RpoS-mediated virulence regulation in Borrelia burgdorferi

RpoS 介导的伯氏疏螺旋体毒力调控

• 批准号: 8259762
• 负责人: Jon Scott Blevins
• 金额: $ 35.35万
• 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/8259762
• 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; Health; 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，既占据节肢动物蜱媒介，也占据哺乳动物宿主。为了在这两种截然不同的环境中生存，细菌经历了重大的适应性变化，其中包括改变几种主要外表面（脂质）蛋白的表达。这种适应性反应由增强子结合蛋白（Rrp2）控制，该蛋白负责激活备选sigma因子级联，其中一个sigma因子（RpoN, sigmaN， CN, C54）调节第二个备选sigma因子（RpoS, sigmaS， CS, C38）的表达。RpoS的表达随后激活几种主要外表面（脂质）蛋白的转录。由于一些rpoS调控的外表面（脂质）蛋白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传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已经进一步了解了伯氏疏螺旋体中rp2/RpoN/RpoS通路的全局基因调控，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。确定对特定调节因子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最终变化。不幸的是，诱导Rrp2/RpoN/RpoS通路的最佳条件非常复杂。因此，我们建议采用另一种方法，即人工诱导感兴趣的调控因子（例如RpoS），然后评估其对基因调控的影响。在Specific Aim 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调控基因做出明智的决定，这将是Specific Aim 3的重点。迄今为止，所有研究Rrp2/RpoN/RpoS通路在伯氏疏螺旋体基因表达中的调控作用的大规模基因表达比较研究都使用了转录微阵列技术。为了增强我们的转录微阵列分析（Specific Aim 1），在Specific Aim 2中，我们将使用二维凝胶电泳（2D-DIGE）来观察从lac诱导表达系统中表达RpoS后伯氏螺旋体蛋白质组的变化，并比较野生型Bb和等基因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，专门针对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地识别和优先考虑rpos调节基因，用于特异性目标3中描述的靶向突变和表型分析。关于rpoS依赖的全球调控的完整程度仍然存在许多问题，并且rpoS调控的许多基因很可能是伯氏疏螺旋体的传播性、传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调控基因的功能，在Specific Aim 3中，我们将使用靶向诱变技术在单个rpos调控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。虽然本目标中研究的大多数基因将使用特异性目标1和2中转录和蛋白质组学研究的数据来确定，但我们已经使用了之前转录微阵列比较的结果来确定两个rpoS调节基因，我们可以在特异性目标1和2中进行分析时对其进行表征。从本提案中描述的Aims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。我们还将能够开始确定单个rpos调节基因在伯氏螺旋体感染生命周期中的功能，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。