Essential Role of Ferritin Pfr in Helicobacter pylori Iron Metabolism and Gastric Colonization

ABSTRACT The reactivity of the essential element iron necessitates a concerted expression of ferritins, which mediate iron storage in a nonreactive state. Here we have further established the role of the Helicobacter pylori ferritin Pfr in iron metabolism and gastric colonization. Iron stored in Pfr enabled H. pylori to multiply under severe iron starvation and protected the bacteria from acid-amplified iron toxicity, as inactivation of the pfr gene restricted growth of H. pylori under these conditions. The lowered total iron content in the pfr mutant, which is probably caused by decreased iron uptake rates, was also reflected by an increased resistance to superoxide stress. Iron induction of Pfr synthesis was clearly diminished in an H. pylori feoB mutant, which lacked high-affinity ferrous iron transport, confirming that Pfr expression is mediated by changes in the cytoplasmic iron pool and not by extracellular iron. This is well in agreement with the recent discovery that iron induces Pfr synthesis by abolishing Fur-mediated repression of pfr transcription, which was further confirmed here by the observation that iron inhibited the in vitro binding of recombinant H. pylori Fur to the pfr promoter region. The functions of H. pylori Pfr in iron metabolism are essential for survival in the gastric mucosa, as the pfr mutant was unable to colonize in a Mongolian gerbil-based animal model. In summary, the pfr phenotypes observed give new insights into prokaryotic ferritin functions and indicate that iron storage and homeostasis are of extraordinary importance for H. pylori to survive in its hostile natural environment.

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

Infection and Immunity ◽

10.1128/iai.68.9.5225-5233.2000 ◽

2000 ◽

Vol 68 (9) ◽

pp. 5225-5233 ◽

Cited By ~ 22

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.

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Colonization and Inflammation Deficiencies in Mongolian Gerbils Infected by Helicobacter pylori Chemotaxis Mutants

Infection and Immunity ◽

10.1128/iai.73.3.1820-1827.2005 ◽

2005 ◽

Vol 73 (3) ◽

pp. 1820-1827 ◽

Cited By ~ 78

Author(s):

David J. McGee ◽

Melanie L. Langford ◽

Emily L. Watson ◽

J. Elliot Carter ◽

Yu-Ting Chen ◽

...

Keyword(s):

Helicobacter Pylori ◽

Immune Cell ◽

Response Regulator ◽

Critical Role ◽

Mongolian Gerbils ◽

Wild Type ◽

In The Wild ◽

H Pylori

ABSTRACT Helicobacter pylori causes disease in the human stomach and in mouse and gerbil stomach models. Previous results have shown that motility is critical for H. pylori to colonize mice, gerbils, and other animal models. The role of chemotaxis, however, in colonization and disease is less well understood. Two genes in the H. pylori chemotaxis pathway, cheY and tlpB, which encode the chemotaxis response regulator and a methyl-accepting chemoreceptor, respectively, were disrupted. The cheY mutation was complemented with a wild-type copy of cheY inserted into the chromosomal rdxA gene. The cheY mutant lost chemotaxis but retained motility, while all other strains were motile and chemotactic in vitro. These strains were inoculated into gerbils either alone or in combination with the wild-type strain, and colonization and inflammation were assessed. While the cheY mutant completely failed to colonize gerbil stomachs, the tlpB mutant colonized at levels similar to those of the wild type. With the tlpB mutant, there was a substantial decrease in inflammation in the gerbil stomach compared to that with the wild type. Furthermore, there were differences in the numbers of each immune cell in the tlpB-mutant-infected stomach: the ratio of lymphocytes to neutrophils was about 8 to 1 in the wild type but only about 1 to 1 in the mutant. These results suggest that the TlpB chemoreceptor plays an important role in the inflammatory response while the CheY chemotaxis regulator plays a critical role in initial colonization. Chemotaxis mutants may provide new insights into the steps involved in H. pylori pathogenesis.

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Antisense RNA Modulation of Alkyl Hydroperoxide Reductase Levels in Helicobacter pylori Correlates with Organic Peroxide Toxicity but Not Infectivity

Journal of Bacteriology ◽

10.1128/jb.00012-07 ◽

2007 ◽

Vol 189 (9) ◽

pp. 3359-3368 ◽

Cited By ~ 18

Author(s):

Matthew A. Croxen ◽

Peter B. Ernst ◽

Paul S. Hoffman

Keyword(s):

Helicobacter Pylori ◽

Rna Interference ◽

Gene Function ◽

Antisense Rna ◽

In Vitro Growth ◽

Protein Levels ◽

Alkyl Hydroperoxide ◽

Gastric Colonization ◽

H Pylori

ABSTRACT Much of the gene content of the human gastric pathogen Helicobacter pylori (∼1.7-Mb genome) is considered essential. This view is based on the completeness of metabolic pathways, infrequency of nutritional auxotrophies, and paucity of pathway redundancies typically found in bacteria with larger genomes. Thus, genetic analysis of gene function is often hampered by lethality. In the absence of controllable promoters, often used to titrate gene function, we investigated the feasibility of an antisense RNA interference strategy. To test the antisense approach, we targeted alkyl hydroperoxide reductase (AhpC), one of the most abundant proteins expressed by H. pylori and one whose function is essential for both in vitro growth and gastric colonization. Here, we show that antisense ahpC (as-ahpC) RNA expression from shuttle vector pDH37::as-ahpC achieved an ∼72% knockdown of AhpC protein levels, which correlated with increased susceptibilities to hydrogen peroxide, cumene, and tert-butyl hydroperoxides but not with growth efficiency. Compensatory increases in catalase levels were not observed in the knockdowns. Expression of single-copy antisense constructs (expressed under the urease promoter and containing an fd phage terminator) from the rdxA locus of mouse-colonizing strain X47 achieved a 32% knockdown of AhpC protein levels (relative to wild-type X47 levels), which correlated with increased susceptibility to organic peroxides but not with mouse colonization efficiency. Our studies indicate that high levels of AhpC are not required for in vitro growth or for primary gastric colonization. Perhaps AhpC, like catalase, assumes a greater role in combating exogenous peroxides arising from lifelong chronic inflammation. These studies also demonstrate the utility of antisense RNA interference in the evaluation of gene function in H. pylori.

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Role of the rdxA and frxA genes in oxygen-dependent metronidazole resistance of Helicobacter pylori

Journal of Medical Microbiology ◽

10.1099/jmm.0.45701-0 ◽

2004 ◽

Vol 53 (11) ◽

pp. 1123-1128 ◽

Cited By ~ 29

Author(s):

Monique M Gerrits ◽

Egbert-Jan van der Wouden ◽

Dorine A Bax ◽

Anton A van Zwet ◽

Arnoud HM van Vliet ◽

...

Keyword(s):

Helicobacter Pylori ◽

Cell Viability ◽

Protein Synthesis Inhibitor ◽

Low Oxygen ◽

Anaerobic Conditions ◽

Metronidazole Resistance ◽

Oxygen Conditions ◽

H Pylori

Almost 50 % of all Helicobacter pylori isolates are resistant to metronidazole, which reduces the efficacy of metronidazole-containing regimens, but does not make them completely ineffective. This discrepancy between in vitro metronidazole resistance and treatment outcome may partially be explained by changes in oxygen pressure in the gastric environment, as metronidazole-resistant (MtzR) H. pylori isolates become metronidazole-susceptible (MtzS) under low oxygen conditions in vitro. In H. pylori the rdxA and frxA genes encode reductases which are required for the activation of metronidazole, and inactivation of these genes results in metronidazole resistance. Here the role of inactivating mutations in these genes on the reversibility of metronidazole resistance under low oxygen conditions is established. Clinical H. pylori isolates containing mutations resulting in a truncated RdxA and/or FrxA protein were selected and incubated under anaerobic conditions, and the effect of these conditions on the MICs of metronidazole, amoxycillin, clarithromycin and tetracycline, and cell viability were determined. While anaerobiosis had no effect on amoxycillin, clarithromycin and tetracycline resistance, all isolates lost their metronidazole resistance when cultured under anaerobic conditions. This loss of metronidazole resistance also occurred in the presence of the protein synthesis inhibitor chloramphenicol. Thus, factor(s) that activate metronidazole under low oxygen tension are not specifically induced by low oxygen conditions, but are already present under microaerophilic conditions. As there were no significant differences in cell viability between the clinical isolates, it is likely that neither the rdxA nor the frxA gene participates in the reversibility of metronidazole resistance.

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Transcriptional profiling of Helicobacter pylori Fur- and iron-regulated gene expression

Microbiology ◽

10.1099/mic.0.27404-0 ◽

2005 ◽

Vol 151 (2) ◽

pp. 533-546 ◽

Cited By ~ 116

Author(s):

Florian D. Ernst ◽

Stefan Bereswill ◽

Barbara Waidner ◽

Jeroen Stoof ◽

Ulrike Mäder ◽

...

Keyword(s):

Helicobacter Pylori ◽

Iron Homeostasis ◽

Transcriptional Profiling ◽

Northern Hybridization ◽

Intracellular Iron ◽

Iron Storage ◽

Gastric Colonization ◽

Genes Encoding ◽

H Pylori ◽

Regulated Gene Expression

Intracellular iron homeostasis is a necessity for almost all living organisms, since both iron restriction and iron overload can result in cell death. The ferric uptake regulator protein, Fur, controls iron homeostasis in most Gram-negative bacteria. In the human gastric pathogen Helicobacter pylori, Fur is thought to have acquired extra functions to compensate for the relative paucity of regulatory genes. To identify H. pylori genes regulated by iron and Fur, we used DNA array-based transcriptional profiling with RNA isolated from H. pylori 26695 wild-type and fur mutant cells grown in iron-restricted and iron-replete conditions. Sixteen genes encoding proteins involved in metal metabolism, nitrogen metabolism, motility, cell wall synthesis and cofactor synthesis displayed iron-dependent Fur-repressed expression. Conversely, 16 genes encoding proteins involved in iron storage, respiration, energy metabolism, chemotaxis, and oxygen scavenging displayed iron-induced Fur-dependent expression. Several Fur-regulated genes have been previously shown to be essential for acid resistance or gastric colonization in animal models, such as those encoding the hydrogenase and superoxide dismutase enzymes. Overall, there was a partial overlap between the sets of genes regulated by Fur and those previously identified as growth-phase, iron or acid regulated. Regulatory patterns were confirmed for five selected genes using Northern hybridization. In conclusion, H. pylori Fur is a versatile regulator involved in many pathways essential for gastric colonization. These findings further delineate the central role of Fur in regulating the unique capacity of H. pylori to colonize the human stomach.

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The Role of E-cadherin in Helicobacter pylori-Related Gastric Diseases

Current Drug Metabolism ◽

10.2174/1389200219666180625113010 ◽

2019 ◽

Vol 20 (1) ◽

pp. 23-28

Author(s):

Yunzhan Zhang ◽

Danyan Li ◽

Yunkai Dai ◽

Ruliu Li ◽

Yong Gao ◽

...

Keyword(s):

Helicobacter Pylori ◽

Gastric Mucosa ◽

Precancerous Lesions ◽

Research Literature ◽

Gastric Diseases ◽

H Pylori ◽

The Relationship ◽

E Cadherin

Background: Helicobacter pylori (H. pylori)-related gastric diseases are a series of gastric mucosal disorders associated with H. pylori infection. Gastric cancer (GC) is widely believed to evolve from gastritis and gastric ulcer. As an important adhesion molecule of epithelial cells, E-cadherin plays a key role in the development of gastric diseases. In this review, we aim to seek the characteristic of E-cadherin expression at different stages of gastric diseases. Methods: We searched plenty of databases for research literature about E-cadherin expression in H. pylori-related gastric diseases, and reviewed the relationship of E-cadherin and H. pylori, and the role of E-cadherin at different stages of gastric diseases. Results: H. pylori was shown to decrease E-cadherin expression by various ways in vitro, while most of clinical studies have not found the relationship between H. pylori and E-cadherin expression. It is defined that poor outcome of GC is related to loss expression of E-cadherin, but it is still unclear when qualitative change of E-cadherin expression in gastric mucosa emerges. Conclusion: Expression level of E-cadherin in gastric cells may be a consequence of injury factors and body’s selfrepairing ability. More studies on E-cadherin expression in gastric mucosa with precancerous lesions need to be performed, which may be potential and useful for early detection, prevention and treatment of GC.

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Helicobacter pylori Stimulates Dendritic Cells To Induce Interleukin-17 Expression from CD4+ T Lymphocytes

Infection and Immunity ◽

10.1128/iai.00524-09 ◽

2009 ◽

Vol 78 (2) ◽

pp. 845-853 ◽

Cited By ~ 58

Author(s):

Wafa Khamri ◽

Marjorie M. Walker ◽

Peter Clark ◽

John C. Atherton ◽

Mark R. Thursz ◽

...

Keyword(s):

T Cells ◽

Helicobacter Pylori ◽

Dendritic Cells ◽

Virulence Factors ◽

Human Monocyte ◽

Interleukin 17 ◽

Th17 Response ◽

H Pylori

ABSTRACT Helicobacter pylori is a human gastroduodenal pathogen that leads to active chronic inflammation characterized by T-cell responses biased toward a Th1 phenotype. It has been accepted that H. pylori infection induces a Th17 response. At mucosal sites, dendritic cells (DCs) have the capacity to induce effector T cells. Here, we evaluate the role of DCs in the H. pylori-induced interleukin-17 (IL-17) response. Immunohistochemistry and immunofluorescence were performed on human gastric mucosal biopsy samples and showed that myeloid DCs in H. pylori-infected patients colocalized with IL-23- and that IL-17-producing lymphocytes were present in H. pylori-infected antral biopsy samples. In parallel, human monocyte-derived DCs stimulated in vitro with live H. pylori cells produced significant levels of IL-23 in the absence of IL-12 release. The subsequent incubation of H. pylori-infected DCs with autologous CD4+ T cells led to gamma interferon (IFN-γ) and IL-17 expression. The inhibition of IL-1 and, to a lesser extent, IL-23 inhibited IL-17 production by T cells. Finally, isogenic H. pylori mutant strains not expressing major virulence factors were less effective in inducing IL-1 and IL-23 release by DCs and IL-17 release by T cells than parental strains. Altogether, we can conclude that DCs are potent inducers of IL-23/IL-17 expression following H. pylori stimulation. IL-1/IL-23 as well as H. pylori virulence factors seem to play an important role in mediating this response.

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NF-κB Activation during Acute Helicobacter pylori Infection in Mice

Infection and Immunity ◽

10.1128/iai.01107-07 ◽

2007 ◽

Vol 76 (2) ◽

pp. 551-561 ◽

Cited By ~ 25

Author(s):

Richard L. Ferrero ◽

Patrick Avé ◽

Delphine Ndiaye ◽

Jean-Christophe Bambou ◽

Michel R. Huerre ◽

...

Keyword(s):

Helicobacter Pylori ◽

Transgenic Mice ◽

Nuclear Factor Κb ◽

Helicobacter Felis ◽

Cell Responses ◽

Mucosal Cells ◽

H Pylori ◽

First Time

ABSTRACT Nuclear factor κB (NF-κB) plays a key regulatory role in host cell responses to Helicobacter pylori infection in humans. Although mice are routinely used as a model to study H. pylori pathogenesis, the role of NF-κB in murine cell responses to helicobacters has not been studied in detail. We thus investigated the abilities of different Helicobacter isolates to induce NF-κB-dependent responses in murine gastric epithelial cells (GECs) and in transgenic mice harboring an NF-κB-responsive lacZ reporter gene. H. pylori and Helicobacter felis strains up-regulated the synthesis in mouse GECs of the NF-κB-dependent chemokines KC (CXCL1) and MIP-2 (CXCL2). These responses were cag pathogenicity island (cagPAI) independent and could be abolished by pretreatment with a pharmacological inhibitor of NF-κB. Consistent with the in vitro data, experimental Helicobacter infection of transgenic mice resulted in increased numbers of GECs with nuclear β-galactosidase activity, which is indicative of specific NF-κB activation. The numbers of β-galactosidase-positive cells in mice were significantly increased at day 1 postinoculation with wild-type H. pylori strains harboring or not harboring a functional cagPAI, compared to naive animals (P = 0.007 and P = 0.04, respectively). Strikingly, however, no differences were observed in the levels of gastric NF-κB activation at day 1 postinoculation with H. felis or at day 30 or 135 postinoculation with H. pylori. This work demonstrates for the first time the induction of NF-κB activation within gastric mucosal cells during acute H. pylori infection. Furthermore, the data suggest that helicobacters may be able to regulate NF-κB signaling during chronic infection.

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Proregenerative Activity of IL-33 in Gastric Tissue Cells Undergoing Helicobacter Pylori-Induced Apoptosis

International Journal of Molecular Sciences ◽

10.3390/ijms21051801 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1801

Author(s):

Weronika Gonciarz ◽

Agnieszka Krupa ◽

Magdalena Chmiela

Keyword(s):

Helicobacter Pylori ◽

Metabolic Activity ◽

Tissue Cell ◽

Host Immune System ◽

Gastric Tissue ◽

Antigenic Complex ◽

H Pylori ◽

Induced Apoptosis

Interleukin (IL)-33 is a proinflammatory mediator that alerts the host immune system to disorders in tissue homeostasis. Aim. To understand the role of IL-33 in modulating gastric tissue cell growth affected by Helicobacter pylori (H. pylori). Methods. IL-33 production in guinea pigs (Caviae porcellus) experimentally infected with H. pylori was evaluated by ELISA or immunohistochemical staining. The proregenerative activity of IL-33 was evaluated using gastric epithelial cells and fibroblasts that were naive or transfected with IL-33 siRNA exposed to H. pylori glycine acid extract antigenic complex (GE), as well as by measuring cell migration, proliferation, metabolic activity and apoptosis. Animals infected by H. pylori responded with increased production of IL-33. Also, cells treated in vitro with GE released more IL-33 than cells that were unstimulated. Silencing IL-33 in cells resulted in downregulation of metabolic activity, adhesion, migration and proliferation, especially after treatment with H. pylori GE, as well as upregulation of cells apoptosis associated with caspase 3 increase and Bcl-xL decrease, suggesting proregenerative activity of IL-33. Interestingly, upregulation of cell proliferation by IL-33 was Erk independent. Our results indicate that IL-33 may protect gastric tissue from loss of homeostasis caused by deleterious effects of H. pylori components and the inflammatory response developed during infection.

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Helicobacter pylori Pore-Forming Cytolysin Orthologue TlyA Possesses In Vitro Hemolytic Activity and Has a Role in Colonization of the Gastric Mucosa

Infection and Immunity ◽

10.1128/iai.69.3.1697-1703.2001 ◽

2001 ◽

Vol 69 (3) ◽

pp. 1697-1703 ◽

Cited By ~ 32

Author(s):

M. Celeste Martino ◽

Richard A. Stabler ◽

Zun W. Zhang ◽

Michael J. G. Farthing ◽

Brendan W. Wren ◽

...

Keyword(s):

Helicobacter Pylori ◽

Gastric Mucosa ◽

Hemolytic Activity ◽

Wild Type Strain ◽

Bacterial Species ◽

Adenocarcinoma Cells ◽

Human Gastric Adenocarcinoma ◽

H Pylori

ABSTRACT Hemolysins have been found to possess a variety of functions in bacteria, including a role in virulence. Helicobacter pylori demonstrates hemolytic activity when cultured on unlysed blood agar plates which is increased under iron-limiting conditions. However, the role of an H. pylori hemolysin in virulence is unclear. Scrutiny of the H. pylori 26695 genome sequence suggests the presence of at least two distinct hemolysins, HP1086 and HP1490, in this strain. Previous studies have shown that the in vitro hemolytic activity of H. pylori is reduced when it is coincubated with dextran 5000, suggesting the presence of a pore-forming cytolysin. HP1086 has homology to pore-forming cytolysins (TlyA) from other bacterial species, and the introduction of the clonedH. pylori tlyA gene into a nonhemolyticEscherichia coli strain conferred hemolytic activity. AnH. pylori tlyA defined mutant showed reduced in vitro hemolytic activity, which appears to be due to pore formation, as the hemolytic activity of the wild-type strain is reduced to the same level as the tlyA mutant by the addition of dextran 5000. The mutant also showed reduced adhesion to human gastric adenocarcinoma cells and failed to colonize the gastric mucosa of mice. These data clearly suggest a role in virulence for H. pyloriTlyA, contrary to the suggestion that hemolytic activity is an in vitro phenomenon for this pathogen.

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