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）是一种用于治疗Hp的抗菌剂，但耐药是常见的，也是治疗失败的主要原因。伯格博士的研究表明，这种抗性是一种多基因性状，rdxA基因的零突变会产生低水平的抗性；另一个基因frxA的突变导致更高的耐药性，而更高的耐药性（超耐药性）是由另一个有待鉴定的基因突变引起的（Jeong等人）。[j] .生物医学工程学报，2002(2):319 - 319。我们[Pan和Weissman PNAS: 99:9346(2002)]已经开发了一种方法来全面筛选复杂DNA混合物的突变。在目前的应用中，我们建议将该方法优化并应用于细菌基因组分析，并使用该方法鉴定使Hp对Mtz产生超耐药的突变和基因。该项目的最终目的是为剖析细菌致病性和基因组进化的分子机制提供一个通用的研究工具。这些实验将有助于满足双亲授予DE Berg (co-PI) AI38166的Specific Aim 4[更全面地了解耐药机制]。这个项目是“探索性的”或“发展性的”，相对于母体的资助，因为我们可能需要克服来自重复和不同序列的潜在“噪音”以及其他意想不到的问题，并对微生物系统进行总体优化。尽管如此，这种方法应该能够以传统（例如鸟枪克隆和DNA转化）类型协议无法比拟的效率鉴定导致超抗性的基因，特别是如果不同的基因群在不同的菌株（遗传背景）中引起超抗性。

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).