RpoS-mediated virulence regulation in Borrelia burgdorferi

RpoS 介导的伯氏疏螺旋体毒力调控

• 批准号: 7992838
• 负责人: Jon Scott Blevins
• 金额: $ 35.71万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7992838
• 关键词: Address; Arthritis; Arthropods; Attention; Bacteria; Bacteria sigma factor KatF protein; Black-legged Tick; Borrelia; Borrelia burgdorferi; Candidate Disease Gene; Carditis; Complement; Complex; Data; Environment; Event; Experimental Designs; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Transcription; Individual; Infection; Knowledge; Lead; Lyme Disease; Mediating; Microarray Analysis; Molecular Profiling; Mus; Mutagenesis; Mutation; Nature; Pathogenesis; Pathogenicity; Pathway interactions; Pattern; Phenotype; Post-Transcriptional Regulation; Production; Proteins; Proteome; Proteomics; Regulation; Regulon; Relative (related person); Research; Role; Selection Criteria; Sigma Factor; Surface; System; Techniques; Therapeutic; Therapeutic Intervention; Ticks; Time; Two-Dimensional Gel Electrophoresis; Vaccines; Variant; Virulence; Work; antimicrobial; base; comparative; enhancer binding protein; gel electrophoresis; genetic element; interest; mutant; prevent; protein expression; protein profiling; public health relevance; research study; response; therapeutic target; two-dimensional; vaccine candidate; vector; vector transmission

DESCRIPTION (provided by applicant): During its natural lifecycle, the Lyme disease spirochete, Borrelia burgdorferi, occupies both an arthropod tick vector and a mammalian host. To persist in these two very distinct environments, the bacterium undergoes significant adaptive changes, which includes altering the expression of several major outer surface (lipo)proteins. This adaptive response is controlled by an enhancer-binding protein (Rrp2) that is responsible for activating an alternative sigma factor cascade, in which one sigma factor (RpoN, sigmaN, CN, C54) modulates the expression of a second alternative sigma factor (RpoS, sigmaS, CS, C38). RpoS expression then activates the transcription of several major outer surface (lipo)proteins. Because some of these rpoS-regulated outer surface (lipo)proteins contribute to the virulence of B. burgdorferi, it is not surprising that mutation of rpoS in B. burgdorferi renders the bacterium non-infectious. These findings underline the importance of studying B. burgdorferi RpoS-mediated gene regulation. While previous microarray experiments have furthered our understanding of global gene regulation by the Rrp2/RpoN/RpoS pathway in B. burgdorferi, the experimental design of comparing gene expression patterns between wild type and mutant strains is not ideal. The optimal experimental approach to identify genes that respond to a particular regulator would be to expose the bacteria to the specific condition(s) that activate the pathway and then observe the resulting changes in gene expression. Unfortunately, the conditions that optimally induce the Rrp2/RpoN/RpoS pathway are exceedingly complex. Therefore, we propose to use an alternative approach in which we artificially induce the regulator of interest (e.g. RpoS) and then assess its impact on gene regulation. In Specific Aim 1, we propose to use our newly developed lac inducible expression system to express RpoS and assess its impact on global gene expression in B. burgdorferi using transcriptional microarrays. This approach using the inducible expression system will allow us to identify those genes that are directly and immediately impacted by RpoS and assess whether there is temporal variation in RpoS-dependent gene activation. The information garnered from these studies will further our understanding of RpoS-mediated regulation, and allow us to make informed decisions regarding RpoS-regulated genes that will be the focus of Specific Aim 3. To date, all large-scale comparative gene expression studies investigating the regulatory role of the Rrp2/RpoN/RpoS pathway in B. burgdorferi gene expression have utilized transcriptional microarray techniques. To augment our transcriptional microarray analyses (Specific Aim 1), in Specific Aim 2, we will use two-dimensional difference in-gel electrophoresis (2D-DIGE) to observe changes in the B. burgdorferi proteome following expression of RpoS from our lac inducible expression system, as well as compare protein profiles between wild type Bb and an isogenic rpoS mutant. This will mark the first time that such a broad proteomic assessment has been carried out in B. burgdorferi specifically addressing the impact of the RpoS pathway on protein expression. We anticipate that the combined results from the transcriptional microarray comparison and the 2D-DIGE proteomic analyses will help us definitively identify and prioritize RpoS-regulated genes for the targeted mutational and phenotypic analyses described in Specific Aim 3. Many questions still exist regarding the full extent of RpoS-dependent global regulation, and it is likely that a number of the genes regulated by rpoS are required for the transmissibility, infectivity, and pathogenicity of B. burgdorferi. However, the majority of genes that are under the control of rpoS have no known predicted function. To begin ascribing functions for these RpoS-regulated genes, in Specific Aim 3, we will use targeted mutagenesis to create B. burgdorferi mutants in individual rpoS-regulated genes and then assess the phenotypes of these mutants in the experimental infectious lifecycle of B. burgdorferi. Although the majority of genes to be studied in this Aim will be identified using data from the transcriptional and proteomic studies in Specific Aims 1 and 2, we have used results from our previous transcriptional microarray comparisons to identify two rpoS- regulated genes which we can characterize as we are pursuing the analyses in Specific Aims 1 and 2. The knowledge gained from the Aims described in this proposal will serve to further our understanding of the regulatory events that modulate B. burgdorferi gene expression. We also will be able to begin ascribing functions for individual RpoS-regulated genes in the infectious lifecycle of B. burgdorferi, which will help us identify potential therapeutic targets that can be used to prevent and/or treat Lyme disease. PUBLIC HEALTH RELEVANCE: This project focuses on studying the regulation of pathogenesis and virulence in Borrelia burgdorferi, the bacterium that causes Lyme disease. In nature, this bacterium exists in either an arthropod/tick vector or a mammalian host, and to adapt to these two very diverse environments, the bacterium must undergo significant changes in gene expression. We are working to characterize the regulatory systems in B. burgdorferi that govern this adaptive response, as well as identify the individual genes that are controlled by these systems. Once we have gained a better understanding of regulatory networks and genes that contribute to vector transmission and mammalian infection, we can begin to develop new strategies to prevent (e.g. vaccines) or treat (e.g. antimicrobial therapeutics) Lyme disease.

描述（由申请人提供）：在其自然生命周期中，莱姆病螺旋体伯氏疏螺旋体占据节肢动物蜱媒介和哺乳动物宿主。为了在这两种截然不同的环境中生存，细菌经历了显着的适应性变化，其中包括改变几种主要外表面（脂）蛋白的表达。这种适应性反应由增强子结合蛋白 (Rrp2) 控制，该蛋白负责激活替代 sigma 因子级联，其中一个 sigma 因子 (RpoN、sigmaN、CN、C54) 调节第二个替代 sigma 因子 (RpoS) 的表达、sigmaS、CS、C38)。然后，RpoS 表达激活几种主要外表面（脂）蛋白的转录。由于这些 rpoS 调节的外表面（脂）蛋白中的一些有助于伯氏疏螺旋体的毒力，因此伯氏疏螺旋体中 rpoS 的突变使细菌不具有感染性也就不足为奇了。这些发现强调了研究伯氏疏螺旋体 RpoS 介导的基因调控的重要性。 虽然之前的微阵列实验进一步加深了我们对伯氏疏螺旋体中 Rrp2/RpoN/RpoS 途径的全局基因调控的理解，但比较野生型和突变株之间基因表达模式的实验设计并不理想。识别对特定调节因子作出反应的基因的最佳实验方法是将细菌暴露于激活该途径的特定条件，然后观察由此产生的基因表达变化。不幸的是，最佳诱导 Rrp2/RpoN/RpoS 途径的条件极其复杂。因此，我们建议使用另一种方法，即人工诱导感兴趣的调节因子（例如 RpoS），然后评估其对基因调节的影响。在具体目标 1 中，我们建议使用我们新开发的 lac 诱导表达系统来表达 RpoS，并使用转录微阵列评估其对伯氏疏螺旋体整体基因表达的影响。这种使用诱导表达系统的方法将使我们能够识别那些直接立即受到 RpoS 影响的基因，并评估 RpoS 依赖性基因激活是否存在时间变化。从这些研究中获得的信息将进一步加深我们对 RpoS 介导的调控的理解，并使我们能够对 RpoS 调控的基因做出明智的决定，这将是具体目标 3 的重点。迄今为止，所有大规模比较基因表达研究都在调查Rrp2/RpoN/RpoS 通路在伯氏疏螺旋体基因表达中的调节作用已利用转录微阵列技术。为了增强我们的转录微阵列分析（具体目标 1），在具体目标 2 中，我们将使用二维差异凝胶电泳 (2D-DIGE) 来观察 lac 诱导物表达 RpoS 后伯氏疏螺旋体蛋白质组的变化表达系统，以及比较野生型 Bb 和同基因 rpoS 突变体之间的蛋白质谱。这将标志着首次在伯氏疏螺旋体中进行如此广泛的蛋白质组学评估，专门研究 RpoS 途径对蛋白质表达的影响。我们预计转录微阵列比较和 2D-​​DIGE 蛋白质组分析的综合结果将帮助我们明确识别和优先考虑 RpoS 调节基因，以进行特定目标 3 中描述的靶向突变和表型分析。关于全面范围仍然存在许多问题RpoS 依赖性全局调控，并且很可能许多受 rpoS 调控的基因是伯氏疏螺旋体的传播性、感染性和致病性所必需的。然而，大多数受 rpoS 控制的基因没有已知的预测功能。为了开始确定这些 RpoS 调节基因的功能，在特定目标 3 中，我们将使用定向诱变在单个 rpoS 调节基因中创建伯氏疏螺旋体突变体，然后评估这些突变体在伯氏疏螺旋体实验感染生命周期中的表型。虽然本目标中要研究的大多数基因将使用特定目标 1 和 2 中转录和蛋白质组学研究的数据进行鉴定，但我们已经使用之前的转录微阵列比较的结果来鉴定两个 rpoS 调节的基因，我们可以表征它们因为我们正在进行具体目标 1 和 2 中的分析。从本提案中描述的目标中获得的知识将有助于进一步了解调节伯氏疏螺旋体基因表达的调控事件。我们还将能够开始归因于伯氏疏螺旋体感染生命周期中单个 RpoS 调节基因的功能，这将帮助我们确定可用于预防和/或治疗莱姆病的潜在治疗靶点。 公共健康相关性：该项目重点研究伯氏疏螺旋体（引起莱姆病的细菌）的发病机制和毒力的调节。在自然界中，这种细菌存在于节肢动物/蜱载体或哺乳动物宿主中，为了适应这两种非常不同的环境，细菌必须在基因表达方面发生显着变化。我们正在努力描述伯氏疏螺旋体控制这种适应性反应的调节系统的特征，并鉴定受这些系统控制的单个基因。一旦我们更好地了解了导致媒介传播和哺乳动物感染的调控网络和基因，我们就可以开始制定新的策略来预防（例如疫苗）或治疗（例如抗菌疗法）莱姆病。