Leptospira interrogans Interactions with Endothelial Cells

描述(申请人提供)：钩端螺旋体病是世界上传播最广泛的人畜共患病，在城市贫民窟，特别是在热带地区，正在成为一种严重的传染病。钩端螺旋体属的几个物种可以引起感染，其严重程度从轻微疾病到伴有多器官衰竭的致命出血性疾病不等。我们对钩端螺旋体致病机制的认识存在很大差距，但对宿主细胞表面、细胞外基质成分和可溶性宿主分子的黏附可能是钩端螺旋体向几乎任何组织传播、持续定植某些组织和造成内皮损伤的关键。特别是，致病钩端螺旋体与特定宿主细胞表面受体的结合可能对该细菌在体内引起播散性感染和疾病的能力至关重要。 有免疫能力的宿主。与细胞表面分子的黏附也可能影响直接环境中的宿主细胞(S)如何对病原体的存在做出反应，以及病原体如何侵入组织。然而，在这一点上，对致病钩端螺旋体细胞如何与宿主细胞表面相互作用知之甚少。我们发现问号钩端螺旋体与内皮细胞表面受体的结合比与细胞外基质的结合更有效，VE-钙粘附素参与了这一活动。血管内皮细胞钙粘附素是内皮细胞完整性的主要介质。我们还根据与内皮细胞结合的选择确定了几种细胞特异性粘附素，其中两种粘附素与钙粘附素结合。此外，我们发现问号钩端螺旋体与内皮细胞的直接结合会导致细胞单层的破坏，并伴随着肌动蛋白细胞骨架的变化。这会导致内皮细胞对细菌的渗透性增加，并可能反映 广泛的内皮损伤是钩端螺旋体病病理生理学的一部分。我们现在建议检验这一假设，即特定的问号钩端螺旋体粘附素-VE-钙粘附素相互作用介导细菌与内皮细胞的附着，导致内皮细胞的破坏。在目标1中，我们将测试这一假设，即问号钩端螺旋体使用识别VE-钙粘附素的特定蛋白粘附素来介导与内皮细胞层的附着和破坏。我们将通过几种方法进一步评估我们的候选粘附素，包括确定它们是否增加了哺乳动物细胞对非致病双挠性乳杆菌的黏附活性。在目标2中，我们将利用生化、细胞生物学和遗传学方法来确定问号钩端螺旋体破坏内皮细胞的机制。我们已经确定的细胞结合细菌粘附素在内皮层破坏中的作用将被评估。本文提出的工作将极大地促进我们对致病钩端螺旋体如何与宿主细胞表面受体相互作用导致感染以及这些相互作用如何促进疾病的病理生理的理解，可能为疫苗开发或治疗提供新的候选方案。

DESCRIPTION (provided by applicant): Leptospirosis is the most widespread zoonotic disease worldwide, and is emerging as a significant infectious disease in urban slums, particularly in tropical regions. Several species of the genus Leptospira can cause infection, which can vary in severity from mild illness to fatal hemorrhagic disease with multiple organ failure. There is a significant gap in our understanding of the pathogenic mechanisms of Leptospira species, but adhesion to host cell surfaces, extracellular matrix (ECM) components, and soluble host molecules is likely to be critical for Leptospira species to disseminate to virtually any tissue, persistently colonize certain tissues, and cause endothelial damage. In particular, binding of pathogenic Leptospira species to specific host cell- surface receptors is likely to be critical to the ability of the bacteria to cause disseminated infection and disease in immunocompetent hosts. Adhesion to cell surface molecules may also affect how the host cell(s) in the immediate environment respond to the presence of the pathogen, and how the pathogen invades tissues. At this point, however, little is known about how pathogenic Leptospira cells interact with host cell surfaces. We found that L. interrogans binds to endothelial cell surface receptors more efficiently than to ECM, and that VE-cadherin participates in this activity. VE-cadherin is the major mediator of endothelial integrity. We also identified several cell-specific adhesins based on selection for binding to endothelial cells, two f which bind to cadherins. In addition, we found that direct L. interrogans binding to endothelial cells causes disruption of the cell monolayers, concomitant with changes in the actin cytoskeleton. This leads to increased endothelial permeability to the bacteria, and likely reflects the widespread endothelial damage that is part of the pathophysiology of leptospirosis. We now propose to test the hypothesis that specific L. interrogans adhesin- VE-cadherin interactions mediate bacterial attachment to endothelial cells, resulting in disruption of the endothelium. In Aim 1, we will test the hypothesis that L. interrogans uses specific protein adhesins that recognize VE-cadherin to mediate attachment to, and disruption of, endothelial layers. We will further evaluate our candidate adhesins in several ways, including determining whether they confer increased mammalian cell adhesion activity to non-pathogenic L. biflexa. In Aim 2, we will determine the mechanism of endothelial disruption by L. interrogans using biochemical, cell biological and genetic approaches. The roles of the cell- binding bacterial adhesins we have identified in endothelial layer disruption will be evaluated. The work proposed here will significantly advance our understanding of how pathogenic Leptospira species interact with host cell surface receptors to cause infection and how these interactions contribute to the pathophysiology of disease, may illuminate new candidates for vaccine development or therapeutics.