Functional dissection of the retron St-85 of Salmonella Typhimurium

描述(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，每年导致全球数亿腹泻病例和数十万人死亡。大多数病例源于食用受污染的食品，但也有一些病例来自与受感染动物或人的直接接触。尽管进行了多年的研究，但人们对这种病原体在自然宿主的胃肠道中生存所使用的策略知之甚少。在之前的研究中，我在牛结扎回肠回肠模型中筛选了一批鼠伤寒沙门氏菌的靶基因缺失突变体，这是最接近于人类胃肠炎的模型。在这个模型中选择的31个新的候选突变体中，我鉴定了一个逆转录酶(STM3846，rrtT)。我在牛和小鼠的小肠结肠炎模型中，通过竞争性感染和互补，证实了STM3846缺失突变体的适用性缺陷。在我的初步数据中，我表明这种酶是产生一种多拷贝单链DNA(MsDNA)所必需的，这是一种由85个核苷酸组成的独特的RNA-DNA杂化分子。在许多不同的细菌物种中，msDNA产生的必要元件被编码在一个被称为“反转录”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。尽管经过了30多年的研究，但缺乏不能制造msDNA的突变体的表型是阻碍鉴定这种分子的自然功能的关键障碍。我发现msDNA对鼠伤寒沙门氏菌在哺乳动物肠道中、厌氧条件下和体外低温下的生存是必不可少的。这是在任何细菌有机体中发现的第一批msDNA突变体的表型。有了这些表型，我就可以唯一地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中具有体外和感染期间活性的msDNA部分，(2)通过产生靶向突变并确定突变体MSR在体外和感染期间的功能来研究MSR的功能，以及(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，并检验有关其自然功能的假设。由于msDNA分子本身是全新的，显然在感染过程中是必要的，这项工作将阐明细菌发病机制的一种新范式。这个独一无二的 RNA-DNA杂化分子代表了一种新的抗菌靶，用于治疗这种重要的人畜共患病病原体，要么是通过抑制分子本身，要么是通过抑制其产生所需的逆转录酶。

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.