scholarly journals Cortactin Promotes Effective AGS Cell Scattering by Helicobacter pylori CagA, but Not Cellular Vacuolization and Apoptosis Induced by the Vacuolating Cytotoxin VacA

Pathogens ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 3
Author(s):  
Irshad Sharafutdinov ◽  
Jakob Knorr ◽  
Delara Soltan Esmaeili ◽  
Steffen Backert ◽  
Nicole Tegtmeyer
Keyword(s):  
Helicobacter Pylori ◽  
Cell Movement ◽  
Actin Binding ◽  
Knockout Mutant ◽  
Vacuolating Cytotoxin ◽  
Cell Functions ◽  
Cell Scattering ◽  
Induction Of Apoptosis ◽  
H Pylori ◽  
Cytoskeletal Rearrangements

Cortactin is an actin-binding protein and actin-nucleation promoting factor regulating cytoskeletal rearrangements in eukaryotes. Helicobacter pylori is a gastric pathogen that exploits cortactin to its own benefit. During infection of gastric epithelial cells, H. pylori hijacks multiple cellular signaling pathways, leading to the disruption of key cell functions. Two bacterial virulence factors play important roles in this scenario, the vacuolating cytotoxin VacA and the translocated effector protein CagA of the cag type IV secretion system (T4SS). Specifically, by overruling the phosphorylation status of cortactin, H. pylori alternates the activity of molecular interaction partners of this important protein, thereby manipulating the performance of cytoskeletal rearrangements, endosomal trafficking and cell movement. Based on shRNA knockdown and other studies, it was previously reported that VacA utilizes cortactin for its cellular uptake, intracellular travel and induction of apoptosis by a mitochondria-dependent mechanism, while CagA induces cell scattering, motility and elongation. To investigate the role of cortactin in these phenotypes in more detail, we produced a complete knockout mutant of cortactin in the gastric adenocarcinoma cell line AGS by CRISPR-Cas9. These cells were infected with H. pylori wild-type or various isogenic mutant strains. Unexpectedly, cortactin deficiency did not prevent the uptake and formation of VacA-dependent vacuoles, nor the induction of apoptosis by internalized VacA, while the induction of T4SS- and CagA-dependent AGS cell movement and elongation were strongly reduced. Thus, we provide evidence that cortactin is required for the function of internalized CagA, but not VacA.

scholarly journals Cortactin: A Major Cellular Target of the Gastric Carcinogen Helicobacter pylori

Cancers ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 159 ◽  
Author(s):  
Irshad Sharafutdinov ◽  
Steffen Backert ◽  
Nicole Tegtmeyer
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Cell Movement ◽  
Cell Types ◽  
Eukaryotic Cell ◽  
Actin Binding ◽  
Gastric Diseases ◽  
Cell Functions ◽  
H Pylori ◽  
Cytoskeletal Rearrangements

Cortactin is an actin binding protein and actin nucleation promoting factor regulating cytoskeletal rearrangements in nearly all eukaryotic cell types. From this perspective, cortactin poses an attractive target for pathogens to manipulate a given host cell to their own benefit. One of the pathogens following this strategy is Helicobacter pylori, which can cause a variety of gastric diseases and has been shown to be the major risk factor for the onset of gastric cancer. During infection of gastric epithelial cells, H. pylori hijacks the cellular kinase signaling pathways, leading to the disruption of key cell functions. Specifically, by overruling the phosphorylation status of cortactin, H. pylori alternates the activity of molecular interaction partners of this important protein, thereby manipulating the performance of actin-cytoskeletal rearrangements and cell movement. In addition, H. pylori utilizes a unique mechanism to activate focal adhesion kinase, which subsequently prevents host epithelial cells from extensive lifting from the extracellular matrix in order to achieve chronic infection in the human stomach.

scholarly journals SHP2-independent tyrosine dephosphorylation of cortactin and vinculin during infection with Helicobacter pylori

2020 ◽  
Vol 10 (1) ◽  
pp. 20-27
Author(s):  
Jakob Knorr ◽  
Steffen Backert ◽  
Nicole Tegtmeyer
Keyword(s):  
Helicobacter Pylori ◽  
Host Cell ◽  
Tyrosine Phosphatase ◽  
Actin Binding ◽  
World Population ◽  
Dependent Manner ◽  
Knockout Mutant ◽  
Ags Cells ◽  
Type Iv ◽  
H Pylori

The gastric pathogen Helicobacter pylori colonizes approximately half of the human world population. The bacterium injects the effector protein cytotoxin associated gene A (CagA) via a type-IV secretion system into host epithelial cells, where the protein becomes phosphorylated at specific EPIYA-motifs by cellular kinases. Inside the host cell, CagA can interact with over 25 different proteins in both phosphorylation-dependent and phosphorylation-independent manners, resulting in manipulation of host-cell signaling pathways. During the course of an H. pylori infection, certain host-cell proteins undergo tyrosine dephosphorylation in a CagA-dependent manner, including the actin-binding proteins cortactin and vinculin. A predominant response of intracellular CagA is the binding and activation of tyrosine phosphatase, the human Src-homology-region-2-domain-containing-phosphatase-2 (SHP2). Here, we considered the possibility that activated SHP2 might be responsible for the dephosphorylation of cortactin and vinculin. To investigate this, phosphatase inhibitor studies were performed. Additionally, a complete knockout mutant of SHP2 in AGS cells was created by CRISPR/Cas9 technology, and these cells were infected with H. pylori. However, neither the presence of an inhibitor nor the inactivation of SHP2 prevented the dephosphorylation of cortactin and vinculin upon CagA delivery. Tyrosine dephosphorylation of these proteins is therefore independent of SHP2 and instead must be caused by another, as yet unidentified, protein tyrosine phosphatase.

scholarly journals Helicobacter pylori CagA Induces Cortactin Y-470 Phosphorylation-Dependent Gastric Epithelial Cell Scattering via Abl, Vav2 and Rac1 Activation

Cancers ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 4241
Author(s):  
Nicole Tegtmeyer ◽  
Aileen Harrer ◽  
Klemens Rottner ◽  
Steffen Backert
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cell ◽  
Cell Motility ◽  
Sh2 Domain ◽  
Small Gtpase ◽  
Gastric Epithelial Cell ◽  
Gastric Epithelium ◽  
Actin Binding ◽  
Cell Scattering ◽  
H Pylori

The pathogen Helicobacter pylori is the first reported bacterial type-1 carcinogen playing a role in the development of human malignancies, including gastric adenocarcinoma. Cancer cell motility is an important process in this scenario, however, the molecular mechanisms are still not fully understood. Here, we demonstrate that H. pylori subverts the actin-binding protein cortactin through its type-IV secretion system and injected oncoprotein CagA, e.g., by inducing tyrosine phosphorylation of cortactin at Y-470, which triggers gastric epithelial cell scattering and motility. During infection of AGS cells, cortactin was discovered to undergo tyrosine dephosphorylation at residues Y-421 and Y-486, which is mediated through inactivation of Src kinase. However, H. pylori also profoundly activates tyrosine kinase Abl, which simultaneously phosphorylates cortactin at Y-470. Phosphorylated cortactin interacts with the SH2-domain of Vav2, a guanine nucleotide exchange factor for the Rho-family of GTPases. The cortactin/Vav2 complex then stimulates a previously unrecognized activation cascade including the small GTPase Rac1, to effect actin rearrangements and cell scattering. We hypothesize that injected CagA targets cortactin to locally open the gastric epithelium in order to get access to certain nutrients. This may disturb the cellular barrier functions, likely contributing to the induction of cell motility, which is important in gastric cancer development.

scholarly journals Effect of Helicobacter pylori on Polymorphonuclear Leukocyte Migration across Polarized T84 Epithelial Cell Monolayers: Role of Vacuolating Toxin VacA and cagPathogenicity Island

2000 ◽  
Vol 68 (9) ◽  
pp. 5225-5233 ◽  
Author(s):  
Véronique Hofman ◽  
Vittorio Ricci ◽  
Antoine Galmiche ◽  
Patrick Brest ◽  
Patrick Auberger ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Polymorphonuclear Leukocyte ◽  
Broth Culture ◽  
Intestinal Cell ◽  
T84 Cells ◽  
Vacuolating Cytotoxin ◽  
Intestinal Cell Line ◽  
H Pylori

ABSTRACT Helicobacter pylori infection can induce polymorphonuclear leukocyte (PMNL) infiltration of the gastric mucosa, which characterizes acute chronic gastritis. The mechanisms underlying this process are poorly documented. The lack of an in vitro model has considerably impaired the study of transepithelial migration of PMNL induced by H. pylori. In the present work, we used confluent polarized monolayers of the human intestinal cell line T84 grown on permeable filters to analyze the epithelial PMNL response induced by broth culture filtrates (BCFs) and bacterial suspensions from different strains of H. pylori. We have evaluated the role of the vacuolating cytotoxin VacA and of the cagpathogenicity island (PAI) of H. pylori in PMNL migration via their effects on T84 epithelial cells. We noted no difference in the rates of PMNL transepithelial migration after epithelial preincubation with bacterial suspensions or with BCFs of VacA-negative or VacA-positive H. pylori strains. In contrast, PMNL transepithelial migration was induced after incubation of the T84 cells with cag PAI-positive and cagE-positiveH. pylori strains. Finally, PMNL migration was correlated with a basolateral secretion of interleukin-8 by T84 cells, thus creating a subepithelial chemotactic gradient for PMNL. These data provide evidence that the vacuolating cytotoxin VacA is not involved in PMNL transepithelial migration and that the cag PAI, with a pivotal role for the cagE gene, provokes a transcellular signal across T84 monolayers, inducing a subepithelial PMNL response.

scholarly journals Helicobacter pylori induces intracellular galectin-8 aggregation around damaged lysosomes within gastric epithelial cells in a host O-glycan-dependent manner

Glycobiology ◽  
2018 ◽  
Vol 29 (2) ◽  
pp. 151-162 ◽  
Author(s):  
Fang-Yen Li ◽  
I-Chun Weng ◽  
Chun-Hung Lin ◽  
Mou-Chieh Kao ◽  
Ming-Shiang Wu ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Rhesus Macaques ◽  
Dependent Manner ◽  
Gastric Epithelial Cells ◽  
Vacuolating Cytotoxin ◽  
Infected Cells ◽  
H Pylori ◽  
Autophagy Activity ◽  
Binding Lectin

Abstract Galectin-8, a beta-galactoside-binding lectin, is upregulated in the gastric tissues of rhesus macaques infected with Helicobacter pylori. In this study, we found that H. pylori infection triggers intracellular galectin-8 aggregation in human-derived AGS gastric epithelial cells, and that these aggregates colocalize with lysosomes. Notably, this aggregation is markedly reduced following the attenuation of host O-glycan processing. This indicates that H. pylori infection induces lysosomal damage, which in turn results in the accumulation of cytosolic galectin-8 around damaged lysosomes through the recognition of exposed vacuolar host O-glycans. H. pylori-induced galectin-8 aggregates also colocalize with autophagosomes, and galectin-8 ablation reduces the activation of autophagy by H. pylori. This suggests that galectin-8 aggregates may enhance autophagy activity in infected cells. We also observed that both autophagy and NDP52, an autophagy adapter, contribute to the augmentation of galectin-8 aggregation by H. pylori. Additionally, vacuolating cytotoxin A, a secreted H. pylori cytotoxin, may contribute to the increased galectin-8 aggregation and elevated autophagy response in infected cells. Collectively, these results suggest that H. pylori promotes intracellular galectin-8 aggregation, and that galectin-8 aggregation and autophagy may reciprocally regulate each other during infection.

scholarly journals Serum IgG antibodies against Helicobacter pylori low molecular weight antigens 50kDa, 30kDa and Urease A 26 kDa, along with vacuolating cytotoxin A are associated with the outcome of the infection

2020 ◽  
Vol 77 (4) ◽  
pp. 405-412
Author(s):  
Nebojsa Manojlovic ◽  
Ivana Tufegdzic ◽  
Elizabeta Ristanovic ◽  
Dubravko Bokonjic
Keyword(s):  
Gastric Cancer ◽  
Molecular Weight ◽  
Helicobacter Pylori ◽  
Functional Dyspepsia ◽  
Low Molecular Weight ◽  
Igg Antibodies ◽  
Vacuolating Cytotoxin ◽  
Specific Outcome ◽  
H Pylori ◽  
Topographic Distribution

Background/Aim. We designed and conducted this study due to the fact that results of the previous studies about seroreactivity to low-molecular-weight Helicobacter pylori antigens, cytotoxin-associated gene A (CagA), vacuolating cytotoxin A (VacA) in patients with gastric cancer and peptic ulcer were conflicting. Methods. The Western blot test was performed in 123 patients, 31 with gastric cancer, 31 with duodenal ulcer, 31 with gastric ulcer, 30 with gastritis and functional dyspepsia in order to determine IgG antibodies to H. pylori antigens (CagA, VacA, Heat shock protein 60kDa, Urease B 66 kDa, Flagellin 55kDa, 50kDa, 30 kDa, Urease A 26 kDa, 24 kDa). In this study we analyzed: seroreactivity to H. pylori antigens between group with functional dyspepsia and others; between grades of different histopathological parameters of inflammation of antral and corporal mucosa and between antrum-predominant gastritis and corpus-predominant gastritis + pangastritis groups. Results. It was shown that seropositivity to 50 kDa antigen could be used as a biomarker for functional dyspepsia, seropositivity to 30 kDa antigen for antrumpredominant gastritis and H. pylori colonization in the antrum, to UreaseA26 kDa antigen for pangastritis and corpus-predominant gastritis and degree of inflammation in the corpus. Seropositivity to VacA was the biomarker for gastric cancer and peptic ulcer taken together and inflammation of antral mucosa. Seropositivity to CagA was associated with more intensive inflammation of antral and corporal mucosa, Urease B66 kDa with inflammation of corpus mucosa, but neither of them with specific outcome of H. pylori infection and topographic distribution of gastric inflammation. Conclusion. Serum IgG antibodies to H. pylori antigens 50kDa, and VacA may represent useful biomarkers for the specific outcome of H. pylori infection, while serum antibodies to 30 kDa and UreaseA26 kDa antigens might be used as specific biomarkers for different topographic distribution of inflammation in gastric mucosa.

scholarly journals A Helicobacter pylori TolC Efflux Pump Confers Resistance to Metronidazole

2005 ◽  
Vol 49 (4) ◽  
pp. 1477-1482 ◽  
Author(s):  
Karin van Amsterdam ◽  
Aldert Bart ◽  
Arie van der Ende
Keyword(s):  
Helicobacter Pylori ◽  
Efflux Pump ◽  
Sodium Deoxycholate ◽  
Loss Of Function ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Active Efflux ◽  
Knockout Mutants ◽  
H Pylori ◽  
Susceptibility Profiles

ABSTRACT In Helicobacter pylori, the contribution of efflux proteins to antibiotic resistance is not well established. As translocases that act in parallel may have overlapping substrate specificities, the loss of function of one such translocase may be compensated for by that of another translocase with no effect on susceptibilities to antibiotics. The genome of H. pylori 26695 was assessed for the presence of putative translocases and outer membrane efflux or TolC-like proteins which could interact to form efflux systems involved in drug resistance. Twenty-seven translocases were identified, of which HP1184 was the sole representative of the multidrug and toxic compound extrusion family of translocases and which could thus have a unique substrate specificity. In addition, four TolC-like proteins (HP0605, HP0971, HP1327, and HP1489) were identified. Thus, it is feasible that inactivation of a TolC-like protein would affect the functions of multiple translocases. We aimed to determine whether efflux systems contribute to antimicrobial susceptibility by evaluation of the susceptibility profiles of an HP1184-knockout mutant, four mutants in which one of the four TolC homologs was inactivated, as well as a mutant in which both HP0605 and HP0971 were inactivated. The HP1184- and HP1489-knockout mutants both showed increased susceptibilities to ethidium bromide, while the HP0605-knockout mutant exhibited increased susceptibilities to novobiocin and sodium deoxycholate. The HP0605 and HP0971 double-knockout mutant was also more susceptible to metronidazole, in addition to being susceptible to novobiocin and sodium deoxycholate. Thus, active efflux is an eminent means of resistance to antimicrobials in H. pylori and resembles the situation in other bacteria.

scholarly journals Helicobacter pylori and Complex Gangliosides

2004 ◽  
Vol 72 (3) ◽  
pp. 1519-1529 ◽  
Author(s):  
Niamh Roche ◽  
Jonas Ångström ◽  
Marina Hurtig ◽  
Thomas Larsson ◽  
Thomas Borén ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Sialic Acid ◽  
Mutant Strain ◽  
Carbohydrate Chain ◽  
Proton Nuclear Magnetic Resonance ◽  
Bacterial Cells ◽  
Wild Type ◽  
Knockout Mutant ◽  
Factors Affecting ◽  
H Pylori

ABSTRACT Recognition of sialic acid-containing glycoconjugates by the human gastric pathogen Helicobacter pylori has been repeatedly demonstrated. To investigate the structural requirements for H. pylori binding to complex gangliosides, a large number of gangliosides were isolated and characterized by mass spectrometry and proton nuclear magnetic resonance. Ganglioside binding of sialic acid-recognizing H. pylori strains (strains J99 and CCUG 17874) and knockout mutant strains with the sialic acid binding adhesin SabA or the NeuAcα3Galβ4GlcNAcβ3Galβ4GlcNAcβ-binding neutrophil-activating protein HPNAP deleted was investigated using the thin-layer chromatogram binding assay. The wild-type bacteria bound to N-acetyllactosamine-based gangliosides with terminal α3-linked NeuAc, while gangliosides with terminal NeuGcα3, NeuAcα6, or NeuAcα8NeuAcα3 were not recognized. The factors affecting binding affinity were identified as (i) the length of the N-acetyllactosamine carbohydrate chain, (ii) the branches of the carbohydrate chain, and (iii) fucose substitution of the N-acetyllactosamine core chain. While the J99/NAP− mutant strain displayed a ganglioside binding pattern identical to that of the parent J99 wild-type strain, no ganglioside binding was obtained with the J99/SabA− mutant strain, demonstrating that the SabA adhesin is the sole factor responsible for the binding of H. pylori bacterial cells to gangliosides.

scholarly journals Alterations in Cell Mechanics by Actin Cytoskeletal Changes Correlate with Strain-Specific Rubella Virus Phenotypes for Cell Migration and Induction of Apoptosis

Cells ◽  
2018 ◽  
Vol 7 (9) ◽  
pp. 136 ◽  
Author(s):  
Martin Kräter ◽  
Jiranuwat Sapudom ◽  
Nicole Bilz ◽  
Tilo Pompe ◽  
Jochen Guck ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Cell Migration ◽  
Cell Mechanics ◽  
Rubella Virus ◽  
Cell Movement ◽  
Cell Stiffness ◽  
Filamentous Actin ◽  
Cell Functions ◽  
Viral Spread ◽  
Induction Of Apoptosis

The cellular cytoskeleton is central for key cellular functions, and as such is a marker for diseased and infected cell states. Here we analyzed infection with rubella virus (RV) strains with respect to phenotypes in cellular mechanical properties, cell movement, and viral cytopathogenicity. Real-time deformability cytometry (RT-DC), as a high-throughput platform for the assessment of cell mechanics, revealed a correlation of an increase in cortical filamentous-actin (F-actin) with a higher cellular stiffness. The additional reduction of stress fibers noted for only some RV strains as the most severe actin rearrangement lowered cell stiffness. Furthermore, a reduced collective and single cell migration speed in a wound healing assay was detected in addition to severe changes in cell morphology. The latter was followed by activation of caspase 3/7 as a sign for induction of apoptosis. Our study emphasizes RT-DC technology as a sensitive means to characterize viral cell populations and to implicate alterations of cell mechanical properties with cell functions. These interdependent events are not only promising options to elucidate viral spread and to understand viral pathologies within the infected host. They also contribute to any diseased cell state, as exemplified by RV as a representative agent for cytoskeletal alterations involved in a cytopathological outcome.

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