The RpoN-RpoS regulatory pathway in Borrelia burgdorferi

描述（由申请人提供）：伯氏疏螺旋体（Bb），莱姆病螺旋体，当它在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。在2001年，我们发现另一种sigma因子RpoS控制了关键外膜脂蛋白的合成，这些外膜脂蛋白赋予了Bb的哺乳动物传染性和致病性。我们还发现RpoS本身受另一个sigma因子rpo（）的控制，rpo（）与RpoS启动子结合。我们将这一新的通路命名为“rpo - rpos调控通路”。这个途径是第一个例子，一个可选的sigma因子调控级联控制细菌病原体的毒力。在Bb中，rpo - rpos通路被伴随蜱虫进食的各种环境刺激激活，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因子促进Bb中rpo依赖的rpoS转录。BosR是第一个例子，在任何细菌中，对依赖基因转录必不可少的额外激活剂。该系统代表了细菌中介导的基因控制的新范式。我们进一步确定BosR是一种含Zn+2的dna结合蛋白，它与atttaantttaaat的一个核心序列（“BosR box”）结合。生物信息学和体外电泳迁移转移分析揭示了rpoS中三个潜在的BosR结合位点，其中一个位于rpo结合位点附近。然而，关于BosR如何发挥作用，特别是当Bb处于蜱虫和哺乳动物的原生环境中时，仍有许多未解之谜。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。选定的rpoS启动子突变/缺失将检查其对生长Bb中rpoS表达的影响，选定的rpoS启动子突变也将评估其逃避蜱虫和感染和传播小鼠的能力。在目标2中，我们将使用核磁共振波谱和x射线晶体学对BosR进行结构表征，以检查解释BosR如何协调金属，倾向于形成同型二聚体，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Bb在蜱虫和哺乳动物的原生环境中受BosR、RpoN和RpoS影响的调控；这些以前没有被直接比较过。新的感兴趣的基因靶点也将评估其在Bb生命周期中的作用。这些联合研究将(i)提供潜在阻断Bb传播的新见解，（ii）阐明BosR激活中心rpo - rpos通路或其他rpo -非依赖性基因的Bb毒力新机制；(3)进一步阐明这种新的细菌基因调控模式。由此产生的发现可能导致莱姆病的新干预策略（疫苗，疗法）。

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.