Genetic Mechanisms in Borrelia burgdorferi Pathogenesis

描述（由申请人提供）：伯氏疏螺旋体是莱姆病的病原，是美国最常见的节肢动物传播的传染性病原体，在持续感染的患者中导致了大量的发病率。伯氏疏螺旋体在哺乳动物和节肢动物宿主中都能有效地定植，因此，必须快速调节基因表达以适应这些不同的环境。虽然我们知道一些改变伯氏疏螺旋体基因表达的分子信号，但我们对这种病原体中潜在的毒力决定因素是如何调控的仍然知之甚少。在过去的资助期内，我们已经鉴定了一种调节蛋白，称为BosR，它参与调节伯氏疏螺旋体的氧化应激反应。最近，我们发现与bosR相关的两个基因bb0646和bb0648共享一个转录本，因此包含一个操纵子。这些基因分别编码一种输出脂肪酶（bb0646）和一种丝氨酸/苏氨酸激酶（bb0648），我们认为它们参与了伯氏疏螺旋体的氧化应激反应。核心假设是BosR和相关基因bb0646和bb0648协调一个重要的适应性反应，感知细胞的氧化还原状态。为了解决这一假设，我们提出以下具体目标：(1)表征感染性伯氏疏螺旋体的bosR操纵子。目前的假设是，bosR及其两侧基因bb0646和bb0648对细胞的氧化还原状态做出适当的反应，以对抗节肢动物血餐或哺乳动物先天免疫反应中产生的有毒氧化化合物。我们尚未能够评估BosR在感染性分离株中的作用，可能是因为BosR调节必需基因。在这里，我们将使用最近开发的严格调控诱导系统来产生感染性伯氏疏螺旋体bosR的条件突变体；(2)评估条件突变体的感染性缺陷和bosr调控基因的敲除。目前的假设是，受BosR调控的基因是与氧化应激和感染性相关的重要生理过程所必需的；(3)确定bosr介导的调控机制。工作假设是BosR通过氧化和金属结合改变其调控活性，从而改变其对靶序列的亲和力；(4)破译BB0646和BB0648在伯氏疏螺旋体发病机制中的作用。我们的工作假设是，这两个基因产物分别通过修饰多不饱和脂质底物和协调对氧化应激的全局反应参与宿主适应。来自这些研究的信息将有助于了解伯氏疏螺旋体如何通过BosR、BB0646和BB0648适应宿主的氧化还原状态，并将有助于确定随后的反应如何与这种重要病原体的疾病潜力相关。

DESCRIPTION (provided by applicant): Borrelia burgdorferi, the etiologic agent of Lyme disease, is the most common arthropod- borne infectious agent in the United States, and contributes to a significant amount of morbidity in persistently infected patients. B. burgdorferi is effective at colonizing both mammalian and arthropod hosts and, as such, must modulate gene expression quickly to adapt to these different environments. Although we know some of the molecular signals that alter gene expression in B. burgdorferi, we still understand little regarding how potential virulence determinants are regulated in this pathogen. In the past funding period, we have characterized a regulatory protein, designated BosR, which is involved in regulating the oxidative stress response in B. burgdorferi. Recently, we found that two genes linked to bosR, bb0646 and bb0648, share a transcript and thus comprise an operon. These genes encode for an exported lipase (bb0646) and a serine/threonine kinase (bb0648), respectively, which we suggest are involved in the oxidative stress response in B. burgdorferi. The central hypothesis is that BosR, and the linked genes bb0646 and bb0648, coordinate an important adaptive response that senses the redox status of the cell. To address this hypothesis, we propose the following Specific Aims: (1) Characterize the bosR operon in infectious B. burgdorferi. The working hypothesis is that bosR and its flanking genes, bb0646 and bb0648, respond appropriately to the redox status of the cell to combat toxic oxidizing compounds generated during the arthropod blood meal or the mammalian innate immune response. We have not yet been able to evaluate the role of BosR in infectious isolates, presumably since bosR regulates essential genes. Here we will use a recently developed tightly regulated inducible system to generate a conditional mutant in bosR in infectious B. burgdorferi; (2) Assess the infectivity deficit in conditional mutants and knockouts in BosR-regulated genes. The working hypothesis is that genes regulated by BosR are required for physiologically important processes related to oxidative stress and infectivity; (3) Determine the mechanism of BosR-mediated regulation. The working hypothesis is that BosR alters its regulatory activity via oxidation and metal binding, which changes its avidity for target sequences; and (4) Decipher the role of BB0646 and BB0648 in B. burgdorferi pathogenesis. Our working hypothesis is that both of these gene products are involved in host adaptation by modifying polyunsaturated lipid substrates and coordinating a global response to oxidative stress, respectively. The information from these studies will provide insight into how B. burgdorferi adapts to the redox status of the host via BosR, BB0646, and BB0648, and will help to determine how the ensuing response relates to the disease potential of this important pathogen.