Genetic Mechanisms in Borrelia burgdorferi Pathogenesis

描述(申请人提供)：伯氏疏螺旋体是莱姆病的病原体，是美国最常见的节肢动物传播的感染源，在持续感染的患者中造成了相当大的发病率。伯氏假单胞菌在哺乳动物和节肢动物宿主上都很有效，因此必须迅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杆菌基因表达的分子信号，但对于这种病原菌中潜在的毒力决定因素是如何调控的，我们仍然知之甚少。在过去的资助期间，我们已经鉴定了一种名为BosR的调节蛋白，它参与调节伯氏杆菌的氧化应激反应。最近，我们发现与BosR连锁的两个基因bb0646和bb0648共享一个转录本，从而组成一个操纵子。这些基因分别编码输出脂肪酶(Bb0646)和丝氨酸/苏氨酸激酶(Bb0648)，我们认为这两个基因参与了伯氏杆菌的氧化应激反应。中心假设是，BosR和相关基因bb0646和bb0648协调一项重要的适应性反应，感知细胞的氧化还原状态。为了解决这一假设，我们提出了以下具体目标：(1)表征传染性伯氏杆菌中的BosR操纵子。工作假说是，BosR及其侧翼基因bb0646和bb0648对细胞的氧化还原状态做出适当反应，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。我们还不能评估BosR在感染性分离株中的作用，可能是因为BosR调节必要的基因。在这里，我们将使用最近开发的严格调控的诱导系统来在具有感染性的Burgdorferi中产生BosR中的条件突变；(2)评估在BosR调控的基因中的条件突变和敲除中的传染性缺陷。工作假说是，在与氧化应激和传染性相关的重要生理过程中，需要由BosR调控的基因；(3)确定BosR介导的调控机制。工作假说是，BosR通过氧化和金属结合改变其调节活性，从而改变其对靶序列的亲和力；以及(4)破译BB0646和BB0648在伯氏杆菌致病中的作用。我们的工作假设是，这两种基因产物分别通过修饰多不饱和脂肪底物和协调全球对氧化应激的反应参与宿主适应。这些研究的信息将为伯氏杆菌如何通过BosR、BB0646和BB0648适应宿主的氧化还原状态提供洞察，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有关。公共卫生相关性：伯氏疏螺旋体是莱姆病的病原体，是美国最常见的节肢动物传播的感染源，因此是一个重要的公共卫生问题。这里描述的研究旨在解决伯氏杆菌如何能够在宿主介导的氧化防御的背景下适应扁虱和哺乳动物，以及这种适应性反应如何影响这种细菌在环境中持续存在并导致疾病的能力。

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. PUBLIC HEALTH RELEVANCE: Borrelia burgdorferi, the etiologic agent of Lyme disease, is the most common arthropod-borne infectious agent in the United States, and thus is an important Public Health issue. The studies described herein are designed to address how B. burgdorferi is able to adapt to both ticks and mammals, in the context of host mediated oxidation defenses, and how this adaptive response affects the ability of this bacterium to persist within the environment and cause disease.