In vitro evaluation of the role of antibodies against Helicobacter pylori in inhibiting adherence of the organism to gastric cells.

ABSTRACT The role of the periplasmic α-carbonic anhydrase (α-CA) (HP1186) in acid acclimation of Helicobacter pylori was investigated. Urease and urea influx through UreI have been shown to be essential for gastric colonization and for acid survival in vitro. Intrabacterial urease generation of NH3 has a major role in regulation of periplasmic pH and inner membrane potential under acidic conditions, allowing adequate bioenergetics for survival and growth. Since α-CA catalyzes the conversion of CO2 to HCO3 −, the role of CO2 in periplasmic buffering was studied using an α-CA deletion mutant and the CA inhibitor acetazolamide. Western analysis confirmed that α-CA was bound to the inner membrane. Immunoblots and PCR confirmed the absence of the enzyme and the gene in the α-CA knockout. In the mutant or in the presence of acetazolamide, there was an ∼3 log10 decrease in acid survival. In acid, absence of α-CA activity decreased membrane integrity, as observed using membrane-permeant and -impermeant fluorescent DNA dyes. The increase in membrane potential and cytoplasmic buffering following urea addition to wild-type organisms in acid was absent in the α-CA knockout mutant and in the presence of acetazolamide, although UreI and urease remained fully functional. At low pH, the elevation of cytoplasmic and periplasmic pH with urea was abolished in the absence of α-CA activity. Hence, buffering of the periplasm to a pH consistent with viability depends not only on NH3 efflux from the cytoplasm but also on the conversion of CO2, produced by urease, to HCO3 − by the periplasmic α-CA.

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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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The role of cytosolic phospholipase A2 in gastric damage induced by helicobacter pylori infection: in vivo and in vitro studies

Gastroenterology ◽

10.1016/s0016-5085(00)85063-5 ◽

2000 ◽

Vol 118 (4) ◽

pp. A732-A733

Author(s):

Gerardo Nardone ◽

Eileen Holicky ◽

Jim R. Uhl ◽

Vittorio Colantuoni ◽

Lina Sabatino ◽

...

Keyword(s):

Helicobacter Pylori ◽

Phospholipase A2 ◽

Cytosolic Phospholipase A2 ◽

In Vitro Studies ◽

Helicobacter Pylori Infection ◽

Gastric Damage ◽

Cytosolic Phospholipase

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

Infection and Immunity ◽

10.1128/iai.70.7.3923-3929.2002 ◽

2002 ◽

Vol 70 (7) ◽

pp. 3923-3929 ◽

Cited By ~ 80

Author(s):

Barbara Waidner ◽

Stefan Greiner ◽

Stefan Odenbreit ◽

Holger Kavermann ◽

Jyoti Velayudhan ◽

...

Keyword(s):

Helicobacter Pylori ◽

Iron Metabolism ◽

Essential Element ◽

Iron Starvation ◽

Iron Storage ◽

Gastric Colonization ◽

H Pylori ◽

Iron Pool

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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Role of screening agar plates for in vitro susceptibility testing of Helicobacter pylori in a routine laboratory setting

Journal of Clinical Pathology ◽

10.1136/jcp.54.5.408 ◽

2001 ◽

Vol 54 (5) ◽

pp. 408-411 ◽

Cited By ~ 6

Author(s):

V J Warburton-Timms

Keyword(s):

Helicobacter Pylori ◽

Susceptibility Testing ◽

Routine Laboratory ◽

Laboratory Setting ◽

In Vitro Susceptibility ◽

In Vitro Susceptibility Testing

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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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