Global Scanning for Resistance Mutations in H.pylori

描述(申请人提供)：幽门螺杆菌(Hp)是消化性溃疡疾病的主要原因，也是胃癌的早期危险因素。甲硝唑(MTZ)是一种抗幽门螺杆菌的抗菌剂，但耐药性很常见，是治疗失败的主要原因。D.Berg的研究表明，这种抗药性是一个多基因特征，rdxA基因的零突变赋予低水平抗药性；另一个基因frxA的突变导致更高的抗药性，而更高的抗药性(超抗力)是由另一个有待鉴定的基因的突变造成的(Jeong等人)。J.Bact。182：3219,2000)。我们[潘和魏斯曼PNAS：99：9346(2002)]已经开发出一种全球筛查复杂DNA混合物中突变的方法。在目前的应用中，我们建议优化和应用这种方法来分析细菌基因组，并使用这种方法来识别使Hp对MTZ产生超抗的突变和基因。这个项目的最终目的是为剖析细菌致病和基因组进化的分子机制提供一个通用的研究工具。这些实验将有助于实现父母授予DE Berg(co-PI)的AI38166的具体目标4[以更充分地了解耐药性的机制]。这个项目是“探索性的”或“发展性的”，相对于父母的资助，因为我们可能需要克服重复和不同的序列以及其他意想不到的问题带来的潜在“噪音”，并对微生物系统进行总体优化。然而，这种方法应该能够以传统的(如鸟枪克隆和DNA转化)类型的方法所无法比拟的效率识别负责超抗性的基因，特别是如果不同的基因星座导致不同品系的超抗性(遗传背景)。

DESCRIPTION (provided by applicant): Helicobacter pylori (Hp) is a major cause of peptic ulcer disease and an early risk factor for gastric cancer. Metronidazole (Mtz) is an antibacterial agent used against Hp, but resistance is common and is a major reason for treatment failure. Research by D. Berg has shown that this resistance is a polygenic trait and that null mutations in the gene rdxA confers low-level resistance; a mutation in another gene, frxA, results in higher resistance and that higher resistance (hyper-resistance) results from mutations in another genes that remain to be identified (Jeong et al. J. Bact. 182:3219, 2000). We [Pan and Weissman PNAS: 99:9346 (2002)] have developed a method for global screening for mutations in complex DNA mixtures. In the present application we propose to optimize and apply this method for analysis of bacterial genomes, and use the method to identify mutations and genes that make Hp hyper-resistant to Mtz. The final purpose of this project is to provide a general research tool for dissecting the molecular mechanisms of bacterial pathogenicity and genome evolution. These experiments will help meet Specific Aim 4 of parent grant AI38166 to DE Berg (co-PI) [To more fully understand mechanisms of drug resistance]. This project is "exploratory" or "developmental," relative to the parent grant, in that we may need to overcome potential "noise" from duplicate and divergent sequences as well as other unexpected problems, and generally optimize this for microbial systems. Nevertheless, this method should allow identification of the genes responsible for hyper-resistance with an efficiency that could not be matched by traditional (e.g. shotgun cloning and DNA transformation) type protocols, especially if different constellations of genes cause hyperR in different strains (genetic backgrounds).