Outer Membrane Proteins of Pathogenic Leptospira Species

描述（由申请方提供）：钩端螺旋体病是一种常见的人畜共患疾病，通常通过大鼠接触传播给人类。人类的钩端螺旋体感染经常导致以肝功能障碍、肾衰竭和肺出血为特征的暴发性、危及生命的疾病。钩端螺旋体病正在世界上经历快速城市化的地区出现。迫切需要研究来了解这些致病性螺旋体如何与它们的哺乳动物宿主相互作用。本研究的重点是阐明外膜蛋白介导的致病和免疫的分子机制。 我们的实验室，在这一领域的领导者，已经制定了膜分离和识别表面暴露的外膜蛋白的策略。我们已经确定了一个家庭的钩端螺旋体免疫球蛋白样重复（Lig）蛋白的目标是由人类免疫反应钩端螺旋体病。当毒性钩端螺旋体暴露于宿主生理水平的渗透压（~ 300 mOsm/升）时，LigA和LigB的表达显著增加，而LipL 36和IPS表达下调，已知这些变化与哺乳动物宿主的感染有关。通过渗透压的调节表明Lig表达发生在感染过程的早期。我们最近表明，LigA和LigB是纤连蛋白和纤维蛋白原结合蛋白，表明这些蛋白质参与宿主组织定植。在LigA和LigB中，纤连蛋白和纤维蛋白原结合结构域重叠，表明蛋白质-蛋白质相互作用的新机制。 具体目标1描述了旨在确定Lig蛋白在钩端螺旋体与粘连蛋白、纤维蛋白原和层粘连蛋白等粘附基质分子结合中的作用的研究。我们将研究的假设，LigA和LigB是混杂的粘附素能够结合多个粘附基质分子使用一个单一的粘附域。具体目标2的目标是确定在感染过程中暴露于钩端螺旋体表面的OMPs表达上调，使用免疫组织化学，逆转录酶PCR，膜分离和表面暴露分析。特异性目的3将确定哪种α-上调的OMP是钩端螺旋体病仓鼠模型中的保护性免疫原。我们将使用杀菌活性、生长抑制、调理吞噬作用和粘附抑制试验来检查针对β-上调的外膜蛋白的抗体介导保护的机制。

DESCRIPTION (provided by applicant): Leptospirosis is a common zoonotic disease, typically transmitted to humans by rat exposure. Leptospiral infection in humans frequently results in fulminant, life-threatening illness characterized by liver dysfunction, kidney failure, and pulmonary hemorrhage. Leptospirosis is emerging in areas of the world undergoing rapid urbanization. Research is urgently needed to understand how these pathogenic spirochetes interact with their mammalian hosts. The focus of this proposal is to elucidate the molecular mechanisms of pathogenesis and immunity mediated by outer membrane proteins (OMPs). Our laboratory, a leader in this field, has developed strategies for membrane fractionation and identification of surface-exposed OMPs. We have identified a family of leptospiral immunoglobulin-like repeat (Lig) proteins that are targeted by the human immune response to leptospirosis. When virulent leptospires are exposed to host-physiologic levels of osmolarity (-300 mOsm/liter), expression of LigA and LigB is dramatically increased, while LipL36 and IPS expression are downregulated, changes known to be associated with infection of the mammalian host. Regulation by osmolarity indicates that Lig expression occurs early in the infectious process. We have recently shown that the LigA and LigB are fibronectin- and fibrinogen-binding proteins, indicating that these proteins are involved in host tissue colonization. In both LigA and LigB, the fibronectin- and fibrinogen-binding domains overlap, indicating a novel mechanism of protein-protein interaction. Specific Aim 1 describes studies designed to determine the role of Lig proteins in leptospiral binding to adhesive matrix molecules including fibronectin, fibrinogen, and laminin. We will examine the hypothesis that LigA and LigB are promiscuous adhesins able to bind multiple adhesive matrix molecules using a single adhesion domain. The goal of Specific Aim 2 is to identify osmo-upregulated OMPs exposed on the leptospiral surface during infection using immuno-histochemistry, reverse-transcriptase PCR, membrane fractionation, and surface-exposure analysis. Specific Aim 3 will determine which osmo-upregulated OMPs are protective immunogens in the hamster model of leptospirosis. We will examine the mechanism(s) by which antibodies to osmo-upregulated OMPs mediate protection using assays of bactericidal activity, growth inhibition, opsonophagocytosis, and adherence inhibition.