Nickel Metabolism in Helicobacter pylori

描述(申请人提供)：化脓性幽门螺杆菌是一种螺旋细菌，定植于人类的胃粘膜，导致多种胃病，包括消化性溃疡、慢性胃炎、粘膜相关淋巴瘤以及下胃和十二指肠腺癌。两种含镍的酶，尿素酶和氢酶，对细菌的粘膜定植能力是重要的。本文提出的目标是了解镍的封存、储存、代谢和金属调节步骤，这些步骤涉及这两种含镍酶的合成/成熟。从对其他生物的研究中，序列确定的辅助蛋白(由ure和Hyp基因编码)有望形成两种镍酶成熟所需的镍隔离和能量利用(GTP水解)复合体。在镍存在的情况下，这些络合物有助于将金属动员到最终的沉淀物、尿素酶或氢酶中。根据我们对纯化的镍结合蛋白和GTPase辅助蛋白及其相互作用的了解，我们将提出一条顺序的镍动员转移途径。假设的镍转移步骤将通过将选定的纯辅助蛋白混合在一起进行测试。一些特定的镍结合蛋白(从基因组序列中预测)在镍隔离、动态平衡和调节中的作用将通过定向突变来解决。了解幽门螺杆菌中镍的代谢、动态平衡和调控的具体方法包括纯化向镍酶输送镍的复合体、鉴定蛋白质的蛋白质组学分析以及对镍补充反应调节的转录本的基因组分析。最后，预计会影响细菌镍营养状态(即镍稳态)的蛋白质中的基因定向突变将被鉴定，以了解三种特定的(但研究较少的)镍结合蛋白的作用。DNA结合和镍敏感蛋白(NikR)的作用尤其令人感兴趣，因为它可能在金属动态平衡中发挥全球调节作用，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.