Analysis of the Borrelia burgdorferi Integrin Ligand P66

描述（由申请方提供）：莱姆病螺旋体通过蜱叮咬从接种部位传播，并且能够持续存在，尽管有宿主免疫反应，这表明在感染期间与哺乳动物细胞的相互作用持续发生。B。伯氏菌与几种整合素结合，这些整合素对不同的细胞功能至关重要，包括维持组织完整性和参与免疫反应。我们确认了B的身份。burgdorferi表面蛋白P66作为3链整合素的配体。我们生成了B。burgdorferi p66突变体，并表明这些突变体在小鼠中不具有感染性，但在蜱中具有感染性。p66突变体在小鼠中从感染部位仅可培养1-2天，并且即使通过蜱传递也不具有感染性。p66突变体确实在植入腹膜腔的透析膜室的保护环境中存活，并且在血餐后的蜱中存活。因此，p66突变体不仅仅在细胞完整性或基本代谢功能方面受到损害。通过将p66恢复到染色体上来补充突变体，从而恢复小鼠的感染性。P66影响培养中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（如吞噬作用和细胞形状和组织完整性的维持）受P66影响的假设一致。我们的结果也与P66在调节血管通透性中的作用以及B的存在一致。蜱中的伯氏疏螺旋体增加了饱腹体重，表明响应于B的血管通透性增加。burgdorferi可能对蜱虫和细菌都有潜在的好处。我们的目标是确定B。Burgdorferi克服了建立持久性、传播性感染的宿主障碍，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B是必需的。由于操纵宿主细胞功能如吞噬作用和维持组织屏障，哺乳动物宿主中的伯氏螺旋体感染。我们将使用三种方法来检验我们的假设：目的1：检验p66突变体在接种部位被先天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P66是否是B所必需的。以避免在接种部位被巨噬细胞和树突细胞吞噬。目的2：检验p66突变体在进出脉管系统方面有缺陷的假设。我们将使用成像来分析和量化B。burgdorferi与脉管系统的相互作用，并将量化B。burgdorferi对已知引诱剂的趋化性。目的3：检验P66对B的能力至关重要的3-链结合的假设。引起感染。结合目的1和2，阻断整联蛋白功能的试剂和在P66中表达定点突变的细菌将用于研究P66在哺乳动物感染中的机制作用。这些研究将确定如何B。Burgdorferi克服了建立感染的宿主障碍，这对自然界中的生物体是至关重要的，并且可能提出预防和治疗莱姆病的新途径。

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.