The RpoN-RpoS regulatory pathway in Borrelia burgdorferi

伯氏疏螺旋体中的 RpoN-RpoS 调控途径

• 批准号: 9208731
• 负责人: MICHAEL V. NORGARD
• 金额: $ 45.34万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9208731
• 关键词: ATP phosphohydrolase; Arthropods; Bacteria; Bacteria sigma factor KatF protein; Bacterial Genes; Binding; Binding Proteins; Binding Sites; Bioinformatics; Biological; Borrelia; Borrelia burgdorferi; Borrelia oxidative stress regulator; Complex; Consensus Sequence; DNA; DNA-Binding Proteins; DNA-Directed RNA Polymerase; Data; Deletion Mutation; Dialysis procedure; Disease; Electrophoretic Mobility Shift Assay; Employee Strikes; Environment; Funding; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Greater sac of peritoneum; Human; Immunoblotting; Implant; In Vitro; Infection; Intervention; Lead; Life Cycle Stages; Lipoproteins; Lyme Disease; Mammals; Mediating; Membrane; Metals; Methods; Molecular Conformation; Mus; NMR Spectroscopy; Names; Nature; Pathogenesis; Pathogenicity; Pathway interactions; Phase; Process; Proteins; Quantitative Reverse Transcriptase PCR; Rattus; Regulatory Pathway; Regulon; Reporter; Research; Role; Sigma Factor; Stimulus; Structure; System; Therapeutic; Ticks; Tissue-Specific Gene Expression; Vaccines; Virulence; X-Ray Crystallography; base; biological adaptation to stress; enhancer binding protein; enzootic; feeding; genetic manipulation; insight; interest; mutant; novel; pathogen; promoter; protein protein interaction; public health relevance; structural biology; three dimensional structure; tool; trait; transcriptome sequencing; transmission process; uptake

DESCRIPTION (provided by applicant): Borrelia burgdorferi (Bb), the Lyme disease spirochete, undergoes dramatic adaptive changes as it cycles in nature between its diverse tick and mammalian hosts. In 2001, we discovered that the alternative sigma factor RpoS (� controls the synthesis of key outer membrane lipoproteins that confer mammalian infectivity and pathogenicity to Bb. We additionally discovered that RpoS itself is controlled by another alternative sigma factor, RpoN (� �), that binds to the rpoS promoter. We named this novel pathway in Bb the "RpoN-RpoS regulatory pathway". This pathway is the first example of an alternative sigma factor regulatory cascade controlling virulence in a bacterial pathogen. The RpoN-RpoS pathway is activated in Bb by various environmental stimuli that accompany tick feeding, and is sustained during Bb's mammalian infection phase. Over the past funding interval, we made the additional striking discovery that in addition to the enhancer- binding protein (Rrp2) needed for RpoN activation, another protein known as BosR (BB0647) functions as a second activator to promote RpoN-dependent rpoS transcription in Bb. BosR is the first example, in any bacterium, of an additional activator essential for �-dependent gene transcription. This system represents a new paradigm of �-mediated gene control in bacteria. We have further determined that BosR is a Zn+2- containing DNA-binding protein that binds to a core sequence ("BosR box") of ATTTAANTTAAAT. Bioinformatics and in vitro electrophoretic mobility shift assays have revealed three potential BosR binding sites within rpoS, one of which is located immediately adjacent to the RpoN binding site. However, there remain many unanswered questions concerning how BosR functions, particularly when Bb is in its native environments of ticks and mammals. In Aim 1 of this proposal, we shall assess the biological role(s) of the three BosR binding sites in rpoS. Selected rpoS promoter mutations/deletions will be examined for their influence on rpoS expression in growing Bb, and selected rpoS promoter mutants also will be assessed for their ability to escape from ticks and infect and disseminate in mice. In Aim 2, we shall structurally character BosR, using both NMR spectroscopy and X-ray crystallography, to examine parameters explaining how BosR coordinates metal, has a predilection to form homodimers, participates in protein-protein interactions, and how it binds to DNA conformationally. In Aim 3, we shall determine the regulons influenced by BosR, RpoN, and RpoS when Bb is within its native settings of ticks and mammals; these have not been previously compared directly. New gene targets of interest also will be assessed for their roles in the life cycle of Bb. These combined studies will (i) provide new insights into potentially blocking the transmission of Bb, (ii) clarify the novel mechanism by which BosR activates the central RpoN-RpoS pathway or other RpoN-independent genes for Bb's virulence; and (iii) further elucidate this new paradigm of �-mediated bacterial gene control. Resultant findings could lead to new intervention strategies (vaccines, therapeutics) for Lyme disease.

描述(申请人提供)：伯氏疏螺旋体(BB)，莱姆病螺旋体，经历了戏剧性的适应性变化，因为它在自然界中在不同的扁虱和哺乳动物宿主之间循环。2001年，我们发现替代的西格玛因子rpos(�)控制着关键外膜脂蛋白的合成，这些外膜蛋白赋予哺乳动物感染性和致病性。我们还发现，RpoS本身受另一种可供选择的sigma因子RpoN(��)控制，它与Rpos启动子结合。我们将这一新途径命名为“RpoN-rpos调控途径”。该途径是控制细菌病原体毒力的另一种西格玛因子调控级联反应的第一个例子。在BB中，RpoN-rpos通路被各种环境刺激激活，并在BB的哺乳动物感染阶段持续存在。在过去的资助期间，我们有了另一个惊人的发现，除了RpoN激活所需的增强子结合蛋白(RRP2)外，另一种被称为BosR(BB0647)的蛋白作为第二激活剂在BB中促进RpoN依赖的rpos转录。在任何细菌中，BosR是第一个对依赖�的基因转录必不可少的额外激活剂的例子。该系统代表了细菌中�介导的基因控制的新范例。我们进一步确定BosR是一个含锌离子的DNA结合蛋白，与ATTTAANTTAAAT的核心序列(“BosR box”)结合。生物信息学和体外电泳迁移率改变分析显示，rpos内有三个潜在的BosR结合部位，其中一个位于RpoN结合部位附近。然而，关于BosR如何发挥作用仍有许多悬而未决的问题，特别是当BB处于扁虱和哺乳动物的自然环境中时。在本提案的目标1中，我们将评估三个BosR结合位点在rpos中的生物学作用(S)。选定的rpos启动子突变/缺失将被检测它们对生长中的BB中rpos表达的影响，而选定的rpos启动子突变也将被评估它们逃脱蜱虫并在小鼠中感染和传播的能力。在目标2中，我们将使用核磁共振光谱和X射线结晶学对BosR进行结构表征，以检查解释BosR如何与金属配位、倾向于形成同源二聚体、参与蛋白质-蛋白质相互作用以及它如何与DNA构象结合的参数。在目标3中，我们将确定当BB处于扁虱和哺乳动物的天然环境中时，受BosR、RpoN和rpos影响的调节子；这些以前没有被直接比较过。还将评估新的感兴趣的基因靶点在BB生命周期中的作用。这些联合研究将(I)为潜在地阻断BB的传播提供新的见解；(Ii)阐明BosR激活中枢RpoN-rpos途径或其他RpoN不依赖于RpoN的基因以促进BB毒力的新机制；以及(Iii)进一步阐明�介导的细菌基因控制的这一新范式。其结果可能导致莱姆病的新干预策略(疫苗、疗法)。