The Heart of OspC as a Borrelia burgdorferi Adhesin

描述（由申请人提供）：伯氏疏螺旋体，莱姆病的病原体，建立持续感染，可影响关节，心脏，皮肤和神经系统。这种螺旋体建立感染的能力需要1)被感染的蜱虫从接种部位传播，2)适应哺乳动物环境，3)尽管宿主免疫反应，它仍然存在。在感染的每个步骤和阶段中，与哺乳动物细胞的相互作用不断发生。通过噬菌体展示筛选，我们发现伯氏疏螺旋体外膜蛋白P66与3链整合素结合。在活体噬菌体展示选择中，我们还发现OspC在活体小鼠内皮中作为一种未知受体的粘附素。携带OspC序列的噬菌体克隆是体内筛选率最高的单组，大多数克隆在心脏中被筛选。OspC是莱姆病伯氏疏螺旋体的一种蛋白质，当细菌从蜱虫传播到哺乳动物时，这种蛋白质就会表达出来。此前，另一组研究人员发现，OspC对小鼠感染的早期阶段至关重要。另一组研究表明，scid小鼠可以通过过度表达其他脂蛋白来部分克服OspC的缺乏。这项工作还表明，OspC似乎对细菌向心脏的正常传播至关重要，而我们的噬菌体克隆最常被选择在心脏组织中。我们的数据也与第三组先前的工作一致，表明心脏中的伯氏疏螺旋体比其他组织中的细菌表达更高水平的ospC。我们的总体假设是OspC的粘附素活性对其在伯氏疏螺旋体生命中的关键功能至关重要。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识别出一种位于伯氏疏螺旋体定植的心脏区域的受体。其次，在小鼠播散性感染的建立过程中，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B. burgdorferi菌株将在细胞培养和靶向小鼠心脏及其特定区域的粘附活性中进行测试。将使用相同的方法生成和测试表达OspC突变等位基因的其他菌株。在这组实验中，我们建议使用我们独特的工具来进一步定义，在生化水平上，OspC在伯氏疏螺旋体感染中的作用。我们期望这项工作能够设计出治疗方法，在蜱叮咬后甚至在未接种疫苗的人和动物中阻止伯氏疏螺旋体的传播。

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.