Transcriptional Regulation of the Nickel and Iron Metabolism in Helicobacter pylori

Up to 50 % of the world's population is infected with Helicobacter pylori. Colonization of the mucus layer of the human stomach by H. pylori, is lifelong unless treated with antibiotics (26). H. pylori, which is a neutralophilic bacterium, survives in the mucus layer of the human stomach with the help of the enzyme urease. Urease is an enzyme that converts urea into ammonium and carbon dioxide, thereby keeping the intracellular and periplasmic pH at neutral. It is estimated that up to 10% of the whole cell protein consists of this nickelcofactored enzyme (19). The nickel necessary to activate the urease is thought to come from foodsources, such as nuts, tea and cereals, which are rich in nickel (1). Metal ions like nickel or iron can be dangerous for bacteria, as they can react with oxygen in order to create reactive oxygen species that in turn can destroy macromolecules like nucleic acids, proteins and cell wall components (27). Therefore, the bacterial metal metabolism has to be tightly regulated. In H. pylori, regulatory proteins are scarce. Only two metal-regulatory proteins are known, the ferric uptake regulator Fur (4), and the nickel responsive regulator NikR (31). Fur is a regulatory protein that can sense and bind intracellular ferrous ions, and subsequently displays iron-dependent binding to conserved promoter sequences (Fur boxes) of its target genes (17). The classical regulation is repression of iron uptake genes in iron-replete conditions (15, 17, 33). Unlike all other Fur homologs known so far, H. pylori Fur can also bind to Fur-boxes in an iron free form (apo-Fur), as was shown for pfr (16) and sodB (Chapter 3). The second metal-dependent regulatory protein is NikR, the nickel responsive regulator, which belongs to the family of Ribbon-Helix-Helix regulatory proteins (9). NikR is directly involved in the regulation of acid resistance via urease and nickel-uptake (Chapter 4), and was previously demonstrated to mediate regulation of the ferric uptake regulator Fur (7, 12, 29). The aim of this thesis was to gain further insight into the transcriptional regulation the ferric uptake regulator Fur and the nickel responsive regulator NikR

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

Comparison of genotyping using pooled DNA samples (allelotyping) and individual genotyping using the affymetrix genome-wide human SNP array 6.0

Background: Genome-wide association studies (GWAS) using array-based genotyping technology are widely used to identify genetic loci associated with complex diseases or other phenotypes. The costs of GWAS projects based on individual genotyping are still comparatively high and increase with the size of study populations. Genotyping using pooled DNA samples, as also being referred as to allelotyping approach, offers an alternative at affordable costs. In the

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