Nickel Metabolism in Helicobacter pylori

描述（由申请人提供）：幽门螺杆菌是一种螺旋菌，定植于人类胃粘膜，导致多种胃病，包括消化性溃疡、慢性胃炎、粘膜相关淋巴瘤和下胃和十二指肠腺癌。两种含镍的酶，尿素酶和氢化酶，对细菌的粘膜定殖能力很重要。这里提出的目标是了解镍螯合，存储，代谢和金属调节步骤参与的两个含镍酶的合成/成熟。从与其他生物的研究，序列鉴定的辅助蛋白（由ure和hyp基因编码）预计将形成镍螯合和能量利用（GTP水解）的两个镍酶的成熟所需的复合物。在镍的存在下，这些络合物促进金属进入最终的汇，脲酶或氢化酶的动员。从我们的知识纯化镍结合和GTdR辅助蛋白和它们的相互作用，一个连续的镍动员转移途径将被提出。将通过将选定的纯辅助蛋白混合在一起来测试假设的Ni转移步骤。一些特定的镍结合蛋白（从基因组序列预测）在镍螯合，稳态和调节中的作用将通过靶向诱变来解决。本文介绍了了解镍在H.幽门螺杆菌的研究将包括纯化将镍传递给镍酶的复合物、鉴定蛋白质的蛋白质组学分析以及对响应于镍补充而调节的转录物的基因组分析。最后，将表征预期影响细菌的镍营养状态（即镍稳态）的蛋白质中的基因定向突变体，以了解三种特定（但研究不足）镍结合蛋白的作用。DNA结合和镍敏感蛋白（NikR）的作用特别令人感兴趣，因为它可能在金属稳态中发挥全局调节作用，进而影响细胞代谢的许多过程。

DESCRIPTION (provided by applicant): 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.