ACID INDUCIBLE RESPONSES IN H PYLORI

幽门螺杆菌的酸诱导反应

• 批准号: 6374009
• 负责人: CATHERINE C MCGOWAN
• 金额: $ 15.01万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6374009
• 关键词: Helicobacter; acidity /alkalinity; bacterial proteins; flow cytometry; gene expression; gene induction /repression; genetic promoter element; genetic regulation; lipopolysaccharides; subtraction hybridization

DESCRIPTION (Adapted from the Applicant's Abstract): Helicobacter pylori are curved Gram-negative bacteria that are now recognized as the cause of chronic superficial gastritis in humans, and this organism plays an important etiologic role in the pathogenesis of peptic ulcer disease and distal gastric adenocarcinoma. H. pylori colonizes the mucus layer overlying the gastric epithelium, where it is exposed to a wide range of pH values. Urease activity is required for H. pylori survival at low pH, and its presence is essential for colonization. However, little else is known about the mechanisms which allow H. pylori to survive in acidic environments. In previous studies, the investigators have used the strategy of subtractive RNA hybridization to identify an acid-inducible gene in H. pylori, wbcJ, that is essential for O-antigen expression and which contributes to acid survival of the organism. The long-term goal of this study is to isolate and identify additional acid-inducible factors in H. pylori which are required for establishment and maintenance of infection. The hypothesis of this study is that H. pylori possesses a regulated, inducible acid stress response system. The specific aims are 1) to study the transcriptional regulation of the acid-inducible H. pylori gene, wbcJ and to characterize its role in LPS biosynthesis and structure, 2) to identify other H. pylori genes whose expression is increased in response to acidic pH, and 3) to isolate acid-inducible promoters in H. pylori. To accomplish the first objective, analyses will be done to characterize the promoter region and genetic organization of wbcJ and its co-transcribed genes. Transcriptional wbcJ fusions then will be constructed using xylE to examine the expression of wbcJ under different environmental conditions in vitro. The LPS structure of wbcJ mutants will be analyzed to determine the role of this gene in H. pylori LPS biosynthetic pathways. The method of subtractive RNA hybridization will be used to isolate H. pylori genes whose expression is increased during growth at acidic pH. Genes identified by this approach will be cloned and disrupted using insertional mutagenesis, which will permit the study of their role in H. pylori acid survival and colonization. As an alternate strategy to identifying acid-regulated genes, we will employ the technique of differential fluorescence induction to enrich for promoters that are up-regulated following exposure to acidic pH, utilizing GFP and a fluorescence-activated cell sorter. The proposed work will further elucidate the mechanisms used by H. pylori to colonize and persist within the gastric environment and will lead to improved understanding of how H. pylori causes disease.

描述（改编自申请人的摘要）：幽门螺杆菌是 弯曲的革兰氏阴性细菌现在被认为是慢性疾病的原因 人类浅表性胃炎，这种微生物起着重要的病因作用 在消化性溃疡病和远端胃的发病机制中的作用 腺癌。幽门螺杆菌定植于胃粘液层 上皮细胞，暴露于各种 pH 值。脲酶活性 是幽门螺杆菌在低 pH 值下生存所必需的，它的存在对于幽门螺杆菌在低 pH 值下生存至关重要。 殖民化。然而，关于 H. 幽门螺杆菌在酸性环境中生存。在之前的研究中， 研究人员使用消减RNA杂交策略 鉴定出幽门螺杆菌中的酸诱导基因 wbcJ，该基因对于 O-抗原表达有助于生物体的酸生存。 这项研究的长期目标是分离和识别额外的 幽门螺杆菌中的酸诱导因子，这是建立和形成所需的 维持感染。本研究的假设是幽门螺杆菌 拥有一个可调节的、诱导性的酸应激反应系统。具体目标 1) 研究酸诱导型幽门螺杆菌的转录调控 基因，wbcJ 并表征其在 LPS 生物合成和结构中的作用，2) 鉴定其他幽门螺杆菌基因，其表达会因响应而增加 酸性 pH 值，3) 分离幽门螺杆菌中的酸诱导启动子。到 完成第一个目标后，将进行分析来描述 wbcJ 及其共转录基因的启动子区域和遗传组织。 然后将使用 xylE 构建转录 wbcJ 融合来检查 体外不同环境条件下wbcJ的表达情况。脂多糖 将分析 wbcJ 突变体的结构以确定该基因的作用 幽门螺杆菌 LPS 生物合成途径。 RNA消减法 杂交将用于分离幽门螺杆菌基因，其表达为 在酸性 pH 条件下生长期间增加。通过这种方法鉴定的基因将 使用插入诱变进行克隆和破坏，这将允许研究 它们在幽门螺杆菌存活和定殖中的作用。作为替补 识别酸调节基因的策略，我们将采用以下技术 差异荧光诱导富集启动子 暴露于酸性 pH 后上调，利用 GFP 和 荧光激活细胞分选仪。拟议的工作将进一步阐明 幽门螺杆菌在胃内定殖和持续存在的机制 环境，并将导致更好地了解幽门螺杆菌如何引起 疾病。