The RpoN-RpoS regulatory pathway in Borrelia burgdorferi

描述（由申请人提供）：莱姆病螺旋体伯氏疏螺旋体（Bb）在其不同蜱和哺乳动物宿主之间自然循环时经历显著的适应性变化。在2001年，我们发现，替代西格玛因子RpoS（β）控制关键外膜脂蛋白的合成，赋予哺乳动物对Bb的感染性和致病性。我们还发现，RpoS本身是由另一种选择性的sigma因子RpoN（β）控制的，它与rpoS启动子结合。我们将Bb中的这种新途径命名为“RpoN-RpoS调节途径”。该途径是控制细菌病原体中毒力的替代σ因子调节级联的第一个实例。RpoN-RpoS通路在Bb中被伴随蜱虫进食的各种环境刺激激活，并且在Bb的哺乳动物感染阶段持续。在过去的资助期间，我们取得了额外的惊人发现，除了RpoN激活所需的增强子结合蛋白（Rrp 2）之外，另一种称为BosR（BB 0647）的蛋白质作为第二激活剂发挥作用，以促进Bb中RpoN依赖性rpoS转录。BosR是细菌中第一个对β依赖性基因转录至关重要的额外激活剂的例子。该系统代表了细菌中β介导的基因控制的新范式。我们进一步确定了BosR是一种含Zn+2的DNA结合蛋白，其结合ATTTAANTTAAAT的核心序列（“BosR盒”）。生物信息学和体外电泳迁移率变动分析揭示了三个潜在的BosR结合位点内的RpoS，其中之一是位于紧邻的RpoN结合位点。然而，关于BosR如何发挥作用，特别是当Bb处于蜱和哺乳动物的原生环境中时，仍然存在许多未解答的问题。在本提案的目标1中，我们将评估rpoS中三个BosR结合位点的生物学作用。将检查选定的rpoS启动子突变/缺失对生长Bb中rpoS表达的影响，还将评估选定的rpoS启动子突变体逃离蜱虫以及在小鼠中感染和传播的能力。在目标2中，我们将使用NMR光谱学和X射线晶体学对BosR的结构进行表征，以检查解释BosR如何与金属配位的参数，具有形成同源二聚体的偏好，参与蛋白质-蛋白质相互作用，以及它如何与DNA构象结合。在目标3中，我们将确定当Bb在蜱和哺乳动物的天然环境中时，BosR，RpoN和RpoS影响的调节子;这些以前没有直接比较。还将评估感兴趣的新基因靶点在Bb生命周期中的作用。这些综合研究将（i）为潜在阻断Bb的传播提供新的见解，（ii）阐明BosR激活中心RpoN-RpoS途径或其他RpoN非依赖性基因的新机制，以实现Bb的毒力;（iii）进一步阐明β介导的细菌基因控制的新范式。由此产生的发现可能导致莱姆病的新干预策略（疫苗，治疗）。

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.