Mechanistic and Functional Analysis of a Putative Regulatory Factor in the Lyme Disease Spirochete

摘要 伯氏疏螺旋体引起莱姆病的能力高度依赖于其建立 在进入哺乳动物宿主后成功感染。此外，莱姆病病原体在 自然界完全依赖于它的地方性生命周期，包括壁虱媒介和宿主。这个 在这两个截然不同的环境之间过渡需要快速适应变化的能力 在基因表达上。我们实验室最近的研究提供了证据，证明BBD07的功能产品 Lp17基因间隔区是一个对寄主适应起重要作用的非编码小RNA(SRNA)。尽管如此 尽管如此，我们对bbd07介导的机制方面的理解仍然存在着根本的差距。 基因调控，而受其调控作用的基因尚不清楚。我们的长期目标是 确定和表征伯氏杆菌适应硬蜱和哺乳动物宿主所需的因素 环境。本申请的总体目标是确定bbd07对于监管的重要性 伯氏杆菌寄主定植的重要基因。根据初步数据，核心假设是 这一建议是bbd07作为调节已知对宿主很重要的基因所必需的sRNA。 适应。拟议研究的理论基础是bbd07 rna及其下的基因产物 调节影响是开发抗人类感染治疗药物的潜在目标，通过 病原体，以及其地方性生活史的中断。因此，拟议的研究与此相关。 NIH使命的一部分，与发展基础知识有关，这可能有助于减少 人类疾病和残疾的负担。根据已公布的和初步的数据，我们的假设将是 通过追求以下两个具体目标进行测试：1)确定bbd07和 其他靶基因的同一性，以及2)在bbd07表达和Ospc之间建立关联 寄主感染期间的抑制。在第一个目标下，bbd07 RNA的5‘端将以种族为特征， 将绘制sRNA-mRNA靶向相互作用图，并将使用iTRAQ分析来确定大规模 Bbd07缺失对伯氏杆菌蛋白质组的影响。在第二个目标下，bbd07的相对水平 和OspC在小鼠和扁虱宿主中的表达将被量化，并使用 免疫荧光法。提出的工作具有创新性，因为它利用了一种新的基于rna-seq的 一种全局定位伯氏杆菌sRNA-mRNA靶向相互作用的方法，这是首次对伯氏杆菌sRNA-mRNAs进行突变分析。 BBD07评估其对宿主适应相关基因调控的各自重要性。当应用时， 从拟议的研究中获得的知识有可能阐明治疗和治疗的新药物靶点。 预防莱姆病。

Summary The ability of Borrelia burgdorferi to cause Lyme disease is highly dependent on its capacity to establish a successful infection upon entering the mammalian host. Moreover, survival of the Lyme disease pathogen in nature is completely dependent on its enzootic life cycle involving both a tick vector and reservoir host. The transition between these two very different environments requires the ability to rapidly adapt through changes in gene expression. Recent studies in our lab have provided evidence that the functional product of the bbd07 intergenic region of lp17 acts as a small non-coding RNA (sRNA) important for host adaptation. Despite this advance, there remains a fundamental gap in our understanding of the mechanistic aspects of bbd07-mediated gene regulation, and the genes subjected to its regulatory effects are unknown. Our long-term goals are to identify and characterize factors necessary for B. burgdorferi adaptation to the tick and mammalian host environments. The overall objective of this application is to establish the importance of bbd07 for regulation of genes important for host colonization by B. burgdorferi. Based on preliminary data, the central hypothesis of this proposal is that bbd07 acts as a sRNA essential for regulation of genes known to be important for host adaptation. The rationale for the proposed research is that bbd07 RNA and the gene products under its regulatory influence represent potential targets for the development of therapeutics against human infection by the pathogen, as well as disruption of its enzootic life cycle. Thus, the proposed research is relevant to that part of NIH’s mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human illness and disability. Guided by published and preliminary data, our hypothesis will be tested by pursuing the following two specific aims: 1) Determine the regulatory mechanism of bbd07 and identity of additional target genes, and 2) Establish a correlation between bbd07 expression and ospC repression during host infection. Under the first aim, the 5’ end of bbd07 RNA will be characterized by RACE, sRNA-mRNA target interactions will be mapped, and iTRAQ analysis will be used to determine the large-scale effects of bbd07 deletion on the B. burgdorferi proteome. Under the second aim, the relative levels of bbd07 and ospC expression in the murine and ticks hosts will be quantified, and OspC production visualized using an immunofluorescence assay. The proposed work is innovative because it utilizes a novel RNA-seq-based method to globally map sRNA-mRNA target interactions in B. burgdorferi, and is the first mutational analysis of bbd07 to assess its respective importance for regulation of genes involved in host adaptation. When applied, the knowledge gained from the proposed studies has the potential to elucidate new drug targets to treat and prevent Lyme disease.