RpoS-mediated virulence regulation in Borrelia burgdorferi

RpoS 介导的伯氏疏螺旋体毒力调控

• 批准号: 8722793
• 负责人: Jon Scott Blevins
• 金额: $ 3.92万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-23 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8722793
• 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 Profile; 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; 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; genetic element; interest; mutant; prevent; protein expression; protein profiling; 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.

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同时占据节肢动物蜱虫载体和哺乳动物宿主。为了在这两种截然不同的环境中生存，细菌经历了显著的适应性变化，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（Rrp 2）控制，该增强子结合蛋白负责激活替代σ因子级联，其中一个σ因子（RpoN、σ N、CN、C54）调节第二个替代σ因子（RpoS、σ S、CS、C38）的表达。然后RpoS表达激活几种主要外表面（脂）蛋白的转录。因为这些rpos调节的外表面（脂）蛋白中的一些有助于B的毒力。burgdorferi，B. Burgdorferi使细菌不具有感染性。这些发现强调了学习B的重要性。burgdorferi RpoS介导的基因调控。虽然先前的微阵列实验已经进一步加深了我们对B中Rrp 2/RpoN/RpoS通路的整体基因调控的理解。Burgdorferi等人的研究表明，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。鉴定对特定调节剂有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下，然后观察基因表达的变化。不幸的是，最佳诱导Rrp 2/RpoN/RpoS途径的条件非常复杂。因此，我们建议使用一种替代方法，在这种方法中，我们人工诱导感兴趣的调节因子（例如RpoS），然后评估其对基因调控的影响。在具体目标1中，我们建议使用我们新开发的乳糖诱导表达系统来表达RpoS，并评估其对B中全局基因表达的影响。burgdorferi使用转录微阵列。这种使用诱导型表达系统的方法将使我们能够识别那些直接和立即受到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。到目前为止，所有大规模的比较基因表达研究调查Rrp 2/RpoN/RpoS途径在B中的调节作用。Burgdorferi基因表达利用了转录微阵列技术。为了增强我们的转录微阵列分析（特异性目标1），在特异性目标2中，我们将使用二维差异凝胶电泳（2D-DIGE）来观察B的变化。从我们的lac诱导型表达系统表达RpoS后的burgdorferi蛋白质组，以及比较野生型Bb和同基因rpoS突变体之间的蛋白质谱。这将标志着第一次在B中进行如此广泛的蛋白质组学评估。burgdorferi的研究，特别是解决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蛋白质组学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，用于特定目标3中描述的靶向突变和表型分析。关于RpoS依赖的全局调节的完整程度仍然存在许多问题，并且很可能由RpoS调节的许多基因是B的传播性、感染性和致病性所必需的。burgdorferi。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，我们将使用靶向诱变来创建B。在单个rpos调节基因中检测伯氏螺旋体突变体，然后在B的实验感染生命周期中评估这些突变体的表型。burgdorferi。尽管在本目标中要研究的大多数基因将使用来自特定目标1和2中的转录和蛋白质组学研究的数据来鉴定，但是我们已经使用来自我们之前的转录微阵列比较的结果来鉴定两个rpoS调节基因，我们可以在我们进行特定目标1和2中的分析时对其进行表征。从本提案中描述的目的中获得的知识将有助于我们进一步理解调节B的调节事件。burgdorferi基因表达。我们也将能够开始归属于功能的个别Rpos调控基因在传染性生命周期的B。这将有助于我们确定可用于预防和/或治疗莱姆病的潜在治疗靶点。