IL-10 deficiency reduces Helicobacter pylori gastric colonization in C57BL/6 mice

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.

Download Full-text

Systematic review: are probiotics useful in controlling gastric colonization by Helicobacter pylori?

Alimentary Pharmacology & Therapeutics ◽

10.1111/j.1365-2036.2006.02868.x ◽

2006 ◽

Vol 23 (8) ◽

pp. 1077-1086 ◽

Cited By ~ 143

Author(s):

M. GOTTELAND ◽

O. BRUNSER ◽

S. CRUCHET

Keyword(s):

Systematic Review ◽

Helicobacter Pylori ◽

Gastric Colonization

Download Full-text

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.

Download Full-text

Channel-mediated potassium uptake in Helicobacter pylori is essential for gastric colonization

The EMBO Journal ◽

10.1038/sj.emboj.7601471 ◽

2006 ◽

Vol 26 (1) ◽

pp. 232-241 ◽

Cited By ~ 21

Author(s):

Kerstin Stingl ◽

Sonja Brandt ◽

Eva-Maria Uhlemann ◽

Roland Schmid ◽

Karlheinz Altendorf ◽

...

Keyword(s):

Helicobacter Pylori ◽

Potassium Uptake ◽

Gastric Colonization

Download Full-text

Identification and Characterization of Helicobacter pylori Genes Essential for Gastric Colonization

Journal of Experimental Medicine ◽

10.1084/jem.20021531 ◽

2003 ◽

Vol 197 (7) ◽

pp. 813-822 ◽

Cited By ~ 185

Author(s):

Holger Kavermann ◽

Brendan P. Burns ◽

Katrin Angermüller ◽

Stefan Odenbreit ◽

Wolfgang Fischer ◽

...

Keyword(s):

Helicobacter Pylori ◽

Bacterial Infections ◽

Meriones Unguiculatus ◽

Type Iv ◽

Gastric Colonization ◽

Colonization Factors ◽

Essential Function ◽

H Pylori ◽

Transformation Competence

Helicobacter pylori causes one of the most common, chronic bacterial infections and is a primary cause of severe gastric disorders. To unravel the bacterial factors necessary for the process of gastric colonization and pathogenesis, signature tagged mutagenesis (STM) was adapted to H. pylori. The Mongolian gerbil (Meriones unguiculatus) was used as model system to screen a set of 960 STM mutants. This resulted in 47 H. pylori genes, assigned to 9 different functional categories, representing a set of biological functions absolutely essential for gastric colonization, as verified and quantified for many mutants by competition experiments. Identification of previously known colonization factors, such as the urease and motility functions validated this method, but also novel and several hypothetical genes were found. Interestingly, a secreted collagenase, encoded by hp0169, could be identified and functionally verified as a new essential virulence factor for H. pylori stomach colonization. Furthermore, comB4, encoding a putative ATPase being part of a DNA transformation-associated type IV transport system of H. pylori was found to be absolutely essential for colonization, but natural transformation competence was apparently not the essential function. Thus, this first systematic STM application identified a set of previously unknown H. pylori colonization factors and may help to potentiate the development of novel therapies against gastric Helicobacter infections.

Download Full-text

The Novel Helicobacter pylori CznABC Metal Efflux Pump Is Required for Cadmium, Zinc, and Nickel Resistance,Urease Modulation, and Gastric Colonization

Infection and Immunity ◽

10.1128/iai.02025-05 ◽

2006 ◽

Vol 74 (7) ◽

pp. 3845-3852 ◽

Cited By ~ 86

Author(s):

Frank Nils Stähler ◽

Stefan Odenbreit ◽

Rainer Haas ◽

Julia Wilrich ◽

Arnoud H. M. Van Vliet ◽

...

Keyword(s):

Helicobacter Pylori ◽

Metal Ion ◽

Insertional Mutagenesis ◽

Urease Activity ◽

Ion Homeostasis ◽

Metal Ion Homeostasis ◽

Gastric Colonization ◽

Nickel Homeostasis ◽

H Pylori ◽

Cadmium Zinc

ABSTRACT Maintaining metal homeostasis is crucial for the adaptation of Helicobacter pylori to the gastric environment. Iron, copper, and nickel homeostasis has recently been demonstrated to be required for the establishment of H. pylori infection in animal models. Here we demonstrate that the HP0969-0971 gene cluster encoding the Czc-type metal export pump homologs HP0969, HP0970, and the H. pylori-specific protein HP0971 forms part of a novel H. pylori metal resistance determinant, which is required for gastric colonization and for the modulation of urease activity. Insertional mutagenesis of the HP0971, HP0970, or HP0969 genes in H. pylori reference strain 26695 resulted in increased sensitivity to cadmium, zinc, and nickel (czn), suggesting that the encoded proteins constitute a metal-specific export pump. Accordingly, the genes were designated cznC (HP0971), cznB (HP0970), and cznA (HP0969). The CznC and CznA proteins play a predominant role in nickel homeostasis, since only the cznC and cznA mutants but not the cznB mutant displayed an 8- to 10-fold increase in urease activity. Nickel-specific affinity chromatography demonstrated that recombinant versions of CznC and CznB can bind to nickel and that the purified CznB protein interacted with cadmium and zinc, since both metals competitively inhibited nickel binding. Finally, single cznA, cznB, and cznC mutants did not colonize the stomach in a Mongolian gerbil-based animal model. This demonstrates that the metal export functions of H. pylori cznABC are essential for gastric colonization and underlines the extraordinary importance of metal ion homeostasis for the survival of H. pylori in the gastric environment.

Download Full-text

Helicobacter pylori detected in pharyngeal and laryngeal pathologies in patients with proven gastric colonization

Head & Neck ◽

10.1002/hed.23492 ◽

2013 ◽

Vol 36 (11) ◽

pp. 1562-1566 ◽

Cited By ~ 5

Author(s):

Jonas Fellmann ◽

Jan U. Weisert ◽

Alex Soltermann ◽

Grégoire Morand ◽

Laura Morra ◽

...

Keyword(s):

Helicobacter Pylori ◽

Gastric Colonization

Download Full-text

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.

Download Full-text