Metaplasia/patologia , Pessoa de Meia-Idade , Neoplasias Gástricas/patologia
9.
EMBO J ; 37(13)2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29858229

RESUMO

The human gastric pathogen Helicobacter pylori is a major causative agent of gastritis, peptic ulcer disease, and gastric cancer. As part of its adhesive lifestyle, the bacterium targets members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family by the conserved outer membrane adhesin HopQ. The HopQ-CEACAM1 interaction is associated with inflammatory responses and enables the intracellular delivery and phosphorylation of the CagA oncoprotein via a yet unknown mechanism. Here, we generated crystal structures of HopQ isotypes I and II bound to the N-terminal domain of human CEACAM1 (C1ND) and elucidated the structural basis of H. pylori specificity toward human CEACAM receptors. Both HopQ alleles target the ß-strands G, F, and C of C1ND, which form the trans dimerization interface in homo- and heterophilic CEACAM interactions. Using SAXS, we show that the HopQ ectodomain is sufficient to induce C1ND monomerization and thus providing H. pylori a route to influence CEACAM-mediated cell adherence and signaling events.


Assuntos
Antígenos CD/fisiologia , Proteínas de Bactérias/fisiologia , Moléculas de Adesão Celular/fisiologia , Helicobacter pylori/fisiologia , Animais , Antígenos CD/química , Proteínas de Bactérias/química , Células CHO , Moléculas de Adesão Celular/química , Linhagem Celular Tumoral , Cricetulus , Humanos , Multimerização Proteica
11.
Sci Rep ; 7(1): 13636, 2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-29057967

RESUMO

Helicobacter pylori γ-glutamyl transferase (gGT) is a key bacterial virulence factor that is not only important for bacterial gastric colonization but also related to the development of gastric pathology. Despite accumulating evidence for pathogenic and immunologic functions of H. pylori gGT, it is still unclear how it supports gastric colonization and how its specific effects on the host's innate and adaptive immune responses contribute to colonization and pathology. We have compared mice showing similar bacterial load after infection with gGT-proficient or gGT-deficient H. pylori to analyse the specific role of the enzyme during infection. Our data indicate that H. pylori gGT supports initial colonization. Nevertheless, bacteria lacking gGT can still colonize and persist. We observed that the presence of gGT during infection favoured a proinflammatory innate and adaptive immune response. Notably, H. pylori gGT activity was linked to increased levels of IFNγ, which were attributed to a differential recruitment of CD8+ T cells to the stomach. Our data support an essential role for H. pylori gGT in gastric colonization and further suggest that gGT favours infiltration of CD8+ cells to the gastric mucosa, which might play an important and yet overlooked role in the pathogenesis of H. pylori.


Assuntos
Proteínas de Bactérias/metabolismo , Linfócitos T CD8-Positivos/imunologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/enzimologia , Fatores de Virulência/metabolismo , gama-Glutamiltransferase/metabolismo , Imunidade Adaptativa , Animais , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/microbiologia , Modelos Animais de Doenças , Feminino , Infecções por Helicobacter/enzimologia , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/crescimento & desenvolvimento , Imunidade Inata , Interferon gama/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estômago/enzimologia , Estômago/imunologia , Estômago/microbiologia , Estômago/patologia
12.
Curr Top Microbiol Immunol ; 400: 53-71, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28124149

RESUMO

Helicobacter pylori infection is commonly acquired during childhood, can persist lifelong if not treated, and can cause different gastric pathologies, including chronic gastritis, peptic ulcer disease, and eventually gastric cancer. H. pylori has developed a number of strategies in order to cope with the hostile conditions found in the human stomach as well as successful mechanisms to evade the strong innate and adaptive immune responses elicited upon infection. Thus, by manipulating innate immune receptors and related signaling pathways, inducing tolerogenic dendritic cells and inhibiting effector T cell responses, H. pylori ensures low recognition by the host immune system as well as its persistence in the gastric epithelium. Bacterial virulence factors such as cytotoxin-associated gene A, vacuolating cytotoxin A, or gamma-glutamyltranspeptidase have been extensively studied in the context of bacterial immune escape and persistence. Further, the bacterium possesses other factors that contribute to immune evasion. In this chapter, we discuss in detail the main evasion and persistence strategies evolved by the bacterium as well as the specific bacterial virulence factors involved.


Assuntos
Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Evasão da Resposta Imune , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Mucosa Gástrica/imunologia , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/fisiologia , Humanos , Fatores de Virulência/genética , Fatores de Virulência/imunologia
13.
Gut ; 66(8): 1369-1381, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-27196595

RESUMO

OBJECTIVE: Lymphotoxin ß receptor (LTßR) signalling has been implicated in inflammation-associated tumour development in different tissues. We have analysed the role of LTßR and alternative NF-κB signalling in Helicobacter pylori-mediated gastric inflammation and pathology. DESIGN: We analysed several ligands and receptors of the alternative NF-κB pathway, RelB, p52 nuclear translocation and target genes in tissue samples of H. pylori-infected patients with different degrees of gastritis or early gastric tumours by in situ hybridisation, immunohistochemistry, Western blot and real-time PCR analyses. Molecular mechanisms involved in LTßR activation by H. pylori were assessed in vitro using human gastric cancer cell lines and distinct H. pylori isolates. The effects of blocking or agonistically activating LTßR on gastric pathology during challenge with a human pathogenic H. pylori strain were studied in a mouse model. RESULTS: Among the tested candidates, LT was significantly increased and activated alternative NF-κB signalling was observed in the gastric mucosa of H. pylori-infected patients. H. pyloriinduced LTßR-ligand expression in a type IV secretion system-dependent but CagA-independent manner, resulting in activation of the alternative NF-κB pathway, which was further enhanced by blocking canonical NF-κB during infection. Blocking LTßR signalling in vivo suppressed H. pylori-driven gastritis, whereas LTßR activation in gastric epithelial cells of infected mice induced a broadened pro-inflammatory chemokine milieu, resulting in exacerbated pathology. CONCLUSIONS: LTßR-triggered activation of alternative NF-κB signalling in gastric epithelial cells executes H. pylori-induced chronic gastritis, representing a novel target to restrict gastric inflammation and pathology elicited by H. pylori, while exclusively targeting canonical NF-κB may aggravate pathology by enhancing the alternative pathway.


Assuntos
Quimiocinas/metabolismo , Gastrite/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori , Receptor beta de Linfotoxina/metabolismo , NF-kappa B/metabolismo , Sistemas de Secreção Tipo IV/metabolismo , Animais , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Quimiocina CCL2/metabolismo , Quimiocina CCL20/metabolismo , Quimiocina CXCL10/metabolismo , Células Epiteliais/metabolismo , Feminino , Mucosa Gástrica/metabolismo , Gastrite/microbiologia , Infecções por Helicobacter/complicações , Humanos , Receptor beta de Linfotoxina/antagonistas & inibidores , Receptor beta de Linfotoxina/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro , Transdução de Sinais , Fator de Transcrição RelB/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
15.
Nat Microbiol ; 2: 16189, 2016 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-27748768

RESUMO

Helicobacter pylori specifically colonizes the human gastric epithelium and is the major causative agent for ulcer disease and gastric cancer development. Here, we identify members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family as receptors of H. pylori and show that HopQ is the surface-exposed adhesin that specifically binds human CEACAM1, CEACAM3, CEACAM5 and CEACAM6. HopQ-CEACAM binding is glycan-independent and targeted to the N-domain. H. pylori binding induces CEACAM1-mediated signalling, and the HopQ-CEACAM1 interaction enables translocation of the virulence factor CagA into host cells and enhances the release of pro-inflammatory mediators such as interleukin-8. Based on the crystal structure of HopQ, we found that a ß-hairpin insertion (HopQ-ID) in HopQ's extracellular 3+4 helix bundle domain is important for CEACAM binding. A peptide derived from this domain competitively inhibits HopQ-mediated activation of the Cag virulence pathway, as genetic or antibody-mediated abrogation of the HopQ function shows. Together, our data suggest the HopQ-CEACAM1 interaction to be a potentially promising novel therapeutic target to combat H. pylori-associated diseases.


Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Moléculas de Adesão Celular/metabolismo , Helicobacter pylori/fisiologia , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno , Adesinas Bacterianas/química , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Cristalografia por Raios X , Humanos , Interleucina-8/metabolismo , Ligação Proteica , Conformação Proteica , Transporte Proteico , Virulência
16.
PLoS One ; 11(5): e0154643, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27138472

RESUMO

The Dsb protein family is responsible for introducing disulfide bonds into nascent proteins in prokaryotes, stabilizing the structure of many proteins. Helicobacter pylori HP0231 is a Dsb-like protein, shown to catalyze disulfide bond formation and to participate in redox homeostasis. Notably, many H. pylori virulence factors are stabilized by the formation of disulfide bonds. By employing H. pylori HP0231 deficient strains we analyzed the effect of lack of this bacterial protein on the functionality of virulence factors containing putative disulfide bonds. The lack of H. pylori HP0231 impaired CagA translocation into gastric epithelial cells and reduced VacA-induced cellular vacuolation. Moreover, H. pylori HP0231 deficient bacteria were not able to colonize the gastric mucosa of mice, probably due to compromised motility. Together, our data demonstrate an essential function for H. pylori HP0231 in gastric colonization and proper function of bacterial virulence factors related to gastric pathology.


Assuntos
Helicobacter pylori/patogenicidade , Estômago/microbiologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Helicobacter pylori/metabolismo , Helicobacter pylori/fisiologia , Transporte Proteico , Vacúolos/microbiologia , Virulência
17.
J Immunol ; 196(10): 4246-52, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27183641

RESUMO

Helicobacter pylori infection is characterized by chronic persistence of the bacterium. Different virulence factors, including H. pylori γ-glutamyltranspeptidase (gGT), have been reported to induce tolerogenicity by reprogramming dendritic cells (DCs). gGT is present in all bacterial isolates, indicating an important role for gGT in the course of infection. In the current study, we have analyzed the effect of H. pylori gGT on human DCs and the subsequent adaptive immune response. We show that glutamate produced due to H. pylori gGT enzymatic activity tolerizes DCs by inhibiting cAMP signaling and dampening IL-6 secretion in response to the infection. Together, our results provide a novel molecular mechanism by which H. pylori manipulates the host's immune response to persist within its host.


Assuntos
Proteínas de Bactérias/imunologia , Células Dendríticas/imunologia , Helicobacter pylori/enzimologia , Receptores de Glutamato/metabolismo , gama-Glutamiltransferase/imunologia , Imunidade Adaptativa , Células Cultivadas , AMP Cíclico/metabolismo , Infecções por Helicobacter/imunologia , Humanos , Interleucina-6/imunologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Fatores de Virulência/imunologia
18.
Sci Signal ; 8(393): ra90, 2015 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-26350900

RESUMO

Given its fundamental role in development and cancer, the Wnt-ß-catenin signaling pathway is tightly controlled at multiple levels. RING finger protein 43 (RNF43) is an E3 ubiquitin ligase originally found in stem cells and proposed to inhibit Wnt signaling by interacting with the Wnt receptors of the Frizzled family. We detected endogenous RNF43 in the nucleus of human intestinal crypt and colon cancer cells. We found that RNF43 physically interacted with T cell factor 4 (TCF4) in cells and tethered TCF4 to the nuclear membrane, thus silencing TCF4 transcriptional activity even in the presence of constitutively active mutants of ß-catenin. This inhibitory mechanism was disrupted by the expression of RNF43 bearing mutations found in human gastrointestinal tumors, and transactivation of the Wnt pathway was observed in various cells and in Xenopus embryos when the RING domain of RNF43 was mutated. Our findings indicate that RNF43 inhibits the Wnt pathway downstream of oncogenic mutations that activate the pathway. Mimicking or enhancing this inhibitory activity of RNF43 may be useful to treat cancers arising from aberrant activation of the Wnt pathway.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Membrana Nuclear/metabolismo , Proteínas Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Humanos , Mutação , Membrana Nuclear/genética , Proteínas Oncogênicas/genética , Fator de Transcrição 4 , Fatores de Transcrição/genética , Transcrição Genética , Xenopus laevis , beta Catenina/genética
19.
Cell Microbiol ; 17(1): 51-61, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25087912

RESUMO

Helicobacter pylori (H. pylori) is a human-specific pathogen that has evolved to cope with the immune response elicited against the infection. We previously reported that H. pylori γ-glutamyltranspeptidase (gGT) impairs T-lymphocyte proliferation and thus might act as immune regulatory factor. In this study, we analysed the underlying mechanism and its implications for H. pylori persistence. We found that H. pylori gGT compromised T-cell proliferation, activation and effector cytokine expression by specifically depriving the extracellular space of glutamine. When assessing signalling cascades and transcription factors affected by H. pylori gGT, we found that expression of cMyc and IRF4, both required for metabolic adaptation of T-lymphocytes, was highly sensitive to extracellular glutamine levels and downregulated upon gGT treatment. Moreover, we could confirm decreased IRF4 expression in T-lymphocytes infiltrating the stomach of infected individuals. Thus, our results suggest that H. pylori gGT-mediated glutamine deprivation in the gastric mucosa may suppress T-cell function thereby contributing to bacterial persistence.


Assuntos
Helicobacter pylori/enzimologia , Fatores Reguladores de Interferon/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Virulência/metabolismo , gama-Glutamiltransferase/metabolismo , Proliferação de Células , Citocinas/metabolismo , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Glutamina/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Ativação Linfocitária
20.
J Immunol ; 193(7): 3566-76, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25172489

RESUMO

Infection with the gram-negative bacterium Helicobacter pylori is the most prevalent chronic bacterial infection, affecting ∼50% of the world's population, and is the main risk factor of gastric cancer. The proinflammatory cytokine IL-1ß plays a crucial role in the development of gastric tumors and polymorphisms in the IL-1 gene cluster leading to increased IL-1ß production have been associated with increased risk for gastric cancer. To be active, pro-IL-1ß must be cleaved by the inflammasome, an intracellular multiprotein complex implicated in physiological and pathological inflammation. Recently, H. pylori was postulated to activate the inflammasome in murine bone marrow-derived dendritic cells; however, the molecular mechanisms as well as the bacterial virulence factor acting as signal 2 activating the inflammasome remain elusive. In this study, we analyzed the inflammasome complex regulating IL-1ß upon H. pylori infection as well as the molecular mechanisms involved. Our results indicate that H. pylori-induced IL-1ß secretion is mediated by activation of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 inflammasome. We also show that reactive oxygen species, potassium efflux, and lysosomal destabilization are the main cellular mechanisms responsible of nucleotide-binding oligomerization domain family, pyrin domain-containing 3 inflammasome activation upon H. pylori infection, and identify vacuolating cytotoxin A and cag pathogenicity island as the bacterial virulence determinants involved. Moreover, in vivo experiments indicate an important role for the inflammasome in the onset and establishment of H. pylori infection and in the subsequent inflammatory response of the host.


Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Ilhas Genômicas/imunologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Imunidade Inata , Inflamassomos/imunologia , Interleucina-1beta/imunologia , Animais , Proteínas de Bactérias/genética , Feminino , Ilhas Genômicas/genética , Infecções por Helicobacter/genética , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Humanos , Masculino , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR
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