Genetic and Biochemical Analysis of DNA Exchange in Helicobacter pylori

幽门螺杆菌 DNA 交换的遗传和生化分析

• 批准号: 8028833
• 负责人: Marion Shonn Dorer
• 金额: $ 3.23万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8028833
• 关键词: Genetic; Biochemical; Analysis; DNA; Exchange

DESCRIPTION (provided by applicant): Helicobacter pylori is a major public health problem: at least 50% of the world population is persistently infected with this bacterium, including at least 30% of the United States population [1]. Inflammation caused by H. pylori puts the infected population at increased risk for chronic gastritis, peptic ulcer disease, and gastric cancers, the second leading cause of cancer-related deaths in the world [2]. Genetic variability in the bacterium explains, in part, this diverse disease outcome: patients are typically infected with more than one distinct strain [3,4,5] and population studies show that up to 25% of genes are variably present between strains [6,7]. My overall research goal is to investigate both mechanisms for generating diversity between H. pylori strains and the function variable genes. Genetic diversity is influenced by environmental factors, since the chronic host inflammatory response damages bacterial DNA [8]. My studies show that DNA damage alters transcription of at least 25% of the H. pylori genome, and importantly induces genes required for natural competence, the ability to take up exogenous DNA. These findings led to the hypothesis that the DNA damage regulated gene set includes novel natural competence genes. Moreover, I find that exogenous DNA is a key transcriptional regulator: increased uptake of exogenous DNA triggers the same transcriptional program as DNA damage and natural competence is required to sustain transcription of the DNA damage program. The only source of DNA in these experiments was donor cells, so I hypothesized that a DNA damage regulated gene is required to obtain DNA from donor cells. Since lytic activities mediate competence induced predation in Streptococcus pneumoniae [9], I deleted a phage T4 lysozome homolog that is induced by DNA damage in H. pylori and found that the lys gene is required to obtain DNA from donor cells. The lys gene is present in 35% of H. pylori strains [6] and thus its role in promoting genetic exchange may contribute to variability in inflammation and progression to ulcer. In Specific Aim 1, I propose biochemical and localization studies to understand the Lys mechanism of action as well as genetic study of two genes that form an operon with lys. In Specific Aim 2, I will use our sequenced-defined library of transposon mutants to identify DNA damage regulated genes required to obtain DNA from donor cells and for natural competence. If the proposed aims are achieved, I will demonstrate that H. pylori has an active mechanism for obtaining DNA from donor cells and identify new components of several steps natural competence. These studies lay a foundation for a future R01, which will focus on mechanistic studies of natural competence and the role of genetic variation in persistent colonization and progression of inflammation to ulcer disease. These outcomes fit the mission of NIDDK to understand and treat the causes of ulcer disease. PUBLIC HEALTH RELEVANCE: Peptic Ulcers are not caused by stress, but rather infection with a bacterium called Helicobacter pylori, which also causes gastric cancer. Studying the bacterium will help us develop new drugs that will rid people of the infection and reduce greatly their risk of developing ulcers or stomach cancer.

描述（由申请人提供）：幽门螺杆菌是一个主要的公共卫生问题：至少有50％的世界人口持续感染了这种细菌，其中包括至少30％的美国人口[1]。由幽门螺杆菌引起的炎症使感染人群患慢性胃炎，消化性溃疡病和胃癌的风险增加，这是世界上与癌症相关死亡的第二大主要原因[2]。细菌中的遗传变异性部分解释了这种多样的疾病结果：患者通常感染多种不同的菌株[3,4,5]，人群研究表明，菌株之间最多可变的基因可变[6,7]。我的总体研究目标是研究两种机制，用于在幽门螺杆菌菌株和功能变量基因之间产生多样性。遗传多样性受环境因素的影响，因为慢性宿主炎症反应会损害细菌DNA [8]。我的研究表明，DNA损伤会改变至少25％的幽门螺杆菌基因组的转录，并且重要的是诱发自然能力所需的基因，即接受外源性DNA的能力。这些发现导致了以下假设：DNA损伤调节基因集包括新的自然能力基因。此外，我发现外源性DNA是一个关键的转录调节剂：增加摄取外源性DNA的摄取剂触发与DNA损伤相同的转录程序，并且需要自然能力，以维持DNA损伤程序的转录。这些实验中DNA的唯一来源是供体细胞，因此我假设需要一个DNA损伤调节基因才能从供体细胞中获得DNA。由于裂解活性介导了肺炎链球菌诱导的捕食[9]，因此我删除了噬菌体T4溶菌酶同源物，该噬菌体在幽门螺杆菌中是由DNA损伤诱导的，发现LYS基因是从供体细胞中获得DNA的。 LYS基因存在于35％的幽门螺杆菌菌株中[6]，因此其在促进遗传交换中的作用可能导致炎症和发展为溃疡的变异性。在特定的目标1中，我提出了生化和定位研究，以了解LYS作用机理以及对形成LYS操纵子的两个基因的遗传研究。在特定的目标2中，我将使用我们测序定义的转座子突变体库来识别从供体细胞获得DNA所需的DNA损伤调节基因和自然能力。如果实现了所提出的目标，我将证明幽门螺杆菌具有从供体细胞中获得DNA并确定多个步骤自然能力的新组件的活跃机制。这些研究为未来的R01奠定了基础，该研究将重点介绍自然能力的机理研究以及遗传变异在持续定殖和炎症向溃疡疾病的发展中的作用。这些结果符合NIDDK的使命，以了解和治疗溃疡疾病的原因。 公共卫生相关性：消化性溃疡不是由压力引起的，而是被称为幽门螺杆菌的细菌感染，这也会引起胃癌。研究细菌将有助于我们开发新药物，这些新药将消除感染的人并大大降低患溃疡或胃癌的风险。