scholarly journals Complex Cellular Responses of Helicobacter pylori-Colonized Gastric Adenocarcinoma Cells

2011 ◽  
Vol 79 (6) ◽  
pp. 2362-2371 ◽  
Author(s):  
Sabine Schneider ◽  
Gert Carra ◽  
Ugur Sahin ◽  
Benjamin Hoy ◽  
Gabriele Rieder ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Host Cell ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Cellular Responses ◽  
Host Cells ◽  
Human Gastric Mucosa ◽  
Content Type ◽  
H Pylori

ABSTRACTHelicobacter pyloriis an important class I carcinogen that persistently infects the human gastric mucosa to induce gastritis, gastric ulceration, and gastric cancer.H. pyloripathogenesis strongly depends on pathogenic factors, such as VacA (vacuolating cytotoxin A) or a specialized type IV secretion system (T4SS), which injects the oncoprotein CagA (cytotoxin-associated gene A product) into the host cell. Since access to primary gastric epithelial cells is limited, many studies on the complex cellular and molecular mechanisms ofH. pyloriwere performed in immortalized epithelial cells originating from individual human adenocarcinomas. The aim of our study was a comparative analysis of 14 different human gastric epithelial cell lines after colonization withH. pylori. We found remarkable differences in host cell morphology, extent of CagA tyrosine phosphorylation, adhesion to host cells, vacuolization, and interleukin-8 (IL-8) secretion. These data might help in the selection of suitable cell lines to study host cell responses toH. pyloriin vitro, and they imply that different host cell factors are involved in the determination ofH. pyloripathogenesis. A better understanding ofH. pylori-directed cellular responses can provide novel and more balanced insights into the molecular mechanisms ofH. pylori-dependent pathogenesisin vivoand may lead to new therapeutic approaches.

scholarly journals Lactobacilli Reduce Helicobacter pylori Attachment to Host Gastric Epithelial Cells by Inhibiting Adhesion Gene Expression

2016 ◽  
Vol 84 (5) ◽  
pp. 1526-1535 ◽  
Author(s):  
Nele de Klerk ◽  
Lisa Maudsdotter ◽  
Hanna Gebreegziabher ◽  
Sunil D. Saroj ◽  
Beatrice Eriksson ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Binding Capacity ◽  
Host Cells ◽  
Gastric Epithelial Cells ◽  
Content Type ◽  
Effector Molecule ◽  
H Pylori

The human gastrointestinal tract, including the harsh environment of the stomach, harbors a large variety of bacteria, of whichLactobacillusspecies are prominent members. The molecular mechanisms by which species of lactobacilli interfere with pathogen colonization are not fully characterized. In this study, we aimed to study the effect of lactobacillus strains upon the initial attachment ofHelicobacter pylorito host cells. Here we report a novel mechanism by which lactobacilli inhibit adherence of the gastric pathogenH. pylori. In a screen withLactobacillusisolates, we found that only a few could reduce adherence ofH. pylorito gastric epithelial cells. Decreased attachment was not due to competition for space or to lactobacillus-mediated killing of the pathogen. Instead, we show that lactobacilli act onH. pyloridirectly by an effector molecule that is released into the medium. This effector molecule acts onH. pyloriby inhibiting expression of the adhesin-encoding genesabA. Finally, we verified that inhibitory lactobacilli reducedH. pyloricolonization in anin vivomodel. In conclusion, certainLactobacillusstrains affect pathogen adherence by inhibitingsabAexpression and thereby reducingH. pyloribinding capacity.

scholarly journals The Helicobacter pylori Autotransporter ImaA Tempers the Bacterium's Interaction with α5β1 Integrin

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
William E. Sause ◽  
Daniela Keilberg ◽  
Soufiane Aboulhouda ◽  
Karen M. Ottemann
Keyword(s):  
Helicobacter Pylori ◽  
Host Cell ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Wild Type ◽  
Content Type ◽  
Type Iv ◽  
H Pylori ◽  
Α5β1 Integrin ◽  
Cell Interface

ABSTRACT The human pathogen Helicobacter pylori uses the host receptor α5β1 integrin to trigger inflammation in host cells via its cag pathogenicity island (cag PAI) type IV secretion system (T4SS). Here, we report that the H. pylori ImaA protein (HP0289) decreases the action of the cag PAI T4SS via tempering the bacterium's interaction with α5β1 integrin. Previously, imaA-null mutants were found to induce an elevated inflammatory response that was dependent on the cag PAI T4SS; here we extend those findings to show that the elevated response is independent of the CagA effector protein. To understand how ImaA could be affecting cag PAI T4SS activity at the host cell interface, we utilized the Phyre structural threading program and found that ImaA has a region with remote homology to bacterial integrin-binding proteins. This region was required for ImaA function. Unexpectedly, we observed that imaA mutants bound higher levels of α5β1 integrin than wild-type H. pylori, an outcome that required the predicted integrin-binding homology region of ImaA. Lastly, we report that ImaA directly affected the amount of host cell β1 integrin but not other cellular integrins. Our results thus suggest a model in which H. pylori employs ImaA to regulate interactions between integrin and the T4SS and thus alter the host inflammatory strength.

scholarly journals The Helicobacter pylori CZB Cytoplasmic Chemoreceptor TlpD Forms an Autonomous Polar Chemotaxis Signaling Complex That Mediates a Tactic Response to Oxidative Stress

2016 ◽  
Vol 198 (11) ◽  
pp. 1563-1575 ◽  
Author(s):  
Kieran D. Collins ◽  
Tessa M. Andermann ◽  
Jenny Draper ◽  
Lisa Sanders ◽  
Susan M. Williams ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Helicobacter Pylori ◽  
Molecular Mechanisms ◽  
Reactive Oxygen ◽  
Host Cells ◽  
Oxygen Species ◽  
Content Type ◽  
Signaling Complex ◽  
H Pylori

ABSTRACTCytoplasmic chemoreceptors are widespread among prokaryotes but are far less understood than transmembrane chemoreceptors, despite being implicated in many processes. One such cytoplasmic chemoreceptor isHelicobacter pyloriTlpD, which is required for stomach colonization and drives a chemotaxis response to cellular energy levels. Neither the signals sensed by TlpD nor its molecular mechanisms of action are known. We report here that TlpD functions independently of the other chemoreceptors. When TlpD is the sole chemoreceptor, it is able to localize to the pole and recruits CheW, CheA, and at least two CheV proteins to this location. It loses the normal membrane association that appears to be driven by interactions with other chemoreceptors and with CheW, CheV1, and CheA. These results suggest that TlpD can form an autonomous signaling unit. We further determined that TlpD mediates a repellent chemotaxis response to conditions that promote oxidative stress, including being in the presence of iron, hydrogen peroxide, paraquat, and metronidazole. Last, we found that all testedH. pyloristrains express TlpD, whereas other chemoreceptors were present to various degrees. Our data suggest a model in which TlpD coordinates a signaling complex that responds to oxidative stress and may allowH. pylorito avoid areas of the stomach with high concentrations of reactive oxygen species.IMPORTANCEHelicobacter pylorisenses its environment with proteins called chemoreceptors. Chemoreceptors integrate this sensory information to affect flagellum-based motility in a process called chemotaxis. Chemotaxis is employed during infection and presumably aidsH. pyloriin encountering and colonizing preferred niches. A cytoplasmic chemoreceptor named TlpD is particularly important in this process, and we report here that this chemoreceptor is able to operate independently of other chemoreceptors to organize a chemotaxis signaling complex and mediate a repellent response to oxidative stress conditions.H. pyloriencounters and must cope with oxidative stress during infection due to oxygen and reactive oxygen species produced by host cells. TlpD's repellent response may allow the bacteria to escape niches experiencing inflammation and elevated reactive oxygen species (ROS) production.

scholarly journals Differential Roles of ASK1 and TAK1 in Helicobacter pylori-Induced Cellular Responses

2013 ◽  
Vol 81 (12) ◽  
pp. 4551-4560 ◽  
Author(s):  
Yoku Hayakawa ◽  
Yoshihiro Hirata ◽  
Hiroto Kinoshita ◽  
Kosuke Sakitani ◽  
Hayato Nakagawa ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Signaling Pathway ◽  
Critical Role ◽  
Cellular Responses ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Content Type ◽  
H Pylori ◽  
Jnk Activation

ABSTRACTThe mitogen-activated protein kinase (MAPK) signaling pathway regulates various cellular functions, including those induced byHelicobacter pylori. TAK1 is an upstream MAPK kinase kinase (MAP3K) required forH. pylori-induced MAPK and NF-κB activation, but it remains unclear whether other MAP3Ks are involved inH. pylori-induced cellular responses. In this study, we focused on the MAP3K ASK1, which plays a critical role in gastric tumorigenesis. In gastric epithelial cells,H. pyloriactivates ASK1 in a reactive oxygen species (ROS)- andcagpathogenicity island-dependent manner, and ASK1 regulates sustained JNK activation and apoptosis induced byH. pylori. In contrast, TAK1 regulatesH. pylori-mediated early JNK activation and cytokine production. We also found reciprocal regulation between ASK1 and TAK1 inH. pylori-related responses, whereby inhibition of TAK1 or downstream p38 MAPK activates ASK1 through ROS production, and ASK1 suppresses TAK1 and downstream NF-κB activation. We identified ROS/ASK1/JNK as a new signaling pathway induced byH. pylori, which regulates apoptotic cell death. The balance of ASK1-induced apoptosis and TAK1-induced antiapoptotic or inflammatory responses may determine the fate of epithelial cells infected withH. pyloriand thus be involved in the pathogenesis of gastritis and gastric cancer.

scholarly journals Chemokines and Antimicrobial Peptides Have acag-Dependent Early Response to Helicobacter pylori Infection in Primary Human Gastric Epithelial Cells

2014 ◽  
Vol 82 (7) ◽  
pp. 2881-2889 ◽  
Author(s):  
Pascale Mustapha ◽  
Isabelle Paris ◽  
Magali Garcia ◽  
Cong Tri Tran ◽  
Julie Cremniter ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Inflammatory Response ◽  
Antimicrobial Peptides ◽  
Virulence Factors ◽  
Inflammatory Mediator ◽  
Host Cells ◽  
Gastric Epithelial Cells ◽  
Content Type ◽  
H Pylori

ABSTRACTHelicobacter pyloriinfection systematically causes chronic gastric inflammation that can persist asymptomatically or evolve toward more severe gastroduodenal pathologies, such as ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. Thecagpathogenicity island (cagPAI) ofH. pyloriallows translocation of the virulence protein CagA and fragments of peptidoglycan into host cells, thereby inducing production of chemokines, cytokines, and antimicrobial peptides. In order to characterize the inflammatory response toH. pylori, a new experimental protocol for isolating and culturing primary human gastric epithelial cells was established using pieces of stomach from patients who had undergone sleeve gastrectomy. Isolated cells expressed markers indicating that they were mucin-secreting epithelial cells. Challenge of primary epithelial cells withH. pyloriB128 underscored early dose-dependent induction of expression of mRNAs of the inflammatory mediators CXCL1 to -3, CXCL5, CXCL8, CCL20, BD2, and tumor necrosis factor alpha (TNF-α). In AGS cells, significant expression of only CXCL5 and CXCL8 was observed following infection, suggesting that these cells were less reactive than primary epithelial cells. Infection of both cellular models withH. pyloriB128ΔcagM, acagPAI mutant, resulted in weak inflammatory-mediator mRNA induction. At 24 h after infection of primary epithelial cells withH. pylori, inflammatory-mediator production was largely due tocagPAI substrate-independent virulence factors. Thus,H. pyloricagPAI substrate appears to be involved in eliciting an epithelial response during the early phases of infection. Afterwards, other virulence factors of the bacterium take over in development of the inflammatory response. Using a relevant cellular model, this study provides new information on the modulation of inflammation duringH. pyloriinfection.

scholarly journals Uptake of Helicobacter pylori Vesicles Is Facilitated by Clathrin-Dependent and Clathrin-Independent Endocytic Pathways

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Annelie Olofsson ◽  
Lars Nygård Skalman ◽  
Ikenna Obi ◽  
Richard Lundmark ◽  
Anna Arnqvist
Keyword(s):  
Helicobacter Pylori ◽  
Host Cell ◽  
Host Cells ◽  
Gastric Epithelial Cells ◽  
Host Cell Membrane ◽  
Content Type ◽  
Effector Molecules ◽  
Chemical Inhibition ◽  
H Pylori ◽  
Endocytic Pathways

ABSTRACTBacteria shed a diverse set of outer membrane vesicles that function as transport vehicles to deliver effector molecules and virulence factors to host cells.Helicobacter pyloriis a gastric pathogen that infects half of the world’s population, and in some individuals the infection progresses into peptic ulcer disease or gastric cancer. Here we report that intact vesicles fromH. pyloriare internalized by clathrin-dependent endocytosis and further dynamin-dependent processes, as well as in a cholesterol-sensitive manner. We analyzed the uptake ofH. pylorivesicles by gastric epithelial cells using a method that we refer to as quantification of internalized substances (qIS). The qIS assay is based on a near-infrared dye with a cleavable linker that enables the specific quantification of internalized substances after exposure to reducing conditions. Both chemical inhibition and RNA interference in combination with the qIS assay showed thatH. pylorivesicles enter gastric epithelial cells via both clathrin-mediated endocytosis and additional endocytic processes that are dependent on dynamin. Confocal microscopy revealed thatH. pylorivesicles colocalized with clathrin and dynamin II and with markers of subsequent endosomal and lysosomal trafficking. Interestingly, however, knockdown of components required for caveolae had no significant effect on internalization and knockdown of components required for clathrin-independent carrier (CLIC) endocytosis increased internalization ofH. pylorivesicles. Furthermore, uptake of vesicles by both clathrin-dependent and -independent pathways was sensitive to depletion, but not sequestering, of cholesterol in the host cell membrane suggesting that membrane fluidity influences the efficiency ofH. pylorivesicle uptake.IMPORTANCEBacterial vesicles act as long-distance tools to deliver toxins and effector molecules to host cells. Vesicles can cause a variety of host cell responses via cell surface-induced cell signaling or internalization. Vesicles of diverse bacterial species enter host cells via different endocytic pathways or via membrane fusion. With the combination of a fluorescence-based quantification assay that quantifies internalized vesicles in a large number of cells and either chemical inhibition or RNA interference, we show that clathrin-mediated endocytosis is the major pathway for uptake ofHelicobacter pylorivesicles and that lipid microdomains of the host cell membrane affect uptake of vesicles via clathrin-independent pathways. Our results provide important insights about membrane fluidity and its important role in the complex process that directs theH. pylorivesicle to a specific endocytic pathway. Understanding the mechanisms that operate in vesicle-host interactions is important to fully recognize the impact of vesicles in pathogenesis.

scholarly journals Helicobacter pylori Exploits Cholesterol-Rich Microdomains for Induction of NF-κB-Dependent Responses and Peptidoglycan Delivery in Epithelial Cells

2010 ◽  
Vol 78 (11) ◽  
pp. 4523-4531 ◽  
Author(s):  
Melanie L. Hutton ◽  
Maria Kaparakis-Liaskos ◽  
Lorinda Turner ◽  
Ana Cardona ◽  
Terry Kwok ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Host Cell ◽  
Severe Disease ◽  
Host Cells ◽  
Type Iv ◽  
Increased Risk ◽  
Proinflammatory Responses ◽  
H Pylori ◽  
Cytoskeletal Rearrangements

ABSTRACT Infection with Helicobacter pylori cag pathogenicity island (cagPAI)-positive strains is associated with more destructive tissue damage and an increased risk of severe disease. The cagPAI encodes a type IV secretion system (TFSS) that delivers the bacterial effector molecules CagA and peptidoglycan into the host cell cytoplasm, thereby inducing responses in host cells. It was previously shown that interactions between CagL, present on the TFSS pilus, and host α5β1 integrin molecules were critical for CagA translocation and the induction of cytoskeletal rearrangements in epithelial cells. As the α5β1 integrin is found in cholesterol-rich microdomains (known as lipid rafts), we hypothesized that these domains may also be involved in the induction of proinflammatory responses mediated by NOD1 recognition of H. pylori peptidoglycan. Indeed, not only did methyl-β-cyclodextrin depletion of cholesterol from cultured epithelial cells have a significant effect on the levels of NF-κB and interleukin-8 (IL-8) responses induced by H. pylori bacteria with an intact TFSS (P < 0.05), but it also interfered with TFSS-mediated peptidoglycan delivery to cells. Both of these effects could be restored by cholesterol replenishment of the cells. Furthermore, we demonstrated for the first time the involvement of α5β1 integrin in the induction of proinflammatory responses by H. pylori. Taking the results together, we propose that α5β1 integrin, which is associated with cholesterol-rich microdomains at the host cell surface, is required for NOD1 recognition of peptidoglycan and subsequent induction of NF-κB-dependent responses to H. pylori. These data implicate cholesterol-rich microdomains as a novel platform for TFSS-dependent delivery of bacterial products to cytosolic pathogen recognition molecules.

scholarly journals Helicobacter pylori Infections in the Bronx, New York: Surveying Antibiotic Susceptibility and Strain Lineage by Whole-Genome Sequencing

2019 ◽  
Vol 58 (3) ◽  
Author(s):  
Rajagopalan Saranathan ◽  
Michael H. Levi ◽  
Alice R. Wattam ◽  
Adel Malek ◽  
Emmanuel Asare ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Molecular Mechanisms ◽  
Kappa Coefficient ◽  
23S Rrna ◽  
Whole Genome ◽  
Content Type ◽  
Phenotypic Resistance ◽  
H Pylori

ABSTRACT The emergence of drug resistance in Helicobacter pylori has resulted in a greater need for susceptibility-guided treatment. While the alleles associated with resistance to clarithromycin and levofloxacin have been defined, there are limited data regarding the molecular mechanisms underlying resistance to other antimicrobials. Using H. pylori isolates from 42 clinical specimens, we compared phenotypic and whole-genome sequencing (WGS)-based detection of resistance. Phenotypic resistance correlated with the presence of alleles of 23S rRNA (A2142G/A2143G) for clarithromycin (kappa coefficient, 0.84; 95% confidence interval [CI], 0.67 to 1.0) and gyrA (N87I/N87K/D91Y/D91N/D91G/D99N) for levofloxacin (kappa coefficient, 0.90; 95% CI, 0.77 to 1.0). Phenotypic resistance to amoxicillin in three isolates correlated with mutations in pbp1, pbp2, and/or pbp3 within coding regions near known amoxicillin binding motifs. All isolates were phenotypically susceptible to tetracycline, although four bore a mutation in 16S rRNA (A926G). For metronidazole, nonsense mutations and R16H substitutions in rdxA correlated with phenotypic resistance (kappa coefficient, 0.76; 95% CI, 0.56 to 0.96). Previously identified mutations in the rpoB rifampin resistance-determining region (RRDR) were not present, but 14 novel mutations outside the RRDR were found in rifampin-resistant isolates. WGS also allowed for strain lineage determination, which may be important for future studies in associating precise MICs with specific resistance alleles. In summary, WGS allows for broad analyses of H. pylori isolates, and our findings support the use of WGS for the detection of clarithromycin and levofloxacin resistance. Additional studies are warranted to better define mutations conferring resistance to amoxicillin, tetracycline, and rifampin, but combinatorial analyses for rdxA gene truncations and R16H mutations have utility for determining metronidazole resistance.

scholarly journals Role of Interleukin-32 in Helicobacter pylori-Induced Gastric Inflammation

2012 ◽  
Vol 80 (11) ◽  
pp. 3795-3803 ◽  
Author(s):  
Kosuke Sakitani ◽  
Yoshihiro Hirata ◽  
Yoku Hayakawa ◽  
Takako Serizawa ◽  
Wachiko Nakata ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
Gastric Mucosa ◽  
Cell Lines ◽  
Inflammatory Diseases ◽  
Nuclear Factor Κb ◽  
Gastric Disease ◽  
Ags Cells ◽  
Content Type ◽  
H Pylori

ABSTRACTHelicobacter pyloriinfection is associated with gastritis and gastric cancer. AnH. pylorivirulence factor, thecagpathogenicity island (PAI), is related to host cell cytokine induction and gastric inflammation. Since elucidation of the mechanisms of inflammation is important for therapy, the associations between cytokines and inflammatory diseases have been investigated vigorously. Levels of interleukin-32 (IL-32), a recently described inflammatory cytokine, are increased in various inflammatory diseases, such as rheumatoid arthritis and Crohn's disease, and in malignancies, including gastric cancer. In this report, we examined IL-32 expression in human gastric disease. We also investigated the function of IL-32 in activation of the inflammatory cytokines in gastritis. IL-32 expression paralleled human gastric tissue pathology, with low IL-32 expression inH. pylori-uninfected gastric mucosa and higher expression levels in gastritis and gastric cancer tissues.H. pyloriinfection increased IL-32 expression in human gastric epithelial cell lines.H. pylori-induced IL-32 expression was dependent on the bacterialcagPAI genes and on activation of nuclear factor κB (NF-κB). IL-32 expression induced byH. pyloriwas not detected in the supernatant of AGS cells but was found in the cytosol. Expression of theH. pylori-induced cytokines CXCL1, CXCL2, and IL-8 was decreased in IL-32-knockdown AGS cell lines compared to a control AGS cell line. We also found that NF-κB activation was decreased inH. pylori-infected IL-32-knockdown cells. These results suggest that IL-32 has important functions in the regulation of cytokine expression inH. pylori-infected gastric mucosa.

scholarly journals Phylogeographic Origin of Helicobacter pylori Determines Host-Adaptive Responses upon Coculture with Gastric Epithelial Cells

2013 ◽  
Vol 81 (7) ◽  
pp. 2468-2477 ◽  
Author(s):  
Alexander Sheh ◽  
Rupesh Chaturvedi ◽  
D. Scott Merrell ◽  
Pelayo Correa ◽  
Keith T. Wilson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Gastric Disease ◽  
Gastric Epithelial Cells ◽  
Gastric Cancer Risk ◽  
Content Type ◽  
H Pylori ◽  
Induced Apoptosis

ABSTRACTWhileHelicobacter pyloriinfects over 50% of the world's population, the mechanisms involved in the development of gastric disease are not fully understood. Bacterial, host, and environmental factors play a role in disease outcome. To investigate the role of bacterial factors inH. pyloripathogenesis, global gene expression of sixH. pyloriisolates was analyzed during coculture with gastric epithelial cells. Clustering analysis of six Colombian clinical isolates from a region with low gastric cancer risk and a region with high gastric cancer risk segregated strains based on their phylogeographic origin. One hundred forty-six genes had increased expression in European strains, while 350 genes had increased expression in African strains. Differential expression was observed in genes associated with motility, pathogenicity, and other adaptations to the host environment. European strains had greater expression of the virulence factorscagA,vacA, andbabBand were associated with increased gastric histologic lesions in patients. In AGS cells, European strains promoted significantly higher interleukin-8 (IL-8) expression than did African strains. African strains significantly induced apoptosis, whereas only one European strain significantly induced apoptosis. Our data suggest that gene expression profiles of clinical isolates can discriminate strains by phylogeographic origin and that these profiles are associated with changes in expression of the proinflammatory and protumorigenic cytokine IL-8 and levels of apoptosis in host epithelial cells. These findings support the hypothesis that bacterial factors determined by the phylogeographic origin ofH. pyloristrains may promote increased gastric disease.

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