Identification of Desmoglein-2 as a novel target of Helicobacter pylori HtrA in epithelial cells

Abstract Background High temperature requirement A (HtrA) is an active serine protease secreted by the group-I carcinogen Helicobacter pylori (H. pylori). The human cell adhesion protein and tumor suppressor E-cadherin (hCdh1) expressed on the surface of gastric epithelial cells was identified as the first HtrA substrate. HtrA-mediated hCdh1 cleavage and subsequent disruption of intercellular adhesions are considered as important steps in H. pylori pathogenesis. In this study, we performed a proteomic profiling of H. pylori HtrA (HpHtrA) to decipher the complex mechanism of H. pylori interference with the epithelial barrier integrity. Results Using a proteomic approach we identified human desmoglein-2 (hDsg2), neuropilin-1, ephrin-B2, and semaphorin-4D as novel extracellular HpHtrA substrates and confirmed the well characterized target hCdh1. HpHtrA-mediated hDsg2 cleavage was further analyzed by in vitro cleavage assays using recombinant proteins. In infection experiments, we demonstrated hDsg2 shedding from H. pylori-colonized MKN28 and NCI-N87 cells independently of pathogen-induced matrix-metalloproteases or ADAM10 and ADAM17. Conclusions Characterizing the substrate specificity of HpHtrA revealed efficient hDsg2 cleavage underlining the importance of HpHtrA in opening intercellular junctions.

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Helicobacter pylori Induces Gastric Epithelial Cell Apoptosis in Association with Increased Fas Receptor Expression

Infection and Immunity ◽

10.1128/iai.67.8.4237-4242.1999 ◽

1999 ◽

Vol 67 (8) ◽

pp. 4237-4242 ◽

Cited By ~ 106

Author(s):

Nicola L. Jones ◽

Andrew S. Day ◽

Hilary A. Jennings ◽

Philip M. Sherman

Keyword(s):

Cell Death ◽

Helicobacter Pylori ◽

Epithelial Cells ◽

Receptor Expression ◽

Gastric Epithelial Cells ◽

Ags Cells ◽

Fas Receptor ◽

Epithelial Cell Apoptosis ◽

H Pylori

ABSTRACT The mechanisms involved in mediating the enhanced gastric epithelial cell apoptosis observed during infection withHelicobacter pylori in vivo are unknown. To determine whether H. pylori directly induces apoptosis of gastric epithelial cells in vitro and to define the role of the Fas-Fas ligand signal transduction cascade, human gastric epithelial cells were infected with H. pylori for up to 72 h under microaerophilic conditions. As assessed by both transmission electron microscopy and fluorescence microscopy, incubation with acagA-positive, cagE-positive, VacA-positive clinical H. pylori isolate stimulated an increase in apoptosis compared to the apoptosis of untreated AGS cells (16.0% ± 2.8% versus 5.9% ± 1.4%, P < 0.05) after 72 h. In contrast, apoptosis was not detected following infection withcagA-negative, cagE-negative, VacA-negative clinical isolates or a Campylobacter jejuni strain. In addition to stimulating apoptosis, infection with H. pylorienhanced Fas receptor expression in AGS cells to a degree comparable to that of treatment with a positive control, gamma interferon (12.5 ng/ml) (148% ± 24% and 167% ± 24% of control, respectively). The enhanced Fas receptor expression was associated with increased sensitivity to Fas-mediated cell death. Ligation of the Fas receptor with an agonistic monoclonal antibody resulted in an increase in apoptosis compared to the apoptosis of cells infected with the bacterium alone (38.5% ± 7.1% versus 16.0% ± 2.8%,P < 0.05). Incubation with neutralizing anti-Fas antibody did not prevent apoptosis of H. pylori-infected cells. Taken together, these findings demonstrate that the gastric pathogen H. pylori stimulates apoptosis of gastric epithelial cells in vitro in association with the enhanced expression of the Fas receptor. These data indicate a role for Fas-mediated signaling in the programmed cell death that occurs in response toH. pylori infection.

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Helicobacter pylori Alters Exogenous Antigen Absorption and Processing in a Digestive Tract Epithelial Cell Line Model

Infection and Immunity ◽

10.1128/iai.66.12.5785-5791.1998 ◽

1998 ◽

Vol 66 (12) ◽

pp. 5785-5791 ◽

Cited By ~ 33

Author(s):

Tamara Matysiak-Budnik ◽

Kathleen Terpend ◽

Sophie Alain ◽

Marie-José Sanson le Pors ◽

Jehan-Francois Desjeux ◽

...

Keyword(s):

Helicobacter Pylori ◽

Intercellular Junctions ◽

Paracellular Pathway ◽

Broth Culture ◽

Epithelial Cell Line ◽

Epithelial Barrier Function ◽

Ussing Chambers ◽

H Pylori ◽

Culture Supernatants

ABSTRACT To study the influence of Helicobacter pylori on epithelial barrier function, bacteria, bacterial sonicates, or broth culture supernatants were incubated for 24 h with HT29-19A intestinal cells grown as monolayers. Subsequently, the monolayers were mounted in Ussing chambers, and electrical resistance (R), fluxes of 22Na (JNa) and14C-mannitol (JMan) (markers of the paracellular pathway), and fluxes of horseradish peroxidase (HRP) in total (J3H-HRP), intact (JHRPi), and degraded forms were measured. H. pylori did not induce any modification of the paracellular pathway (R = 148 ± 10 versus 174 ± 16 Ω · cm2; JNa = 4.16 ± 0.44 versus 3.51 ± 0.41 μEq/h · cm2; JMan = 0.081 ± 0.01 versus 0.058 ± 0.009 μmol/h · cm2), nor did it modify J3H-HRP (2,201 ± 255 versus 2,110 ± 210 ng/h · cm2 forH. pylori-infected and control cells, respectively). However, in the presence of H. pylori, we observed a significant increase in JHRPi (520 ± 146 versus 171 ± 88 ng/h · cm2). This effect was not dependent of thecag status of the strain and was not reproduced by the sonicates or the culture supernatants. It was related to the presence of urease, since a urease-negative mutant of H. pylori did not induce this effect. Ammonia and bafilomycin A1, two agents known to increase the endolysosomal pH, reproduced the increase in JHRPi. In conclusion, H. pylori does not affect directly the integrity of intercellular junctions of epithelial cells in vitro, but it increases the passage of intact HRP, probably by inhibition of the intralysosomal degradation due to the release of ammonia. The increased transport of intact macromolecules may contribute to the induction and maintenance of gastric inflammation by H. pylori.

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Helicobacter pylori Colonization Drives Urokinase Receptor (uPAR) Expression in Murine Gastric Epithelium During Early Pathogenesis

Microorganisms ◽

10.3390/microorganisms8071019 ◽

2020 ◽

Vol 8 (7) ◽

pp. 1019

Author(s):

Warner Alpízar-Alpízar ◽

Mette E. Skindersoe ◽

Lone Rasmussen ◽

Mette C. Kriegbaum ◽

Ib J. Christensen ◽

...

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Epithelial Cells ◽

Protein Expression ◽

De Novo ◽

Urokinase Receptor ◽

Gastric Epithelium ◽

Upar Expression ◽

H Pylori

(1) Background: Persistent Helicobacter pylori infection is the most important risk factor for gastric cancer. The urokinase receptor (uPAR) is upregulated in lesions harboring cancer invasion and inflammation. Circumstantial evidence tends to correlate H. pylori colonization with increased uPAR expression in the human gastric epithelium, but a direct causative link has not yet been established in vivo; (2) Methods: In a mouse model of H. pylori-induced gastritis, we investigated the temporal emergence of uPAR protein expression in the gastric mucosa in response to H. pylori (SS1 strain) infection; (3) Results: We observed intense uPAR immunoreactivity in foveolar epithelial cells of the gastric corpus due to de novo synthesis, compared to non-infected animals. This uPAR induction represents a very early response, but it increases progressively over time as do infiltrating immune cells. Eradication of H. pylori infection by antimicrobial therapy causes a regression of uPAR expression to its physiological baseline levels. Suppression of the inflammatory response by prostaglandin E2 treatment attenuates uPAR expression. Notwithstanding this relationship, H. pylori does induce uPAR expression in vitro in co-cultures with gastric cancer cell lines; (4) Conclusions: We showed that persistent H. pylori colonization is a necessary event for the emergence of a relatively high uPAR protein expression in murine gastric epithelial cells.

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T4SS-dependent TLR5 activation by Helicobacter pylori infection

Nature Communications ◽

10.1038/s41467-019-13506-6 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 7

Author(s):

Suneesh Kumar Pachathundikandi ◽

Nicole Tegtmeyer ◽

Isabelle Catherine Arnold ◽

Judith Lind ◽

Matthias Neddermann ◽

...

Keyword(s):

Helicobacter Pylori ◽

Epithelial Cells ◽

Pathogenic Bacteria ◽

Type Iv Secretion ◽

Downstream Signaling ◽

Type Iv ◽

Efficient Control ◽

H Pylori ◽

Highly Virulent

AbstractToll-like receptor TLR5 recognizes a conserved domain, termed D1, that is present in flagellins of several pathogenic bacteria but not in Helicobacter pylori. Highly virulent H. pylori strains possess a type IV secretion system (T4SS) for delivery of virulence factors into gastric epithelial cells. Here, we show that one of the H. pylori T4SS components, protein CagL, can act as a flagellin-independent TLR5 activator. CagL contains a D1-like motif that mediates adherence to TLR5+ epithelial cells, TLR5 activation, and downstream signaling in vitro. TLR5 expression is associated with H. pylori infection and gastric lesions in human biopsies. Using Tlr5-knockout and wild-type mice, we show that TLR5 is important for efficient control of H. pylori infection. Our results indicate that CagL, by activating TLR5, may modulate immune responses to H. pylori.

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Helicobacter pylori Caga Protein Can Be Tyrosine Phosphorylated in Gastric Epithelial Cells

Journal of Experimental Medicine ◽

10.1084/jem.191.4.593 ◽

2000 ◽

Vol 191 (4) ◽

pp. 593-602 ◽

Cited By ~ 336

Author(s):

Momoyo Asahi ◽

Takeshi Azuma ◽

Shigeji Ito ◽

Yoshiyuki Ito ◽

Hiroyuki Suto ◽

...

Keyword(s):

Helicobacter Pylori ◽

Epithelial Cells ◽

Gastric Epithelial Cells ◽

Phosphorylated Proteins ◽

Caga Protein ◽

Host Cytoplasm ◽

Cytotoxin Associated Gene A ◽

Tyrosine Phosphorylated Protein ◽

H Pylori

Attachment of Helicobacter pylori to gastric epithelial cells induces various cellular responses, including the tyrosine phosphorylation of an unknown 145-kD protein and interleukin 8 production. Here we show that this 145-kD protein is the cagA product of H. pylori, an immunodominant, cytotoxin-associated antigen. Epithelial cells infected with various H. pylori clinical isolates resulted in generation of tyrosine-phosphorylated proteins ranging from 130 to 145 kD in size that were also induced in vitro by mixing host cell lysate with bacterial lysate. When epithelial cells were infected with [35S]methionine-labeled H. pylori, a radioactive 145-kD protein was detected in the immunoprecipitates with antiphosphotyrosine antibody or anti-CagA (cytotoxin-associated gene A) antibody. Consistently, the 145-kD protein recognized by the anti-CagA and antiphosphotyrosine antibodies was induced in epithelial cells after infection of wild-type H. pylori but not the cagA::Km mutant. Furthermore, the amino acid sequence of the phosphorylated 145-kD protein induced by H. pylori infection was identical to the H. pylori CagA sequence. These results reveal that the tyrosine-phosphorylated 145-kD protein is H. pylori CagA protein, which may be delivered from attached bacteria into the host cytoplasm. The identification of the tyrosine-phosphorylated protein will thus provide further insights into understanding the precise roles of CagA protein in H. pylori pathogenesis.

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α-Difluoromethylornithine reduces gastric carcinogenesis by causing mutations in Helicobacter pylori cagY

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814497116 ◽

2019 ◽

Vol 116 (11) ◽

pp. 5077-5085 ◽

Cited By ~ 6

Author(s):

Johanna C. Sierra ◽

Giovanni Suarez ◽

M. Blanca Piazuelo ◽

Paula B. Luis ◽

Dara R. Baker ◽

...

Keyword(s):

Helicobacter Pylori ◽

Epithelial Cells ◽

Oxidative Dna Damage ◽

Mongolian Gerbils ◽

Gastric Epithelial Cells ◽

Oncogenic Signaling ◽

Cytotoxin Associated Gene A ◽

H Pylori ◽

Oncogenic Signaling Pathways

Infection by Helicobacter pylori is the primary cause of gastric adenocarcinoma. The most potent H. pylori virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene cagY encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent α-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces H. pylori-mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect H. pylori pathogenicity. We show that H. pylori output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the cagY gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged cagY or the parental strain in which the wild-type cagY was replaced by cagY with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of H. pylori by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in cagY, demonstrating that DFMO directly affects genomic stability. Deletion of mutS2 abrogated the ability of DFMO to induce cagY rearrangements directly. In conclusion, DFMO-induced oxidative stress in H. pylori leads to genomic alterations and attenuates virulence.

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Helicobacter pylori Infection Stimulates Plasminogen Activator Inhibitor 1 Production by Gastric Epithelial Cells

Infection and Immunity ◽

10.1128/iai.00584-08 ◽

2008 ◽

Vol 76 (9) ◽

pp. 3992-3999 ◽

Cited By ~ 19

Author(s):

A. C. Keates ◽

S. Tummala ◽

R. M. Peek ◽

E. Csizmadia ◽

B. Kunzli ◽

...

Keyword(s):

Helicobacter Pylori ◽

Epithelial Cells ◽

Plasminogen Activator Inhibitor ◽

Gastric Epithelial Cells ◽

Plasminogen Activator Inhibitor 1 ◽

Ags Cells ◽

H Pylori ◽

Activator Inhibitor ◽

Pai 1

ABSTRACT Chronic infection with the gastric pathogen Helicobacter pylori significantly increases the risk of developing atrophic gastritis, peptic ulcer disease, and gastric adenocarcinoma. H. pylori strains that possess the cag pathogenicity island, which translocates CagA into the host cells, augment these risks. The aim of this study was to determine the molecular mechanisms through which H. pylori upregulates the expression of plasminogen activator inhibitor 1 (PAI-1), a member of the urokinase activator system that is involved in tumor metastasis and angiogenesis. Levels of PAI-1 mRNA and protein were examined in tissues from H. pylori-infected patients and in vitro using AGS gastric epithelial cells. In vitro, cells were infected with toxigenic cag-positive or nontoxigenic cag-negative strains of H. pylori or isogenic mutants. The amount of PAI-1 secretion was measured by enzyme-linked immunosorbent assay, and mRNA levels were determined using real-time PCR. The regulation of PAI-1 was examined using the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor and small interfering RNA. Analysis of human biopsy samples revealed an increase in both PAI-1 mRNA and protein levels in patients with H. pylori gastritis compared to those of uninfected controls. Infection of AGS cells with H. pylori significantly increased PAI-1 mRNA expression and the secretion of PAI-1 protein. Moreover, PAI-1 mRNA and protein production was more pronounced when AGS cells were infected by H. pylori strains carrying a functional cag secretion system than when cells were infected by strains lacking this system. PAI-1 secretion was also reduced when cells were infected with either cagE-negative or cagA-negative mutants. The ectopic overexpression of CagA significantly increased the levels of PAI-1 mRNA and protein, whereas blockade of the ERK1/2 pathway inhibited H. pylori-mediated PAI-1 upregulation. These findings suggest that the upregulation of PAI-1 in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.

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Regulation of Human β-Defensins by Gastric Epithelial Cells in Response to Infection with Helicobacter pylori or Stimulation with Interleukin-1

Infection and Immunity ◽

10.1128/iai.68.9.5412-5415.2000 ◽

2000 ◽

Vol 68 (9) ◽

pp. 5412-5415 ◽

Cited By ~ 80

Author(s):

Deborah A. O'Neil ◽

Sheri P. Cole ◽

Edith Martin-Porter ◽

Michael P. Housley ◽

Lide Liu ◽

...

Keyword(s):

Helicobacter Pylori ◽

Epithelial Cells ◽

Interleukin 1 ◽

Peptide Antibiotic ◽

Gastric Epithelial Cells ◽

Proinflammatory Mediators ◽

H Pylori

ABSTRACT Gastric epithelial cells in vitro and in vivo are shown to constitutively express the peptide antibiotic human β-defensin type 1 (hBD-1). In contrast, hBD-2 expression is regulated in gastric epithelial cells and increases in response to infection withHelicobacter pylori or stimulation with the proinflammatory cytokine interleukin-1. These data suggest that hBD-2 is a component of the regulated host gastric epithelial cell response to H. pylori infection and proinflammatory mediators.

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CagA Effector Protein in Helicobacter pylori-Infected Human Gastric Epithelium in Vivo: From Bacterial Core and Adhesion/Injection Clusters to Host Cell Proteasome-Rich Cytosol

Toxins ◽

10.3390/toxins11110618 ◽

2019 ◽

Vol 11 (11) ◽

pp. 618 ◽

Cited By ~ 1

Author(s):

Vittorio Necchi ◽

Vittorio Ricci ◽

Patrizia Sommi ◽

Enrico Solcia

Keyword(s):

Helicobacter Pylori ◽

Epithelial Cells ◽

Host Cell ◽

Experimental Studies ◽

Intracellular Distribution ◽

Gastric Epithelium ◽

Effector Protein ◽

H Pylori

A key role in the carcinogenic action of Helicobacter pylori is played by the effector protein CagA, the first identified oncoprotein of the bacterial world. However, the present knowledge in regard to the bacterial injection of CagA into epithelial cells (through a type IV secretion system) and its intracellular fate is based primarily on experimental studies in vitro. Our study was aimed to investigate, in H. pylori-infected human gastric epithelium, CagA delivery and intracellular distribution in order to identify any in vivo counterpart of the cell injection mechanism described in vitro and any intracellular cytoplasmic site of preferential CagA distribution, thus shedding light on the natural history of CagA in vivo. By transmission electron microscopy and ultrastructural immunocytochemistry (which combine precise molecule localization with detailed analysis of bacterial-host cell interaction and epithelial cell ultrastructure), we investigated endoscopic biopsies of gastric antrum from H. pylori-infected dyspeptic patients. Our findings provide support for CagA direct injection into gastric epithelial cells at bacterial adhesion sites located on the lateral plasma membrane and for its cytosolic intracellular distribution with selective concentration inside peculiar proteasome-rich areas, which might be site not only of CagA degradation but also of CagA-promoted crucial events in gastric carcinogenesis.

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The rod-to-coccoid body transformation in cultures of Helicobacter pylori

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100160686 ◽

1990 ◽

Vol 48 (3) ◽

pp. 626-627

Author(s):

A. R. Crooker ◽

W. G. Kraft ◽

T. L. Beard ◽

M. C. Myers

Keyword(s):

Helicobacter Pylori ◽

Growth Cycle ◽

Negative Bacterium ◽

Body Formation ◽

Coccoid Form ◽

Spiral Form ◽

Prolonged Culture ◽

H Pylori ◽

Upper Gastrointestinal

Helicobacter pylori is a microaerophilic, gram-negative bacterium found in the upper gastrointestinal tract of humans. There is strong evidence that H. pylori is important in the etiology of gastritis; the bacterium may also be a major predisposing cause of peptic ulceration. On the gastric mucosa, the organism exists as a spiral form with one to seven sheathed flagella at one (usually) or both poles. Short spirals were seen in the first successful culture of the organism in 1983. In 1984, Marshall and Warren reported a coccoid form in older cultures. Since that time, other workers have observed rod and coccal forms in vitro; coccoid forms predominate in cultures 3-7 days old. We sought to examine the growth cycle of H. pylori in prolonged culture and the mode of coccoid body formation.

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