Helicobacter pyori is a spiral bacterium that colonizes the gastric mucosa of humans, leading to a variety of gastric diseases that include peptic ulcers, chronic gastritis, mucosal-associated lymphomas, and adenocarcinomas of the lower stomach and duodenum. Two nickel-containing enzymes, urease and hydrogenase, are important for the bacterium's mucosal-colonizing abilities. The goal proposed here is to understand the nickel sequestering, storage, metabolizing and metalloregulatory steps involved in the synthesis/maturation of the two nickel-containing enzymes. From studies with other organisms, the sequence-identified accessory proteins (encoded by ure and hyp genes) would be expected to form nickel sequestering and energy utilizing (GTP hydrolyzing) complexes needed for maturation of the two Ni-enzymes. In the presence of nickel, these complexes facilitate mobilization of the metal into the final sink, urease or hydrogenase. From our knowledge of purified nickel binding and GTPase accessory proteins and their interactions, a sequential Ni-mobilizing transfer pathway will be proposed. The hypothesized Ni-transfer steps will be tested by mixing together selected pure accessory proteins. The roles of some specific nickel-binding proteins (predicted from the genome sequence) in nickel sequestering, homeostasis, and regulation will be addressed via targeted mutagenesis. The specific approaches to understand nickel metabolism, homeostasis, and regulation, in H. pylori will include purification of complexes that deliver nickel to the Ni-enzymes, proteomic analysis to identify proteins, and genomic analysis of transcripts that are regulated in response to nickel supplementation. Finally, gene directed mutants in proteins that are anticipated to affect the nickel nutritional status of the bacterium (i.e. nickel homeostasis) will be characterized to understand the roles of three specific (but poorly studied) nickel binding proteins. The role of a DNA binding and nickel-sensing protein (NikR) is of particular interest as it may play a global regulatory role in metal homeostasis, in turn affecting many processes of cell metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK062852-02
Application #
6872172
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Hamilton, Frank A
Project Start
2004-03-15
Project End
2007-02-28
Budget Start
2005-03-01
Budget End
2006-02-28
Support Year
2
Fiscal Year
2005
Total Cost
$257,600
Indirect Cost
Name
University of Georgia
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
Benoit, St├ęphane L; Seshadri, Susmitha; Lamichhane-Khadka, Reena et al. (2013) Helicobacter hepaticus NikR controls urease and hydrogenase activities via the NikABDE and HH0418 putative nickel import proteins. Microbiology 159:136-46
Benoit, Stephane L; Miller, Erica F; Maier, Robert J (2013) Helicobacter pylori stores nickel to aid its host colonization. Infect Immun 81:580-4
Benoit, St├ęphane L; McMurry, Jonathan L; Hill, Stephanie A et al. (2012) Helicobacter pylori hydrogenase accessory protein HypA and urease accessory protein UreG compete with each other for UreE recognition. Biochim Biophys Acta 1820:1519-25
Hong, Yang; Wang, Ge; Maier, Robert J (2008) The NADPH quinone reductase MdaB confers oxidative stress resistance to Helicobacter hepaticus. Microb Pathog 44:169-74
Benoit, Stephane L; Mehta, Nalini; Weinberg, Michael V et al. (2007) Interaction between the Helicobacter pylori accessory proteins HypA and UreE is needed for urease maturation. Microbiology 153:1474-82
Benoit, Stephane L; Zbell, Andrea L; Maier, Robert J (2007) Nickel enzyme maturation in Helicobacter hepaticus: roles of accessory proteins in hydrogenase and urease activities. Microbiology 153:3748-56
Maier, Robert J; Benoit, Stephane L; Seshadri, Susmitha (2007) Nickel-binding and accessory proteins facilitating Ni-enzyme maturation in Helicobacter pylori. Biometals 20:655-64
Smith, Todd G; Lim, Jae-Min; Weinberg, Michael V et al. (2007) Direct analysis of the extracellular proteome from two strains of Helicobacter pylori. Proteomics 7:2240-5
Hong, Yang; Wang, Ge; Maier, Robert J (2007) A Helicobacter hepaticus catalase mutant is hypersensitive to oxidative stress and suffers increased DNA damage. J Med Microbiol 56:557-62
Weinberg, Michael V; Maier, Robert J (2007) Peptide transport in Helicobacter pylori: roles of dpp and opp systems and evidence for additional peptide transporters. J Bacteriol 189:3392-402

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