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

Common variants in mendelian kidney disease genes and their association with renal function

Many common genetic variants identified by genome-wide association studies for complex traitsmap to genes previously linked to rare inherited Mendelian disorders. A systematic analysis of common single-nucleotide polymorphisms (SNPs) in genes responsible for Mendelian diseases with kidney phenotypes has not been performed. We thus developed a comprehensive database of genes for Mendelian kidney conditions and evaluated the association between common genetic variants within these genes and kidney function in the general population. Using the Online Mendelian Inheritance in Man database, we identified 731 unique disease entries related to specific renal search terms and confirmed a kidney phenotype in 218 of these entries, corresponding to mutations in 258 genes. We interrogated common SNPs (minor allele frequency >5%) within these genes for association with the estimated GFR in 74,354 European-ancestry participants from the CKDGen Consortium. However, the top four candidate SNPs (rs6433115 at LRP2, rs1050700 at TSC1, rs249942 at PALB2, and rs9827843 at ROBO2) did not achieve significance in a stage 2meta-analysis performed in 56,246 additional independent individuals, indicating that these common SNPs are not associated with estimated GFR. The effect of less common or rare variants in these genes on kidney function in the general population and disease-specific cohorts requires further research. Copyrigh