IDENTIFICATION OF HELICOBACTER PYLORI VIRULENCE GENES

描述（改编自申请人的摘要）：H.慢性幽门 感染胃粘膜，并与人类 胃炎、消化性溃疡和胃癌。该提案概述了 功能基因组学为基础的方法，旨在确定和表征H。 pylori毒力基因与人类胃病相关拟议 实验将提供H.幽门螺杆菌基因组， 补充了两个完全测序的H的完整序列。幽门螺杆菌基因组。一 称为“动力学监测逆转录酶启动PCR”的方法; （kRT-PCR）构成了实验计划的基础。这种方法是在 在主要研究者的实验室中，并允许高通量 转录物定量。全基因组转录谱的建议H。 pylori在某些基准状态下。生长阶段，pH值变化， 铁和人上皮细胞对H. pylori基因表达进行分析。 这些生长条件以及cag内的敲除突变 致病岛和特定的转录因子基因内，将 测试它们对H.幽门螺杆菌转录组。候选毒力 从基因组序列中鉴定的基因包括参与分子生物学的那些。 拟态，LPS生物合成，外膜蛋白，候选“相位变异” 基因，以及数百个H.通过比较确定的幽门特异性基因 阁下于幽门螺杆菌基因组序列与其他致病性和非致病性 细菌螺杆pylori转录组将被组织成操纵子。操纵子 结构和协调表达谱应揭示潜在的功能 未知的ORF H.体内幽门螺杆菌将是 使用非人类灵长类动物模型获得。在一个 各种体外条件将被组织成相关数据集， 分析以揭示H.幽门。

DESCRIPTION (Adapted from the Applicant's Abstract): H. pylori chronically infects gastric mucosa and is implicated in the pathogenesis of human gastritis, peptic ulcer disease, and gastric cancer. This proposal outlines a functional genomics-based approach designed to identify and characterize H. pylori virulence genes associated with human gastric disease. The proposed experiments will provide a functional map of the H. pylori genome which complements the complete sequences of two fully sequenced H. pylori genomes. A method termed "kinetically monitored reverse transcriptase-initiated PCR"; (kRT-PCR) forms the basis for the experimental plan. This method was developed in the laboratory of the principal investigator, and permits high throughput transcript quantitation. Genome-wide transcript profiling is proposed for H. pylori in certain benchmark states. The effects of growth phase, pH shift, iron, and human epithelial cells on H. pylori gene expression will be analyzed. These growth conditions, as well as knockout mutations within the cag pathogenicity island and within specific transcription factor genes, will be tested for their effects on the H. pylori transcriptome. Candidate virulence genes identified from the genome sequence include those involved in molecular mimicry, LPS biosynthesis, outer membrane proteins, candidate "phase variation" genes, and several hundred H. pylori-specific genes identified by comparisons of H. pylori genome sequences with those of other pathogenic and non-pathogenic bacteria. The H. pylori transcriptome will be organized into operons. Operon structures and coordinate expression profiles should reveal potential functions for unknown ORFs. Gene expression profiles for H. pylori in vivo will be obtained using a non-human primate model. Transcript profiles obtained under a variety of in vitro conditions will be organized into relational data sets and analyzed to reveal transcriptional paths and networks operative in H. pylori.