Publications by authors named "Raquel Mejias-Luque"

37 Publications

Engagement of CEACAM1 by HopQ Is Important for the Activation of Non-Canonical NF-κB in Gastric Epithelial Cells.

Microorganisms 2021 Aug 16;9(8). Epub 2021 Aug 16.

Institute for Medical Microbiology, Immunology and Hygiene, Technical University Munich, 81675 Munich, Germany.

The gastric pathogen infects half of the world's population and is a major risk factor for gastric cancer development. In order to attach to human gastric epithelial cells and inject the oncoprotein CagA into host cells, utilizes the outer membrane protein HopQ that binds to the cell surface protein CEACAM, which can be expressed on the gastric mucosa. Once bound, activates a number of signaling pathways, including canonical and non-canonical NF-κB. We investigated whether HopQ-CEACAM interaction is involved in activating the non-canonical NF-κB signaling pathway. Different gastric cancer cells were infected with the wild type, or HopQ mutant strains, and the activation of non-canonical NF-κB was related to CEACAM expression levels. The correlation between CEACAM levels and the activation of non-canonical NF-κB was confirmed in human gastric tissue samples. Taken together, our findings show that the HopQ-CEACAM interaction is important for activation of the non-canonical NF-κB pathway in gastric epithelial cells.
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http://dx.doi.org/10.3390/microorganisms9081748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8400456PMC
August 2021

Microbiota alteration at different stages in gastric lesion progression: a population-based study in Linqu, China.

Am J Cancer Res 2021 1;11(2):561-575. Epub 2021 Feb 1.

Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Cancer Epidemiology, Peking University Cancer Hospital & Institute Beijing, China.

In addition to Helicobacter pylori (H.pylori), gastric microbiota may be involved in carcinogenesis process. However, the longitudinal study to assess changes in the gastric microbiota associated with the development of gastric carcinogenesis is still limited. The aim of this study is to explore dynamic microbial alterations in gastric cancer (GC) development based on a 4-year endoscopic follow-up cohort in Linqu County, China. Microbial alterations were investigated by deep sequencing of the microbial 16S ribosomal RNA gene in 179 subjects with various gastric lesions, and validated in paired gastric biopsies prospectively collected before and after lesion progression and in non-progression controls. Significant differences were found in microbial diversity and community structure across various gastric lesions, with 62 candidate differential taxa between at least two lesion groups. Further validations identified Helicobacter, Bacillus, Capnocytophaga and Prevotella to be associated with lesion progression-to-dysplasia (DYS)/GC (all P < 0.05), especially for subjects progressing from intestinal metaplasia (IM) to DYS/GC. The combination of the four genera in a microbial dysbiosis index showed a significant difference after lesion progression-to-DYS/GC compared to controls (P = 0.027). The panel including the four genera identified subjects after progression-to-DYS/GC with an area under the receiver-operating curve (AUC) of 0.941. Predictive significance was found before lesion progression-to-DYS/GC with an AUC = 0.776 and an even better AUC (0.927) for subjects progressing from IM to DYS/GC. Microbiota may play different roles at different stages in gastric carcinogenesis. A panel of bacterial genera associated with gastric lesions may help to assess gastric microbial dysbiosis and show potential predictive values for lesion progression. Our findings provide new clues for the microbial mechanism of H.pylori-associated carcinogenesis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7868750PMC
February 2021

Senescence: A Novel Driver of Helicobacter pylori-Induced Gastric Atrophy.

Cell Mol Gastroenterol Hepatol 2021 28;11(3):887-888. Epub 2020 Dec 28.

Institute for Medical Microbiology, Immunology and Hygiene, School of Medicine, Technical University Munich, Munich, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.jcmgh.2020.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900830PMC
July 2021

Loss of RNF43 Function Contributes to Gastric Carcinogenesis by Impairing DNA Damage Response.

Cell Mol Gastroenterol Hepatol 2021 11;11(4):1071-1094. Epub 2020 Nov 11.

Institute for Medical Microbiology, Immunology and Hygiene, School of Medicine, Technical University of Munich, Munich, Germany. Electronic address:

Background & Aims: RING finger protein 43 (RNF43) is a tumor suppressor that frequently is mutated in gastric tumors. The link between RNF43 and modulation of Wingless-related integration site (WNT) signaling has not been shown clearly in the stomach. Because mutations in RNF43 are highly enriched in microsatellite-unstable gastric tumors, which show defects in DNA damage response (DDR), we investigated whether RNF43 is involved in DDR in the stomach.

Methods: DDR activation and cell viability upon γ-radiation was analyzed in gastric cells where expression of RNF43 was depleted. Response to chemotherapeutic agents 5-fluorouracil and cisplatin was analyzed in gastric cancer cell lines and xenograft tumors. In addition, involvement of RNF43 in DDR activation was analyzed upon Helicobacter pylori infection in wild-type and Rnf43 mice. Furthermore, a cohort of human gastric biopsy specimens was analyzed for RNF43 expression and mutation status as well as for activation of DDR.

Results: RNF43 depletion conferred resistance to γ-radiation and chemotherapy by dampening the activation of DDR, thereby preventing apoptosis in gastric cells. Upon Helicobacter pylori infection, RNF43 loss of function reduced activation of DDR and apoptosis. Furthermore, RNF43 expression correlated with DDR activation in human gastric biopsy specimens, and RNF43 mutations found in gastric tumors conferred resistance to DNA damage. When exploring the molecular mechanisms behind these findings, a direct interaction between RNF43 and phosphorylated H2A histone family member X (γH2AX) was observed.

Conclusions: We identified a novel function for RNF43 in the stomach as a regulator of DDR. Loss of RNF43 function in gastric cells confers resistance to DNA damage-inducing radiotherapy and chemotherapy, suggesting RNF43 as a possible biomarker for therapy selection.
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http://dx.doi.org/10.1016/j.jcmgh.2020.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898035PMC
March 2022

Gut Microbiota-Derived Propionate Regulates the Expression of Reg3 Mucosal Lectins and Ameliorates Experimental Colitis in Mice.

J Crohns Colitis 2020 Oct;14(10):1462-1472

Klinik für Innere Medizin II, Klinikum rechts der Isar, Techn. Univ. Munich, Munich, Germany.

Background And Aims: Regenerating islet-derived protein type 3 [Reg3] lectins are antimicrobial peptides at mucosal surfaces of the gut, whose expression is regulated by pathogenic gut microbes via interleukin-22- or Toll-like receptor signalling. In addition to antimicrobial effects, tissue protection is hypothesized, but has been poorly investigated in the gut.

Methods: We applied antibiotic-induced microbiota perturbations, gnotobiotic approaches and a dextran-sodium sulfate [DSS] colitis model to assess microbial Reg3 regulation in the intestines and its role in colitis. We also used an intestinal organoid model to investigate this axis in vitro.

Results: First, we studied whether gut commensals are involved in Reg3 expression in mice, and found that antibiotic-mediated reduction of Clostridia downregulated intestinal Reg3B. A loss in Clostridia was accompanied by a significant reduction of short-chain fatty acids [SCFAs], and knock-out [KO] mice for SCFA receptors GPR43 and GPR109 expressed less intestinal Reg3B/-G. Propionate was found to induce Reg3 in intestinal organoids and in gnotobiotic mice colonized with a defined, SCFA-producing microbiota. Investigating the role of Reg3B as a protective factor in colitis, we found that Reg3B-KO mice display increased inflammation and less crypt proliferation in the DSS colitis model. Propionate decreased colitis and increased proliferation. Treatment of organoids exposed to DSS with Reg3B or propionate reversed the chemical injury with a loss of expression of the stem-cell marker Lgr5 and Olfm4.

Conclusions: Our results suggest that Clostridia can regulate Reg3-associated epithelial homeostasis through propionate signalling. We also provide evidence that the Reg3-propionate axis may be an important mediator of gut epithelial regeneration in colitis.
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http://dx.doi.org/10.1093/ecco-jcc/jjaa065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8921751PMC
October 2020

Cysteine Residues in Adhesin HopQ are Required for CEACAM-HopQ Interaction and Subsequent CagA Translocation.

Microorganisms 2020 Mar 25;8(4). Epub 2020 Mar 25.

Institute for Medical Microbiology, Immunology and Hygiene, Technical University Munich, 81675 Munich, Germany.

Attachment to the host gastric mucosa is a key step in infection. Recently, a novel adhesin, HopQ, was shown to bind distinct host CEACAM proteins-an interaction that was found to be essential for the translocation of CagA, a key virulence factor of The HopQ-CEACAM1 co-crystal structure revealed a binding mode dependent on loops in HopQ that are clasped by disulfide bonds. In this study, we investigated the importance of these cysteine residues for CEACAM1 engagement by . We observed a loss of CEACAM1 binding and CagA translocation upon disruption of the disulfide bond in loop CL1 (connecting C103 to C132 in HopQ). Deletion of the Dsb-like oxidoreductase HP0231 did not affect cell surface expression of HopQ or alter the interaction of with target cells. Although HP0231 deletion was previously described to impede CagA translocation, our results indicate that this occurs through a HopQ-independent mechanism. Together, our results open up new avenues to therapeutically target the HopQ-CEACAM1 interaction and reduce the burden of pathogenic .
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http://dx.doi.org/10.3390/microorganisms8040465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232459PMC
March 2020

Effect of on gastrointestinal microbiota: a population-based study in Linqu, a high-risk area of gastric cancer.

Gut 2020 09 19;69(9):1598-1607. Epub 2019 Dec 19.

Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Cancer Epidemiology, Peking University Cancer Hospital & Institute, Beijing, China

Objective: Gastrointestinal microbiota may be involved in associated gastric cancer development. The aim of this study was to explore the possible microbial mechanisms in gastric carcinogenesis and potential dysbiosis arising from 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- treatment, relative to 49 negative subjects.

Results: In 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 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 and , , , , were found only in advanced gastric lesion patients, and were absent in normal/superficial gastritis group. Changes in faecal microbiota included increased after successful eradication and more upregulated drug-resistant functional orthologs after failed treatment.

Conclusion: infection contributes significantly to gastric microbial dysbiosis that may be involved in carcinogenesis. Successful eradication potentially restores gastric microbiota to a similar status as found in uninfected individuals, and shows beneficial effects on gut microbiota.
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http://dx.doi.org/10.1136/gutjnl-2019-319696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456744PMC
September 2020

A mass spectrometry guided approach for the identification of novel vaccine candidates in gram-negative pathogens.

Sci Rep 2019 11 22;9(1):17401. Epub 2019 Nov 22.

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany.

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.
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http://dx.doi.org/10.1038/s41598-019-53493-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6874673PMC
November 2019

Exploits the NLRC4 Inflammasome to Dampen Host Defenses.

J Immunol 2019 10 11;203(8):2183-2193. Epub 2019 Sep 11.

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Fakultät für Medizin, Technische Universität München, 81675 Munich, Germany; and

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 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 -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 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, mice failed to produce gastric IL-18 upon infection. Compared with wild type mice, mice controlled better without showing strong inflammation. Moreover, -induced IL-18 inhibits β-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.
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http://dx.doi.org/10.4049/jimmunol.1900351DOI Listing
October 2019

Concomitant Infection of S. mansoni and H. pylori Promotes Promiscuity of Antigen-Experienced Cells and Primes the Liver for a Lower Fibrotic Response.

Cell Rep 2019 07;28(1):231-244.e5

Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2019.05.108DOI Listing
July 2019

Increased LIGHT expression and activation of non-canonical NF-κB are observed in gastric lesions of MyD88-deficient mice upon Helicobacter felis infection.

Sci Rep 2019 05 7;9(1):7030. Epub 2019 May 7.

Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California, USA.

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 β receptor (LTβR). 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.
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http://dx.doi.org/10.1038/s41598-019-43417-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504916PMC
May 2019

Mutated Aggravates -Induced Gastric Pathology.

Cancers (Basel) 2019 Mar 16;11(3). Epub 2019 Mar 16.

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, 81675 Munich, Germany.

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 RNF43 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 , which has been described as one of the major risk factors for gastric cancer. Mice bearing mutant RNF43 showed higher gastritis scores upon infection compared to wild-type mice, accompanied by increased lymphocyte infiltration and levels. Furthermore, infected 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 can worsen induced pathology.
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http://dx.doi.org/10.3390/cancers11030372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468876PMC
March 2019

Loss of endogenous RNF43 function enhances proliferation and tumour growth of intestinal and gastric cells.

Carcinogenesis 2019 06;40(4):551-559

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany.

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.
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http://dx.doi.org/10.1093/carcin/bgy152DOI Listing
June 2019

Association Between Gut Microbiota and -Related Gastric Lesions in a High-Risk Population of Gastric Cancer.

Front Cell Infect Microbiol 2018 19;8:202. Epub 2018 Jun 19.

Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Cancer Epidemiology, Peking University Cancer Hospital and Institute, Beijing, China.

Eradication of 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 -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 , and 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 infection compared with negative subjects. As for the differential bacteria, the average relative abundance of was found to significantly decrease from negative (66.16%) to past infection group (33.01%, = 0.007), as well as from normal (76.49%) to gastritis (56.04%) and metaplasia subjects (46.83%, = 0.027). For and , the average relative abundances showed elevated trends in the past infection group (47.11, 20.53%) compared to negative group (23.44, 9.05%, = 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%, = 0.016 and 0.294, respectively) or metaplasia subjects (32.33, 20.07%, both < 0.05). These findings suggest that the alterations of fecal microbiota, especially the dominant phyla of and , may be involved in the process of -related gastric lesion progression and provide hints for future evaluation of microbial changes after eradication.
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http://dx.doi.org/10.3389/fcimb.2018.00202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6018392PMC
July 2019

adhesin HopQ disrupts dimerization in human CEACAMs.

EMBO J 2018 07 1;37(13). Epub 2018 Jun 1.

Structural and Molecular Microbiology, Structural Biology Research Center, VIB, Brussels, Belgium

The human gastric pathogen 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 specificity toward human CEACAM receptors. Both HopQ alleles target the β-strands G, F, and C of C1ND, which form the 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 a route to influence CEACAM-mediated cell adherence and signaling events.
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http://dx.doi.org/10.15252/embj.201798665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028033PMC
July 2018

Evidence suggests that germline mutations are a rare cause of serrated polyposis.

Gut 2018 12 12;67(12):2230-2232. Epub 2018 Jan 12.

Hereditary Cancer Program, Catalan Institute of Oncology, IDIBELL, Hospitalet de Llobregat, Barcelona, Spain.

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http://dx.doi.org/10.1136/gutjnl-2017-315733DOI Listing
December 2018

Helicobacter pylori γ-glutamyl transferase contributes to colonization and differential recruitment of T cells during persistence.

Sci Rep 2017 10 20;7(1):13636. Epub 2017 Oct 20.

Institut für medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, 81675, Munich, Germany.

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.
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http://dx.doi.org/10.1038/s41598-017-14028-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651840PMC
October 2017

Immune Evasion Strategies and Persistence of Helicobacter pylori.

Curr Top Microbiol Immunol 2017;400:53-71

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany.

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.
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http://dx.doi.org/10.1007/978-3-319-50520-6_3DOI Listing
June 2017

Helicobacter pylori adhesin HopQ engages in a virulence-enhancing interaction with human CEACAMs.

Nat Microbiol 2016 Oct 17;2:16189. Epub 2016 Oct 17.

Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, 81675 Munich, Germany.

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 β-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.
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http://dx.doi.org/10.1038/nmicrobiol.2016.189DOI Listing
October 2016

Lymphotoxin β receptor signalling executes -driven gastric inflammation in a T4SS-dependent manner.

Gut 2017 08 13;66(8):1369-1381. Epub 2016 Apr 13.

Institut für Virologie, Technische Universität München, Helmholtz Zentrum München, Neuherberg, Germany.

Objective: Lymphotoxin β receptor (LTβR) signalling has been implicated in inflammation-associated tumour development in different tissues. We have analysed the role of LTβR and alternative NF-κB signalling in 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 -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 LTβR activation by were assessed in vitro using human gastric cancer cell lines and distinct isolates. The effects of blocking or agonistically activating LTβR on gastric pathology during challenge with a human pathogenic 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 -infected patients. induced LTβR-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 LTβR signalling in vivo suppressed driven gastritis, whereas LTβR activation in gastric epithelial cells of infected mice induced a broadened pro-inflammatory chemokine milieu, resulting in exacerbated pathology.

Conclusions: LTβR-triggered activation of alternative NF-κB signalling in gastric epithelial cells executes -induced chronic gastritis, representing a novel target to restrict gastric inflammation and pathology elicited by , while exclusively targeting canonical NF-κB may aggravate pathology by enhancing the alternative pathway.
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http://dx.doi.org/10.1136/gutjnl-2015-310783DOI Listing
August 2017

Helicobacter pylori γ-Glutamyltranspeptidase Induces Tolerogenic Human Dendritic Cells by Activation of Glutamate Receptors.

J Immunol 2016 05 18;196(10):4246-52. Epub 2016 Apr 18.

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, 81675 Munich, Germany; and

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.
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http://dx.doi.org/10.4049/jimmunol.1501062DOI Listing
May 2016

Helicobacter pylori HP0231 Influences Bacterial Virulence and Is Essential for Gastric Colonization.

PLoS One 2016 3;11(5):e0154643. Epub 2016 May 3.

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154643PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854439PMC
July 2017

The E3 ligase RNF43 inhibits Wnt signaling downstream of mutated β-catenin by sequestering TCF4 to the nuclear membrane.

Sci Signal 2015 Sep 8;8(393):ra90. Epub 2015 Sep 8.

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich 81675, Germany.

Given its fundamental role in development and cancer, the Wnt-β-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 β-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.
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http://dx.doi.org/10.1126/scisignal.aac6757DOI Listing
September 2015

Helicobacter pylori-induced IL-1β secretion in innate immune cells is regulated by the NLRP3 inflammasome and requires the cag pathogenicity island.

J Immunol 2014 Oct 29;193(7):3566-76. Epub 2014 Aug 29.

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, 81675 Munich, Germany; German Centre for Infection Research, Partner Site Munich, 81675 Munich, Germany

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-1β plays a crucial role in the development of gastric tumors and polymorphisms in the IL-1 gene cluster leading to increased IL-1β production have been associated with increased risk for gastric cancer. To be active, pro-IL-1β 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-1β upon H. pylori infection as well as the molecular mechanisms involved. Our results indicate that H. pylori-induced IL-1β 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.
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http://dx.doi.org/10.4049/jimmunol.1400362DOI Listing
October 2014

Involvement of Toll-like receptors on Helicobacter pylori-induced immunity.

PLoS One 2014 25;9(8):e104804. Epub 2014 Aug 25.

Lehrstuhl für Innere Medizin 1, Universität Witten/Herdecke, Wuppertal, Germany.

Dendritic cells (DCs) play a major role in the innate immune response since they recognize a broad repertoire of PAMPs mainly via Toll-like receptors (TLRs). During Helicobacter pylori (H. pylori) infection, TLRs have been shown to be important to control cytokine response particularly in murine DCs. In the present study we analyzed the effect of blocking TLRs on human DCs. Co-incubation of human DCs with H. pylori resulted in the release of the pro-inflammatory cytokines IL-12p70, IL-6 and IL-10. Release of IL-12p70 and IL-10 was predominantly influenced when TLR4 signaling was blocked by adding specific antibodies, suggesting a strong influence on subsequent T cell responses through TLR4 activation on DCs. Co-incubation of H. pylori-primed DC with allogeneic CD4+ T cells resulted in the production of IFN-γ and IL-17A as well as the expression of Foxp3, validating a mixed Th1/Th17 and Treg response in vitro. Neutralization of TLR4 during H. pylori infection resulted in significantly decreased amounts of IL-17A and IFN-γ and reduced levels of Foxp3-expressing and IL-10-secreting T cells. Our findings suggest that DC cytokine secretion induced upon TLR4-mediated recognition of H. pylori influences inflammatory and regulatory T cell responses, which might facilitate the chronic bacterial persistence.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0104804PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143222PMC
May 2015

Helicobacter pylori γ-glutamyltranspeptidase impairs T-lymphocyte function by compromising metabolic adaption through inhibition of cMyc and IRF4 expression.

Cell Microbiol 2015 Jan 30;17(1):51-61. Epub 2014 Aug 30.

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, 81675, Munich, Germany.

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.
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http://dx.doi.org/10.1111/cmi.12335DOI Listing
January 2015

H. pylori virulence factors: influence on immune system and pathology.

Mediators Inflamm 2014 21;2014:426309. Epub 2014 Jan 21.

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, 81675 Munich, Germany.

Helicobacter pylori is the most widespread chronic bacterial agent in humans and is well recognized for its association with ulcer disease and gastric cancer, with both representing major global health and socioeconomic issues. Given the high level of adaptation and the coevolution of this bacterium with its human host, a thorough and multidirectional view of the specific microbiological characteristics of this infection as well as the host physiology is needed in order to develop novel means of prevention of therapy. This review aims to pinpoint some of these potentially important angles, which have to be considered mutually when studying H. pylori's pathogenicity. The host's biological changes due to the virulence factors are a valuable pillar of H. pylori research as are the mechanisms by which bacteria provoke these changes. In this context, necessary adhesion molecules and significant virulence factors of H. pylori are discussed. Moreover, metabolism of the bacteria, one of the most important aspects for a better understanding of bacterial physiology and consequently possible therapeutic and prophylactic strategies, is addressed. On the other hand, we discuss the recent experimental proofs of the "hygiene hypothesis" in correlation with Helicobacter's infection, which adds another aspect of complexity to this infection.
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http://dx.doi.org/10.1155/2014/426309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918698PMC
October 2014

The stem cell factor SOX2 regulates the tumorigenic potential in human gastric cancer cells.

Carcinogenesis 2014 Apr 9;35(4):942-50. Epub 2013 Dec 9.

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, 81675 Munich, Germany.

Gastric cancer (GC) is still one of the most common causes of cancer-related death worldwide, which is mainly attributable to late diagnosis and poor treatment options. Infection with Helicobacter pylori, different environmental factors and genetic alterations are known to influence the risk of developing gastric tumors. However, the molecular mechanisms involved in gastric carcinogenesis are still not fully understood, making it difficult to design targeted therapeutic approaches. Aberrant expression of the specific gastric differentiation marker SOX2 has been observed in stomach cancer. However, the role of SOX2 in gastric tumors has not been well established to date. To elucidate the role of SOX2 in gastric tumorigenesis, SOX2 transcriptional activity was blocked in AZ-521 cells. Interestingly, inhibition of SOX2 reduced cell proliferation and migration, increased apoptosis and induced changes in cell cycle. Blocking of SOX2 also reduced the tumorigenic potential of AZ-521 cells in vivo. In addition, correlation of SOX2 expression and proliferation was observed in a subset of human gastric tumors. Finally, target genes of SOX2 were for the first time identified by RNA microarray in GC cells. Taken together, the results presented here indicate that SOX2 controls several aspects related to GC development and progression by regulating the expression of members of important signaling pathways. These findings could provide new therapeutic options for a subset of GCs exhibiting SOX2 deregulation.
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http://dx.doi.org/10.1093/carcin/bgt410DOI Listing
April 2014

Helicobacter pylori cytotoxin-associated gene A impairs human dendritic cell maturation and function through IL-10-mediated activation of STAT3.

J Immunol 2014 Jan 29;192(1):316-23. Epub 2013 Nov 29.

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, 81675 Munich, Germany;

Helicobacter pylori infection induces chronic gastric inflammation that can progress to cancer. In this process, the virulence factor cytotoxin-associated gene A (CagA) plays a central role by directly altering epithelial cell signaling and inducing a strong Th1 immune response, which contributes to carcinogenesis. It is still barely understood how the bacterium evades clearance despite this solid immune response and persists lifelong. Dendritic cells (DCs) play a major role in determining the adaptive immune response toward H. pylori, and high levels of regulatory T cells have been detected infiltrating the gastric mucosa of H. pylori-infected patients, which contribute to bacterial persistence. Although murine studies indicate that H. pylori induces tolerization of DCs and impairs DC maturation, the virulence determinants involved are still controversial. Moreover, the signaling cascades engaged in human DC tolerization upon H. pylori infection remain unknown. In the current study, we analyzed the effect of H. pylori infection on human DC maturation and function, focusing on the virulence factors implicated and signaling pathways involved. Our results reveal that CagA is crucial for DC tolerization by modulating IL-10 secretion and, in turn, STAT3 phosphorylation, favoring a regulatory T cell immune response. Our findings help to unravel the paradox why CagA-positive strains, although eliciting a stronger inflammatory response, have overcome evolutionary pressure and persisted in their human host.
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http://dx.doi.org/10.4049/jimmunol.1302476DOI Listing
January 2014
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