The Heart of OspC as a Borrelia burgdorferi Adhesin

描述(申请人提供)：伯氏疏螺旋体是莱姆病的病原体，可持续感染，影响关节、心脏、皮肤和神经系统。这种螺旋体建立感染的能力需要1)从受感染的壁虱接种的地方传播，2)适应哺乳动物的环境，3)尽管宿主有免疫反应，但仍要坚持下去。在感染的每个步骤和阶段，与哺乳动物细胞的相互作用不断发生。通过体外噬菌体展示筛选，我们发现伯氏杆菌外膜蛋白p66与3链整合素结合。在活体噬菌体展示选择中，我们还显示OspC在活着的小鼠体内作为一种未知的内皮受体的粘附素。携带OspC序列的噬菌体克隆是体内选择最高的一组克隆，并且大多数克隆是在心脏中选择的。OspC是莱姆病疏螺旋体的一种蛋白质，当细菌从壁虱转移到哺乳动物时表达，此前另一组研究表明，它对小鼠感染的早期阶段至关重要。另一组研究表明，SCID小鼠的OSPC缺陷可以通过其他脂蛋白的过度表达而部分克服。这项工作还揭示了OspC似乎对于细菌向心脏的正常传播是必不可少的，心脏是我们最经常选择噬菌体克隆的组织。我们的数据也与第三个小组之前的工作一致，该工作表明，伯氏杆菌在心脏中表达的SPC水平高于其他被测试组织中的细菌。我们的总体假设是OspC的粘附素活性对其在伯氏假单胞菌生活中的关键功能是重要的。基于我们的结果以及其他三个小组的结果，我们将在本提案中测试这一假设的两个方面。首先，OSPC识别定位于伯氏杆菌定植的心脏区域的受体。第二，在建立小鼠播散性感染时，粘附素的活性和正常功能需要特定的保守氨基酸。表达和不表达OspC的伯氏杆菌菌株将在细胞培养中进行黏附活性测试，并在小鼠的心脏及其特定区域进行靶向。其他表达OSPC突变等位基因的菌株将被产生并使用相同的方法进行测试。在这组实验中，我们建议使用我们独特的工具在生化水平上进一步定义OspC在伯氏杆菌感染中的作用。我们预计，这项工作可能会使设计治疗方法，即使在未接种疫苗的人和动物中，也能阻止壁虱叮咬后伯氏杆菌的传播。 公共卫生相关性：莱姆病现在是北半球最流行的媒介传播疾病，在由于疾病的神经学、心脏和风湿学表现而常见的地区，莱姆病对卫生系统造成重大负担。我们打算深入研究伯氏疏螺旋体是如何与哺乳动物宿主的心脏相互作用的。我们将专注于一种伯氏杆菌蛋白OspC，它是细菌在小鼠中引起正常播散性感染所必需的。我们的总体目标是了解OspC对宿主细胞分子的识别如何允许伯氏杆菌克服宿主屏障，建立持续的、播散性感染，这最终可能导致帮助宿主免疫系统清除作为抗生素治疗辅助手段的机体的策略。

DESCRIPTION (provided by applicant): Borrelia burgdorferi, the causative agent of Lyme disease, establishes persistent infection that can affect the joints, heart, skin, and nervous system. The ability of this spirochete to establish infection demands 1) dissemination from the site of inoculation by an infected tick, 2) adaptation to the mammalian environment, and 3) persistence despite the host immune response. Interactions with mammalian cells occur continually during each of these steps and phases of infection. Using a phage display selection in vitro, we showed that the B. burgdorferi outer membrane protein, P66, binds to the 3-chain integrins. In an in vivo phage display selection, we also showed that OspC serves as an adhesin to an unknown receptor in the endothelium in living mice. Phage clones bearing OspC sequences were the single group most highly selected in vivo, and most of the clones were selected in the heart. OspC is a protein of the Lyme disease Borrelia that is expressed as the bacteria move from tick to mammal, and was previously shown by another group to be critical for the earliest stages of infection of mice. A different group showed that a deficiency in OspC could be partially overcome in scid mice by over-expression of other lipoproteins. This work also revealed that OspC does appear to be essential for normal dissemination of the bacteria to the heart, the tissue in which our phage clones were most frequently selected. Our data are also consistent with previous work by a third group demonstrating that B. burgdorferi in the heart express higher levels of ospC than do the bacteria in other tissues tested. Our overall hypothesis is that the adhesin activity of OspC is important to its critical function in the life of B. burgdorferi. Based on our results as well as those of the three other groups, we will test two facets of this hypothesis in this proposal. The first is that OspC recognizes a receptor localized in regions of the heart colonized by B. burgdorferi. The second is that specific conserved amino acids are required for adhesin activity and normal function in establishment of disseminated infection in mice. B. burgdorferi strains that do and do not express OspC will be tested for adhesion activity in cell culture and targeting of the heart, and particular regions therein, in mice. Additional strains expressing mutated alleles of OspC will be generated and tested using the same approaches. In this set of experiments we propose to employ our unique tools to further define, at the biochemical level, the role of OspC in B. burgdorferi infection. We anticipate that this work may enable the design of therapeutic approaches to halt dissemination of B. burgdorferi after the tick bite even in unvaccinated people and animals. PUBLIC HEALTH RELEVANCE: Lyme disease is now the most prevalent vector-borne illness in the northern hemisphere, and a significant burden on the health system in regions in which it is common due to neurologic, cardiac, and rheumatologic manifestations of disease. We propose to delve into how Borrelia burgdorferi interacts with the heart in a mammalian host. We will focus on one B. burgdorferi protein, OspC, which is essential for the bacteria to cause the normal disseminated infection in mice. Our overall goal is to understand how OspC recognition of host cell molecules allows B. burgdorferi to overcome host barriers to the establishment of persistent, disseminated infection, which could eventually lead to strategies that will help the host immune system clear the organism as adjuncts to antibiotic therapy.