Analysis of the Borrelia burgdorferi Integrin Ligand P66

描述(申请人提供)：莱姆病螺旋体通过扁虱叮咬从接种部位传播，并能够在宿主免疫反应的情况下持续存在，表明在感染过程中与哺乳动物细胞不断发生相互作用。伯氏杆菌与几种整合素结合，这些整合素对多种细胞功能至关重要，包括维持组织完整性和参与免疫反应。我们鉴定了伯氏杆菌表面蛋白P66是3链整合素的配体。我们产生了伯氏杆菌p66-突变体，并表明这些突变体在小鼠中不会感染，但在扁虱中会感染。P66突变体在小鼠的感染部位只能培养1-2天，即使通过扁虱传播也不具传染性。P66-突变体确实在植入腹膜腔的受保护的透析膜室的环境中存活，并在血餐后的扁虱中存活。因此，p66-突变体不仅在细胞完整性或基本代谢功能上受到损害。通过将p66恢复到染色体上来补充突变体，可以恢复小鼠的感染性。P66影响培养的人类细胞对细菌的反应，我们的结果与宿主对病原体的反应，如吞噬和维持细胞形状和组织完整性，是受p66影响的假说。我们的结果也与p66在调节血管通透性方面的作用是一致的，并且伯氏杆菌在硬蜱中的存在增加了耗竭重量，这表明对伯氏杆菌的反应增加的血管通透性可能对扁虱和细菌都有利。我们的目标是确定伯氏杆菌如何克服宿主障碍，建立持续的、传播性感染，这对生物体的生命至关重要。我们的假设是，p66的整合素结合活性对于哺乳动物宿主中的伯氏杆菌是必不可少的，这是由于对宿主细胞功能的操纵，如吞噬和维持组织屏障。我们将使用三种方法验证我们的假设：目标1：测试p66-突变体在接种地点被天然免疫系统的细胞迅速吞噬的假设。我们将使用成像和靶向细胞消融来确定伯氏杆菌是否需要p66来避免接种部位的巨噬细胞和树突状细胞吞噬。目的2：验证p66-突变体在进入和离开血管系统的过程中存在缺陷的假设。我们将使用成像来分析和量化伯氏杆菌与血管系统的相互作用，并将定量伯氏杆菌对已知引诱剂的趋化作用。目的3：验证p66 3链结合对伯氏杆菌感染能力的影响这一假说。与AIMS 1和2一起，阻断整合素功能的试剂和表达p66定点突变的细菌将被用于研究p66在哺乳动物感染中的机制作用。这些研究将确定伯氏杆菌如何克服宿主障碍建立感染，这在自然界中对生物体至关重要，并可能为莱姆病的预防和治疗提供新的途径。

DESCRIPTION (provided by applicant): Lyme disease spirochetes disseminate from the site of inoculation via tick bite, and are able to persist despite the host immune response, indicating that interactions with mammalian cells occur continually during infection. B. burgdorferi binds to several integrins, which are critical to diverse cellular functions including maintenance of tissue integrity and participating in the immune response. We identified the B. burgdorferi surface protein P66 as a ligand for the ¿3-chain integrins. We generated B. burgdorferi p66- mutants, and showed that these mutants are not infectious in mice, but are in ticks. The p66 mutants are cultivable from the site of infection in mice for only 1-2 days, and are not infectious even when delivered by ticks. The p66- mutants do survive in the protected environment of dialysis membrane chambers implanted into the peritoneal cavity, and in the tick after the bloodmeal. Thus, the p66- mutants are not simply compromised in cell integrity or essential metabolic function. Complementation of the mutants by restoration of p66 to the chromosome restores infectivity in mice. P66 affects the responses of human cells in culture to the bacteria, and our results are consistent with the hypothesis that host responses to pathogens, such as phagocytosis and maintenance of cell shape and tissue integrity, are affected by P66. Our results are also consistent with a role for P66 in regulation of vascular permeability, and the presence of B. burgdorferi in ticks increases repletion weight, suggesting that increased vessel permeability in response to B. burgdorferi could potentially benefit the tick as well as the bacterium. Our goal is to determine how B. burgdorferi overcomes host barriers to the establishment of persistent, disseminated infection, which is critical to the life of the organism. Our hypothesis is that the integrin binding activity of P66 is essential to B. burgdorferi in the mammalian host due to manipulation of host cell functions such as phagocytosis and maintenance of tissue barriers. We will test our hypothesis using three approaches: Aim 1: Test the hypothesis that the p66- mutants are rapidly phagocytosed by cells of the innate immune system at the site of inoculation. We will use imaging and targeted cell ablation to determine whether P66 is required for B. burgdorferi to avoid phagocytosis by macrophages and dendritic cells at the site of inoculation. Aim 2: Test the hypothesis that the p66- mutants are defective in crossing into and out of the vasculature. We will use imaging to analyze and quantify B. burgdorferi interactions with the vasculature, and will quantify B. burgdorferi chemotaxis toward known attractants. Aim 3: Test the hypothesis that ¿3-chain binding by P66 is essential to the ability of B. burgdorferi to cause infection. In conjunction with Aims 1 and 2, reagents that block integrin function and bacteria expressing site- directed mutations in P66 will be used to investigate the mechanistic role of P66 in mammalian infection. These studies will define how B. burgdorferi overcomes host barriers to the establishment of infection, which is critical to the organism in nature, and may suggest novel routes to prevention and treatment of Lyme disease.