Barbara Waidner

The Helicobacter pylori Homologue of the Ferric Uptake Regulator Is Involved in Acid Resistance

ABSTRACT The only known niche of the human pathogen Helicobacter pylori is the gastric mucosa, where large fluctuations of pH occur, indicating that the bacterial response and resistance to acid are important for successful colonization. One of the few regulatory proteins in the H. pylori genome is a homologue of the ferric uptake regulator (Fur). In most bacteria, the main function of Fur is the regulation of iron homeostasis. However, in Salmonella enterica serovar Typhimurium, Fur also plays an important role in acid resistance. In this study, we determined the role of the H. pylori Fur homologue in acid resistance. Isogenic fur mutants were generated in three H. pylori strains (1061, 26695, and NCTC 11638). At pH 7 there was no difference between the growth rates of mutants and the parent strains. Under acidic conditions, growth of the fur mutants was severely impaired. No differences were observed between the survival of the fur mutant and parent strain 1061 after acid shock. Addition of extra iron or removal of iron from the growth medium did not improve the growth of the fur mutant at acidic pH. This indicates that the phenotype of the fur mutant at low pH was not due to increased iron sensitivity. Transcription of fur was repressed in response to low pH. From this we conclude that Fur is involved in the growth at acidic pH of H. pylori; as such, it is the first regulatory protein implicated in the acid resistance of this important human pathogen.

Regulation of Ferritin-Mediated Cytoplasmic Iron Storage by the Ferric Uptake Regulator Homolog (Fur) of Helicobacter pylori

Homologs of the ferric uptake regulator Fur and the iron storage protein ferritin play a central role in maintaining iron homeostasis in bacteria. The gastric pathogen Helicobacter pylori contains an iron-induced prokaryotic ferritin (Pfr) which has been shown to be involved in protection against metal toxicity and a Fur homolog which has not been functionally characterized in H. pylori. Analysis of an isogenic fur-negative mutant revealed that H. pylori Fur is required for metal-dependent regulation of ferritin. Iron starvation, as well as medium supplementation with nickel, zinc, copper, and manganese at nontoxic concentrations, repressed synthesis of ferritin in the wild-type strain but not in the H. pylori fur mutant. Fur-mediated regulation of ferritin synthesis occurs at the mRNA level. With respect to the regulation of ferritin expression, Fur behaves like a global metal-dependent repressor which is activated under iron-restricted conditions but also responds to different metals. Downregulation of ferritin expression by Fur might secure the availability of free iron in the cytoplasm, especially if iron is scarce or titrated out by other metals.

Nickel-responsive induction of urease expression in Helicobacter pylori is mediated at the transcriptional level

ABSTRACT The nickel-containing enzyme urease is an essential colonization factor of the gastric pathogen Helicobacter pylori, as it allows the bacterium to survive the acidic conditions in the gastric mucosa. Although urease can represents up to 10% of the total protein content of H. pylori, expression of urease genes is thought to be constitutive. Here it is demonstrated that H. pyloriregulates the expression and activity of its urease enzyme as a function of the availability of the cofactor nickel. Supplementation of brucella growth medium with 1 or 100 μM NiCl2 resulted in up to 3.5-fold-increased expression of the urease subunit proteins UreA and UreB and up to 12-fold-increased urease enzyme activity. The induction was specific for nickel, since the addition of cadmium, cobalt, copper, iron, manganese, or zinc did not affect the expression of urease. Both Northern hybridization studies and a transcriptionalureA::lacZ fusion demonstrated that the observed nickel-responsive regulation of urease is mediated at the transcriptional level. Mutation of the HP1027 gene, encoding the ferric uptake regulator (Fur), did not affect the expression of urease in unsupplemented medium but reduced the nickel induction of urease expression to only twofold. This indicates that Fur is involved in the modulation of urease expression in response to nickel. These data demonstrate nickel-responsive regulation of H. pyloriurease, a phenomenon likely to be of importance during the colonization and persistence of H. pylori in the gastric mucosa.

Iron-Responsive Regulation of the Helicobacter pylori Iron-Cofactored Superoxide Dismutase SodB Is Mediated by Fur

Maintaining iron homeostasis is a necessity for all living organisms, as free iron augments the generation of reactive oxygen species like superoxide anions, at the risk of subsequent lethal cellular damage. The iron-responsive regulator Fur controls iron metabolism in many bacteria, including the important human pathogen Helicobacter pylori, and thus is directly or indirectly involved in regulation of oxidative stress defense. Here we demonstrate that Fur is a direct regulator of the H. pylori iron-cofactored superoxide dismutase SodB, which is essential for the defense against toxic superoxide radicals. Transcription of the sodB gene was iron induced in H. pylori wild-type strain 26695, resulting in expression of the SodB protein in iron-replete conditions but an absence of expression in iron-restricted conditions. Mutation of the fur gene resulted in constitutive, iron-independent expression of SodB. Recombinant H. pylori Fur protein bound with low affinity to the sodB promoter region, but addition of the iron substitute Mn2+ abolished binding. The operator sequence of the iron-free form of Fur, as identified by DNase I footprinting, was located directly upstream of the sodB gene at positions -5 to -47 from the transcription start site. The direct role of Fur in regulation of the H. pylori sodB gene contrasts with the small-RNA-mediated sodB regulation observed in Escherichia coli. In conclusion, H. pylori Fur is a versatile regulator involved in many pathways essential for gastric colonization, including superoxide stress defense.

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.

The Helicobacter pylori CrdRS Two-Component Regulation System (HP1364/HP1365) Is Required for Copper-Mediated Induction of the Copper Resistance Determinant CrdA

Here we describe that the Helicobacter pylori sensor kinase produced by HP1364 and the response regulator produced by HP1365 and designated CrdS and CrdR, respectively, are both required for transcriptional induction of the H. pylori copper resistance determinant CrdA by copper ions. CrdRS-deficient mutants lacked copper induction of crdA expression and were copper sensitive. A direct role of CrdR in transcriptional regulation of crdA was confirmed by in vitro binding of CrdR to the crdA upstream region. A 21-nucleotide sequence located near the crdA promoter was shown to be required for CrdR binding.

A Novel System of Cytoskeletal Elements in the Human Pathogen Helicobacter pylori

Pathogenicity of the human pathogen Helicobacter pylori relies upon its capacity to adapt to a hostile environment and to escape from the host response. Therefore, cell shape, motility, and pH homeostasis of these bacteria are specifically adapted to the gastric mucus. We have found that the helical shape of H. pylori depends on coiled coil rich proteins (Ccrp), which form extended filamentous structures in vitro and in vivo, and are differentially required for the maintenance of cell morphology. We have developed an in vivo localization system for this pathogen. Consistent with a cytoskeleton-like structure, Ccrp proteins localized in a regular punctuate and static pattern within H. pylori cells. Ccrp genes show a high degree of sequence variation, which could be the reason for the morphological diversity between H. pylori strains. In contrast to other bacteria, the actin-like MreB protein is dispensable for viability in H. pylori, and does not affect cell shape, but cell length and chromosome segregation. In addition, mreB mutant cells displayed significantly reduced urease activity, and thus compromise a major pathogenicity factor of H. pylori. Our findings reveal that Ccrp proteins, but not MreB, affect cell morphology, while both cytoskeletal components affect the development of pathogenicity factors and/or cell cycle progression.

Magnesium Uptake by CorA Is Essential for Viability of the Gastric Pathogen Helicobacter pylori

ABSTRACT We show here that Mg2+ acquisition by CorA is essential for Helicobacter pylori in vitro, as corA mutants did not grow in media without Mg2+ supplementation. Complementation analysis performed with an Escherichia coli corA mutant revealed that H. pylori CorA transports nickel and cobalt in addition to Mg2+. However, Mg2+ is the dominant CorA substrate, as the corA mutation affected neither cobalt and nickel resistance nor nickel induction of urease in H. pylori. The drastic Mg2+ requirement (20 mM) of H. pylori corA mutants indicates that CorA plays a key role in the adaptation to the low-Mg2+ conditions predominant in the gastric environment.

An Interferon-γ-binding Protein of Novel Structure Encoded by the Fowlpox Virus

Poxviruses have evolved various strategies to counteract the host immune response, one of which is based on the expression of soluble cytokine receptors. Using various biological assays, we detected a chicken interferon-γ (chIFN-γ)-neutralizing activity in supernatants of fowlpox virus (FPV)-infected cells that could be destroyed by trypsin treatment. Secreted viral proteins were purified by affinity chromatography using matrix-immobilized chIFN-γ, followed by two-dimensional gel electrophoresis. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis indicated that the viral IFN-γ-binding protein in question was encoded by the FPV gene 016. The chicken IFN-γ binding and neutralizing activity of the recombinant FPV016 protein was confirmed using supernatants of cells infected with a recombinant vaccinia virus that lacked its own IFN-γ-binding protein but instead expressed theFPV016 gene. The FPV016 gene product also neutralized the activity of duck and human IFN-γ but failed to neutralize the activity of mouse and rat IFN-γ. Unlike previously known cellular and poxviral IFN-γ receptors, which all contain fibronectin type III domains, the IFN-γ-binding protein of FPV contains an immunoglobulin domain. Remarkably, it exhibits no significant homology to any known viral or cellular protein. Because IFN-γ receptors of birds have not yet been characterized at the molecular level, the possibility remains that FPV016represents a hijacked chicken gene and that avian and mammalian IFN-γ receptors have fundamentally different primary structures.

Metallibacterium scheffleri gen. nov., sp. nov., an alkalinizing gammaproteobacterium isolated from an acidic biofilm

A Gram-stain-negative, non-motile, facultatively anaerobic, acid-tolerant rod, designated strain DKE6(T), was isolated from an acidic biofilm (pH 2.5) harvested in the pyrite mine Drei Kronen und Ehrt in Germany. The isolate grew optimally at pH 5.5, between 25 and 30 °C and only with casein as the carbon and energy source; although a variety of sugars were tested as growth substrates, none supported growth of the isolate. During casein consumption, strain DKE6(T) produced ammonium, which led to an alkalinization of the medium. This is a possible strategy to raise the pH in the direct vicinity of the cell and hence modulate the pH towards the growth optimum. The predominant fatty acids (>5 %) were iso-C11 : 0 3-OH, iso-C15 : 0, iso-C17 : 0 and iso-C17 : 1ω9c. The DNA G+C content was 66.6 %. Strain DKE6(T) was not able to oxidize iron or thiosulfate. Iron reduction was detected. The isolate showed 93.3 % 16S rRNA gene sequence similarity to the most closely related cultivable strain, Dokdonella koreensis DS-123(T), but <93.2 % sequence similarity with other type strains of closely related type species of the Gammaproteobacteria. On the basis of physiological and biochemical data, the isolate is considered to represent a novel species of a new genus in the class Gammaproteobacteria, for which we propose the name Metallibacterium scheffleri gen. nov., sp. nov. The type strain of the type species is DKE6(T) ( = DSM 24874(T) = JCM 17596(T)).