Nickel Homeostasis in Helicobacter Species

Gastric Helicobacter species are adapted to colonize the acidic environment of the stomach. Colonization with H pylori is life long if untreated, and can lead to gastritis, peptic ulcer disease and eventually to gastric cancer. Although H pylori is sensitive to many antibiotics in vitro, only a limited number of antibiotics can be used in vivo while increasing resistance against these therapeutics significantly impairs the treatment of H pylori infection. Metals play an essential role in the metabolism of all living organisms, including gastric Helicobacter species, but can also be deleterious when metal availability is either too low or too high. Therefore cells need to maintain homeostasis of intracellular metal concentrations to allow survival and growth. A better understanding of metal homeostasis in gastric Helicobacter species may allow for the knowledge-led development of therapeutics which are based on disturbing the balance of the intracellular metal concentrations to either toxicity or restriction. The focus of this PhD-thesis is on nickel metabolism, since this metal is the cofactor of the urease enzyme and hydrogenase enzyme, both essential for colonization of gastric Helicobacter species. The high expression level of the urease enzyme mediates acid resistance in the presence of urea, but also necessitates the import of relatively high concentrations of nickel. Although transcriptional regulation by the nickel-dependent regulator NikR has been studied, relative little is known about which proteins are involved in actual transport of nickel. The genome era has opened the possibility of functional genomics investigations, using the information from the genomes of different Helicobacter species. Many of the genes of Helicobacter species do not yet have a predicted function, or have been assigned a putative function only based on homology with genes from other bacterial species. Comparison of the genomic content of different Helicobacter species and transcriptional and functional characterization of the genes putatively involved in nickel homeostasis, as presented in this thesis, will provide more insight in how these bacteria are able to acquire sufficient concentrations of nickel

Cooling curves for neutron stars with hadronic matter and quark matter

The thermal evolution of isothermal neutron stars is studied with matter both in the hadronic phase as well as in the mixed phase of hadronic matter and strange quark matter. In our models, the dominant early-stage cooling process is neutrino emission via the direct Urca process. As a consequence, the cooling curves fall too fast compared to observations. However, when superfluidity is included, the cooling of the neutron stars is significantly slowed down. Furthermore, we find that the cooling curves are not very sensitive to the precise details of the mixing between the hadronic phase and the quark phase and also of the pairing that leads to superfluidity.Comment: 19 pages, 25 figure

NikR mediates nickel-responsive transcriptional induction of urease expression in Helicobacter pylori

The important human pathogen Helicobacter pylori requires the abundant expression and activity of its urease enzyme for colonization of the gastric mucosa. The transcription, expression, and activity of H. pylori urease were previously demonstrated to be induced by nickel supplementation of growth media. Here it is demonstrated that the HP1338 protein, an ortholog of the Escherichia coli nickel regulatory protein NikR, mediates nickel-responsive induction of urease expression in H. pylori. Mutation of the HP1338 gene (nikR) of H. pylori strain 26695 resulted in significant growth inhibition of the nikR mutant in the presence of supplementation with NiCl(2) at > or =100 microM, whereas the wild-type strain tolerated more than 10-fold-higher levels of NiCl(2). Mutation of nikR did not affect urease subunit expression or urease enzyme activity in unsupplemented growth media. However, the nickel-induced increase in urease subunit expression and urease enzyme activity observed in wild-type H. pylori was absent in the H. pylori nikR mutant. A similar lack of nickel responsiveness was observed upon removal of a 19-bp palindromic sequence in the ureA promoter, as demonstrated by using a genomic ureA::lacZ reporter gene fusion. In conclusion, the H. pylori NikR protein and a 19-bp operator sequence in the ureA promoter are both essential for nickel-responsive induction of urease expression in H. pylori

Transcriptional profiling of Helicobacter pylori Fur- and iron-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

The role of the ferric uptake regulator (Fur) in regulation of Helicobacter pylori iron uptake

Background. Availability of the essential nutrient iron is thought to vary greatly in the gastric mucosa, and thus the human gastric pathogen Helicobacter pylori requires regulatory responses to these environmental changes. Bacterial iron-responsive regulation is often mediated by Ferric Uptake Regulator (Fur) homologs, and in this study we have determined the role of H. pylori Fur in regulation of H. pylori iron uptake. Methods. Wild-type H. pylori and fur mutant derivatives were compared after growth in ironrestricted and iron-replete conditions. Iron-uptake was measured using 55Fe-labeled iron, whereas gene expression was mon

Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis

The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C173) of Gal-3 or lysine (K166) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial–host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization

Differential regulation of amidase- and formamidase-mediated ammonia production by the Helicobacter pylori fur repressor.

The production of high levels of ammonia allows the human gastric pathogen Helicobacter pylori to survive the acidic conditions in the human stomach. H. pylori produces ammonia through urease-mediated degradation of urea, but it is also able to convert a range of amide substrates into ammonia via its AmiE amidase and AmiF formamidase enzymes. Here data are provided that demonstrate that the iron-responsive regulatory protein Fur directly and indirectly regulates the activity of the two H. pylori amidases. In contrast to other amidase-positive bacteria, amidase and formamidase enzyme activities were not induced by medium supplementation with their respective substrates, acrylamide and formamide. AmiE protein expression and amidase enzyme activity were iron-repressed in H. pylori 26695 but constitutive in the isogenic fur mutant. This regulation was mediated at the transcriptional level via the binding of Fur to the amiE promoter region. In contrast, formamidase enzyme activity was not iron-repressed but was significantly higher in the fur mutant. This effect was not mediated at the transcriptional level, and Fur did not bind to the amiF promoter region. These roles of Fur in regulation of the H. pylori amidases suggest that the H. pylori Fur regulator may have acquired extra functions to compensate for the absence of other regulatory systems

Metal-responsive gene regulation and metal transport in Helicobacter species

Helicobacter species are among the most successful colonizers of the mammalian gastrointestinal and hepatobiliary tract. Colonization is usually lifelong, indicating that Helicobacter species have evolved intricate mechanisms of dealing with stresses encountered during colonization of host tissues, like restriction of essential metal ions. The recent availability of genome sequences of the human gastric pathogen Helicobacter pylori, the murine enterohepatic pathogen Helicobacter hepaticus and the unannotated genome sequence of the ferret gastric pathogen Helicobacter mustelae has allowed for comparitive genome analyses. In this review we present such analyses for metal transporters, metal-storage and metal-responsive regulators in these three Helicobacter species, and discuss possible contributions of the differences in metal metabolism in adaptation to the gastric or enterohepatic niches occupied by Helicobacter species

An ABC Transporter and a TonB Ortholog Contribute to Helicobacter mustelae Nickel and Cobalt Acquisition▿ †

The genomes of Helicobacter species colonizing the mammalian gastric mucosa (like Helicobacter pylori) contain a large number of genes annotated as iron acquisition genes but only few nickel acquisition genes, which contrasts with the central position of nickel in the urease-mediated acid resistance of these gastric pathogens. In this study we have investigated the predicted iron and nickel acquisition systems of the ferret pathogen Helicobacter mustelae. The expression of the outer membrane protein-encoding frpB2 gene was iron and Fur repressed, whereas the expression of the ABC transporter genes fecD and ceuE was iron and Fur independent. The inactivation of the two tonB genes showed that TonB1 is required for heme utilization, whereas the absence of TonB2 only marginally affected iron-dependent growth but led to reduced cellular nickel content and urease activity. The inactivation of the fecD and ceuE ABC transporter genes did not affect iron levels but resulted in significantly reduced urease activity and cellular nickel content. Surprisingly, the inactivation of the nixA nickel transporter gene affected cellular nickel content and urease activity only when combined with the inactivation of other nickel acquisition genes, like fecD or ceuE. The FecDE ABC transporter is not specific for nickel, since an fecD mutant also showed reduced cellular cobalt levels and increased cobalt resistance. We conclude that the H. mustelae fecDE and ceuE genes encode an ABC transporter involved in nickel and cobalt acquisition, which works independently of the nickel transporter NixA, while TonB2 is required primarily for nickel acquisition, with TonB1 being required for heme utilization