Invasion Dynamics

摘要：项目3：威斯康星医学院 钩端螺旋体病是世界范围内传播最广泛的人畜共患病，其严重程度从轻微到 伴有多器官衰竭的致命出血性疾病。因为血管内皮损伤和血管增多 通透性是钩端螺旋体病的显著特征，我们重点鉴定问号钩端螺旋体粘附素 在培养中介导与人内皮细胞的附着，鉴定与其相关的内皮细胞受体 细菌结合，以及对钩端螺旋体与内皮细胞相互作用的后果的完整性 内皮层。致病性问号钩端螺旋体有效地跨越内皮层(跨内皮细胞迁移)， 而非致病双歧杆菌则不具有致病活性。内皮细胞的主要贡献者--细胞黏附连接 (AJS)和屏障功能，VE-钙粘附素，是问号钩端螺旋体的受体，问号钩端螺旋体引起 AJS，而双弯毛藻则不是。我们鉴定了两种能与纯化的VE-钙粘附素结合的问号钩端螺旋体粘附素 亲和力强。我们将与巴斯德和加州大学洛杉矶分校的团队合作，评估现有的 转座子突变克隆，以及细胞培养中的敲入(功能获得)和敲除突变 用动物模型鉴定和更好地鉴定有助于内皮细胞入侵的钩端螺旋体蛋白 障碍。在这些实验中，我们还将产生用于1)细菌和2)宿主基因分析的样本 表达，以及3)在相对简单的细胞培养模型中产生宿主反应生物标志物。数据集2和 3将与杜克大学小组产生的反应进行比较，以识别和表征产生的反应 在人类感染期间。我们的假设是钩端螺旋体病的内皮损伤是协同作用的结果。 细菌毒力决定因素和内皮细胞反应之间的关系。AIM 1将评估人类内皮细胞 钩端螺旋体不同分离株的细胞反应、侵袭动态和体内组织嗜性。内皮AJ 感染后完整性和细菌跨内皮细胞迁移将与临床和 环境菌株属于高毒和低毒致病种，腐生菌 物种，在接种后的不同时间。上清液将被用来量化细胞因子和其他潜在的 钩端螺旋体感染的生物标志物。细胞层和上清液将用于RNA-Seq以识别变化 在宿主和细菌的基因表达上。致病性、血管内皮细胞相关性的统计分析 损害和宿主反应将确定潜在的生物标志物作为患者血液中钩端螺旋体病的指标 样本，这些样本可以结合Duke小组获得的结果提供改进的诊断信息。 目标2将确定已知的和候选的问号钩端螺旋体黏附蛋白在黏附和 内皮细胞层的侵袭。一些感兴趣的候选粘附素包括一个富含亮氨酸的家族- 巴斯德小组确定的重复(LRR)蛋白，值得注意的是，LRR蛋白在 高致病性钩端螺旋体基因组。我们的工作将在基因组含量、蛋白质功能、 钩端螺旋体病内皮细胞损伤中的内皮反应。

Abstract: Project 3: Medical College of Wisconsin Leptospirosis is the most widespread zoonotic disease worldwide, and varies in severity from mild illness to fatal hemorrhagic disease with multiple organ failure. Since endothelial damage and increased vascular permeability are prominent features of leptospirosis, we focus on identification of L. interrogans adhesins that mediate attachment to human endothelial cells in culture, identification of endothelial cell receptors to which the bacteria bind, and on the consequences of Leptospira-endothelial cell interaction on the integrity of the endothelial layer. Pathogenic L. interrogans crosses endothelial layers (transendothelial migration) efficiently, while the non-pathogenic L. biflexa does not. The major contributor to endothelial cell-cell adherens junctions (AJs) and barrier function, VE-cadherin, is a receptor for L. interrogans, and L. interrogans causes disruption of AJs, while L. biflexa does not. We identified two L. interrogans adhesins that bind to purified VE-cadherin with high affinity. We will collaborate with the Pasteur and UCLA groups to assess phenotypes of existing transposon mutant clones, as well as knock-in (gain of function) and knock-down mutants in cell culture and in animal models to identify and better characterize Leptospira proteins that contribute to invasion of endothelial barriers. In these experiments we will also generate samples for analyses of 1) bacterial and 2) host gene expression, and 3) host response biomarker production in a relatively simple cell culture model. Datasets 2 and 3 will be compared to those generated by the Duke group to identify and characterize the responses generated during human infection. Our hypothesis is that endothelial damage in leptospirosis results from collaboration between bacterial virulence determinants and endothelial cell responses. Aim 1 will assess human endothelial cell responses to, invasion dynamics, and in vivo tissue tropism of, diverse Leptospira isolates. Endothelial AJ integrity and bacterial transendothelial migration will be compared after infection with clinical and environmental isolates belonging to high-virulence and low-virulence pathogenic species, and saprophytic species, at various times post-inoculation. Supernatants will be used to quantify cytokines and other potential biomarkers of Leptospira infection. Cell layers and supernatants will be used for RNA-Seq to identify changes in host and bacterial gene expression. Statistical analyses of associations between pathogenicity, endothelial damage, and host responses will identify potential biomarkers as indicators of leptospirosis in patient blood samples, which could inform improved diagnostics in conjunction with the results obtained by the Duke group. Aim 2 will determine the roles of known and candidate L. interrogans adhesion proteins in adherence to, and invasion of, endothelial cell layers. Some of the candidate adhesins of interest include a family of leucine-rich- repeat (LRR) proteins identified by the Pasteur group, and notably, LRR proteins are over-represented in highly pathogenic Leptospira genomes. Our work will establish ties between genome content, protein function, and endothelial responses in endothelial damage in leptospirosis.