The role of small non-coding RNA in borrelial pathogenesis

 描述(申请人提供)：莱姆病或莱姆疏螺旋体病的病原体，伯氏疏螺旋体，在美国产生的节肢动物传播的感染比任何其他病原体都多，每年诊断超过30万例。在伯氏杆菌感染流行的地区，与这种感染相关的发病率很高，主要表现为心脏、神经或关节炎后遗症。尽管与大多数生命系统相比，伯氏杆菌基因组过于简单，但随着病原体从节肢动物媒介和它的生命周期中占据的哺乳动物物种转移，它擅长改变其基因调控模式。虽然我们了解了几个转录调节因子在这种适应性反应中的作用，但对一些侧翼基因(例如，BosR和DBpA)还有额外的调控层，这些调控还没有被阐明。鉴于它们在许多细菌系统中作为重要的调节效应器出现，包括与毒力基因表达和一些病原体中的蛋白质产生的连锁，我们假设在伯氏杆菌中需要小的非编码RNA(SRNAs)来实现类似的调节功能。使用协作方法，B。 Burgdorferi TN文库与TN-seq以及RNA-seq技术相结合，鉴定了14个可能的sRNA突变体，这些突变体在实验感染后减弱。这些相同的位点编码了先前通过总转录谱的RNA-seq以及sRNA特异性文库鉴定的sRNA。随后在这些部位感染单个TN突变体表明，其中4个突变体的定植能力受到了损害。这项提议旨在 建立sRNAs与它们通过改变靶mRNAs来调节基因表达和/或蛋白质生产的作用之间的联系。为了解决这一问题，我们提出了以下具体目标：(1)将sRNA物种与疏螺旋体的传染性联系起来。将测试旨在消除特定sRNA的精确突变体，以确定它们在体内以空间和时间方式感染疏螺旋体中的作用；以及(2)确定选择性疏导sRNA耗竭的全球调控效果。将使用全球转录和蛋白质图谱来跟踪特定sRNA丢失的影响，以确定sRNA如何影响未连接基因的转录或翻译。本文概述的工作提供了sRNA调控通路和伯氏杆菌感染之间的联系。到目前为止，这种联系还没有深入分析，因此，这是一个机会来确定sRNA如何调节转录或转录后调控途径，从而影响骨骼肌的生理和发病机制。

 DESCRIPTION (provided by applicant): The agent of Lyme disease or Lyme borreliosis, Borrelia burgdorferi, yields more arthropod-borne infections than any other pathogen in the United States with over 300,000 cases diagnosed each year. In areas where B. burgdorferi infections are prevalent, morbidity associated with this infection is high and is seen mostly in th form of cardiac, neurologic, or arthritic sequelae. Although B. burgdorferi has a simplistic genome relative to most living systems, it is adept at altering its gene regulatory pattern as the pathogen moves from the arthropod vector and mammalian species it can occupy during its lifecycle. Although we understand the role of several transcriptional regulators in this adaptive response, there are additional layers of regulation for some borrelial genes (e.g., bosR and dbpA) that have not been elucidated. Given their emergence as important regulatory effectors in many bacterial systems, including a linkage to virulence gene expression and protein production in a number of pathogens, we hypothesized that small, non-coding RNAs (sRNAs) were required for similar regulatory functions in B. burgdorferi. Using a collaborative approach, the B. burgdorferi Tn library coupled with Tn-seq, as well as RNA-seq technologies, identified 14 putative sRNA mutants that were attenuated following experimental infection. These same sites encoded sRNA previously identified via RNA- seq of total transcript profiles as well as libraries specific for sRNAs. Subsequent infections with individual Tn mutants at these sites indicated that 4 of the mutants were impaired in their ability to colonize mice. This proposal is designed to establish a link between the sRNAs and their role in regulating gene expression and/or protein production via alteration of target mRNAs. To address this we propose the following Specific Aims: (1) Link sRNA species to borrelial infectivity. Precise mutants, designed to eliminate specific sRNAs, will be tested to determine their role in borrelial infectivity in vivo in both a spatial and temporal manner; and (2) Determine the global regulatory effect of select borrelial sRNA depletion. The effect of the loss of specific sRNAs will be tracked using global transcript and protein profiles to determine how the sRNA affects transcription or translation of unlinked genes. The work outlined herein provides a link between sRNA regulatory pathways and B. burgdorferi infection. To date, this connection has not analyzed in depth and, as such, represents an opportunity to determine how sRNA modulate either transcriptional or post- transcriptional regulatory pathways that impact borrelial physiology and pathogenesis.