Functional dissection of the retron St-85 of Salmonella Typhimurium

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例死亡。大多数病例来自食用受污染的食品，但也有一些病例来自直接接触受感染的动物或人。尽管经过多年的研究，这种病原体在天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，这是最接近人类胃肠炎的模型。在该模型中选择的31个新的候选突变体中，我发现了一个逆转录酶（STM3846, rrtT）。我在牛和小鼠小肠结肠炎模型中使用竞争性感染和互补证实了STM3846缺失突变体的适合度缺陷。在我的初步数据中，我表明这种酶是产生多拷贝单链DNA （msDNA）所必需的，这是一种独特的85个核苷酸的RNA-DNA杂交分子。在许多不同的细菌物种中，产生msDNA的必要元件被编码在一个被称为“逆转录子”的操纵子中，包括msr（编码RNA引物）、msd（逆转录模板）和一个逆转录酶。尽管已经进行了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条件下的生存至关重要。这些是在任何细菌有机体中鉴定出的msDNA突变体的第一个表型。有了这些表型在手，我有独特的准备来确定这个分子的功能区域，并测试关于其关键分子任务的假设。为了实现这些目标，我将(1)阐明STm中在体外和感染期间都具有活性的msDNA部分，(2)通过产生靶向突变并确定体外和感染期间突变msr的功能来研究msr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，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式。这种独特的

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.