The RpoN-RpoS regulatory pathway in Borrelia burgdorferi

描述（由申请人提供）：伯氏疏螺旋体（Bb）是莱姆病螺旋体，在自然界中不同的蜱虫和哺乳动物宿主之间循环时经历了巨大的适应性变化。 2001 年，我们发现替代 sigma 因子 RpoS（� 控制关键外膜脂蛋白的合成，从而赋予 Bb 哺乳动物感染性和致病性。我们还发现 RpoS 本身受另一个替代 sigma 因子 RpoN (� �) 控制，该因子与 rpoS 启动子结合。我们将 Bb 中的这一新途径命名为“RpoN-RpoS 调节途径”。该途径是替代途径的第一个例子控制细菌病原体毒力的西格玛因子调节级联在 Bb 中被蜱进食时的各种环境刺激所激活，并且在 Bb 的哺乳动物感染阶段持续存在。在过去的资助期间，我们做出了额外的惊人发现，除了 RpoN 激活所需的增强子结合蛋白 (Rrp2) 之外，另一种称为 BosR (BB0647) 的蛋白质也可作为促进 RpoN 依赖性的第二激活剂。 Bb 中的 rpoS 转录是任何细菌中 β 依赖性基因转录所必需的额外激活剂的第一个例子。我们进一步确定 BosR 是一种含有 Zn 2 的 DNA 结合蛋白，可与 ATTTAANTTAAAT 的核心序列（“BosR 盒”）结合。然而，关于 BosR 如何发挥作用，仍有许多未解答的问题，特别是当 Bb 处于蜱和哺乳动物的天然环境中时，我们将评估 rpoS 中三个 BosR 结合位点的生物学作用，并将检查选定的 rpoS 启动子突变/缺失对生长中的 Bb 和选定的 rpoS 启动子的影响。在目标 2 中，我们将使用 NMR 光谱和 X 射线晶体学对 BosR 进行结构表征，以检查解释 BosR 如何与金属配合、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在构象上与 DNA 结合的参数。当 Bb 在蜱和哺乳动物的天然环境中时，这些新的目标基因也将被评估其在 Bb 生命周期中的作用，(i) 为潜在地阻断 Bb 的传播提供新的见解，(ii) 阐明 BosR 激活中央 RpoN-RpoS 途径或其他与 Bb 毒力无关的基因的新机制，以及 (iii) 进一步阐明这一点。介导的细菌基因控制的新范式。由此产生的结果可能会导致莱姆病的新干预策略（疫苗、治疗方法）。

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.