Functional dissection of the retron St-85 of Salmonella Typhimurium

鼠伤寒沙门氏菌逆转录子 St-85 的功能解剖

基本信息

批准号：
9266352
负责人：
Johanna Rebecca Elfenbein
金额：
$ 16.78万
依托单位：
NORTH CAROLINA STATE UNIVERSITY RALEIGH
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-15 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9266352
关键词：
Abdominal Pain Acute Anaerobic Bacteria Animals Bacteria Candidate Disease Gene Cattle Cells Cessation of life Characteristics Clinical Colitis Complement Consumption DNA Data Defect Development Diarrhea Disease Dissection Elderly Elements Enterocolitis Environment Enzymes Escherichia coli Feces Fever Food Contamination Future Gastroenteritis Gastrointestinal tract structure Gene Deletion Gene Expression Gene Targeting Genes Genetic Transcription Goals Growth Hand Histologic Human Hybrids Immune response Immunocompetent In Vitro Individual Infection Intestines Libraries Link Livestock Modeling Molecular Mus Mutagenesis Mutation Nucleotides Open Reading Frames Operon Organism Oxygen Pathogenesis Phenotype Process Production Proteins Proteome Public Health Publishing RNA RNA primers RNA-Directed DNA Polymerase Regulation Regulator Genes Retroelements Reverse Transcription Role Salmonella Salmonella enterica Salmonella typhimurium Sequence Homology Single-Stranded DNA Small RNA Structure Testing United States Untranslated RNA Vibrio Virulence Factors Vomiting Work Zoonoses antimicrobial cold temperature fitness foodborne foodborne illness immunosuppressed insight microbial mouse model mutant novel novel therapeutics pathogen prevent public health relevance stem transcriptome

项目摘要

DESCRIPTION (provided by applicant): Non-typhoidal Salmonellae are the leading cause of bacterial food borne gastroenteritis causing hundreds of millions cases of diarrhea and hundreds of thousands deaths world wide each year. The majority of cases originate from consumption of contaminated food products, but some are from direct contact with infected animals or people. Despite many years of study, the strategies used by this pathogen to survive within the gastrointestinal tract of natural hosts are poorly understood. In prior studies, I screened a pool of targeted gene deletion mutants of Salmonella Typhimurium in the bovine ligated ileal loop model, the model most closely mimicking human gastroenteritis. Among 31 novel candidate mutants under selection in this model, I identified a reverse transcriptase (STM3846, rrtT). I confirmed the fitness defect of a deletion mutant in STM3846 using competitive infection and complementation in both bovine and murine models of enterocolitis. In my preliminary data, I show that this enzyme is required to produce a multi-copy single-stranded DNA (msDNA) that is a unique RNA-DNA hybrid molecule of 85 nucleotides. The necessary elements for msDNA production are encoded in an operon termed a "retron" in many different bacterial species, and include msr (encoding the RNA primer), msd (template for reverse transcription) and a reverse transcriptase. Lack of a phenotype for mutants unable to make msDNA was a critical roadblock preventing identification of the natural function of this molecule despite more than 30 years of study. I have discovered that msDNA is essential for survival of Salmonella Typhimurium in the mammalian intestine, in anaerobic conditions, and at low temperature in vitro. These are the first phenotypes of msDNA mutants identified in any bacterial organism. With these phenotypes in hand, I am uniquely poised to identify functional regions of this molecule and to test hypotheses regarding its critical molecular tasks. To accomplish these goals, I will (1) elucidate the portion of msDNA from STm that has activity both in vitro and during infection, (2) investigate the function of msr by generating targeted mutations and determining the functionality of mutant msr both in vitro and during infection, and (3) determine the effect of expression of msDNA on global transcription and protein levels. This work will determine the key functional regions of msDNA and test hypotheses regarding its natural function. Because the msDNA molecule itself is totally novel and is clearly necessary during infection, this work will illuminate a novel paradigm in bacterial pathogenesis. This unique RNA-DNA hybrid molecule represents a novel antimicrobial target for treatment of this important zoonotic pathogen either through inhibition of the molecule itself or the reverse transcriptase necessary for its production.

描述（由申请人提供）：非伤寒沙门氏菌是细菌性食源性胃肠炎的主要原因，每年在全世界造成数亿例腹泻和数十万人死亡。大多数病例源于食用受污染的食品，但也有一些病例是由于直接接触受感染的动物或人。尽管经过多年的研究，人们对这种病原体在自然宿主胃肠道内生存的策略仍知之甚少。在之前的研究中，我在牛结扎回肠环模型中筛选了鼠伤寒沙门氏菌的靶向基因缺失突变体，该模型最接近人类胃肠炎。在该模型中选择的 31 个新候选突变体中，我鉴定出了一种逆转录酶（STM3846，rrtT）。我在牛和鼠小肠结肠炎模型中利用竞争性感染和互补证实了 STM3846 缺失突变体的适应性缺陷。在我的初步数据中，我表明需要这种酶来产生多拷贝单链 DNA (msDNA)，这是一种独特的 85 个核苷酸的 RNA-DNA 杂合分子。 msDNA 产生所需的元件在许多不同细菌物种中被称为“逆转录子”的操纵子中编码，包括 msr（编码 RNA 引物）、msd（逆转录模板）和逆转录酶。尽管进行了 30 多年的研究，但无法产生 msDNA 的突变体缺乏表型是阻碍鉴定该分子自然功能的一个关键障碍。我发现 msDNA 对于鼠伤寒沙门氏菌在哺乳动物肠道、厌氧条件和体外低温下的生存至关重要。这是在任何细菌生物体中首次发现的 msDNA 突变体表型。有了这些表型，我就可以独特地识别该分子的功能区域并测试有关其关键分子任务的假设。为了实现这些目标，我将 (1) 阐明 STm 中在体外和感染过程中均具有活性的 msDNA 部分，(2) 通过生成靶向突变并确定突变体 msr 的体外和感染功能来研究 msr 的功能。感染期间，(3) 确定 msDNA 表达对整体转录和蛋白质水平的影响。这项工作将确定 msDNA 的关键功能区域并测试有关其自然功能的假设。由于 msDNA 分子本身是全新的，并且在感染过程中显然是必需的，因此这项工作将阐明细菌发病机制的新范例。这种独特的 RNA-DNA杂合分子代表了一种新的抗菌靶点，可以通过抑制分子本身或抑制其产生所需的逆转录酶来治疗这种重要的人畜共患病原体。