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：
9058463
负责人：
Johanna Rebecca Elfenbein
金额：
$ 14.52万
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 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9058463
关键词：
Abdominal Pain Acute Animals Bacteria Candidate Disease Gene Cattle Cells Cessation of life Characteristics Clinical Colitis 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 Genes Genetic Transcription Goals Growth Hand Health Histologic Human Hybrids Immune response Immunocompetent In Vitro Indium 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 antimicrobial cold temperature fitness foodborne foodborne illness immunosuppressed insight microbial mouse model mutant novel novel therapeutics pathogen prevent stem transcriptome

Abdominal Pain Acute Animals Bacteria Candidate Disease Gene Cattle Cells Cessation of life Characteristics Clinical Colitis 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 Genes Genetic Transcription Goals Growth Hand Health Histologic Human Hybrids Immune response Immunocompetent In Vitro Indium 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 antimicrobial cold temperature fitness foodborne foodborne illness immunosuppressed insight microbial mouse model mutant novel novel therapeutics pathogen prevent stem transcriptome

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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）：非类型沙门氏菌是细菌食品传播胃肠炎的主要原因，每年造成数亿例腹泻和数以千计的全球死亡。大多数病例源于消费受污染的食品，但有些来自与受感染的动物或人的直接接触。尽管有多年的研究，但这种病原体在天然宿主的胃肠道中生存的策略知之甚少。在先前的研究中，我筛选了牛腹膜沙门氏菌鼠伤寒的靶向基因缺失突变体池，该模型最紧密地模仿了人类胃炎的模型。在该模型中选择的31种新型候选突变体中，我确定了逆转录酶（STM3846，RRTT）。我确认了使用竞争性感染和小肠结肠炎模型中的竞争性感染和互补的STM3846中缺失突变体的适应性缺陷。在我的初步数据中，我表明这种酶是产生多拷贝的单链DNA（MSDNA），该单链DNA（MSDNA）是独特的RNA-DNA杂交分子的85个核苷酸。 MSDNA产生的必要元素被编码在许多不同细菌物种中称为“反龙”的操纵子中，包括MSR（编码RNA底漆），MSD（逆转录模板）和逆转录酶。缺乏无法制作MSDNA的突变体的表型是一个关键的障碍，尽管研究了30多年，但仍能鉴定出该分子的自然功能。我发现MSDNA对于在厌氧条件下和在低温体外生存对于哺乳动物肠中沙门氏菌伤寒的生存至关重要。这些是在任何细菌生物中鉴定出的MSDNA突变体的第一个表型。借助这些表型，我完全准备识别该分子的功能区域，并就其关键分子任务进行测试。为了实现这些目标，我将（1）阐明来自STM的MSDNA的一部分，在体外和感染过程中具有活性，（2）通过产生靶向突变并确定突变体MSR在体外和感染过程中的功能，以及（3）确定MSDNA表达对全球转录和蛋白质水平的影响。这项工作将确定MSDNA的关键功能区域和关于其自然功能的假设。因为MSDNA分子本身是完全新颖的，并且在感染过程中显然是必要的，所以这项工作将阐明细菌发病机理中新型的范式。这个独特 RNA-DNA杂交分子是通过抑制分子本身或其生产所需的逆转录酶来治疗这种重要人畜共患病原体的新型抗菌靶标。