Invasion Dynamics

摘要：项目3：威斯康星州医学院 钩端螺旋体病是世界范围内最广泛的人畜共患疾病，其严重程度从轻度疾病到 致命的出血性疾病，伴有多器官衰竭。由于内皮损伤和血管增加 渗透性是钩端螺旋体病的突出特征，我们着重于鉴定L。问号粘附素， 介导对培养的人内皮细胞的附着，鉴定与其结合的内皮细胞受体， 细菌结合，以及钩端螺旋体-内皮细胞相互作用对细胞完整性的影响。 内皮层致病湖问号线虫有效地穿过内皮层（经内皮迁移）， 而非致病性L. biflexa没有。内皮细胞-细胞粘附连接的主要贡献者 (AJs)和屏障功能，VE-钙粘蛋白，是L.问号和L.问号会导致 AJs，L. biflexa没有。我们发现了两个L。与纯化的VE-钙粘蛋白结合， 高亲和力。我们将与巴斯德和加州大学洛杉矶分校的小组合作，评估现有的 转座子突变体克隆，以及在细胞培养物中的敲入（功能获得）和敲低突变体， 动物模型，以确定和更好地表征有助于侵入内皮细胞的钩端螺旋体蛋白质 隔栏.在这些实验中，我们还将产生用于分析1）细菌和2）宿主基因的样品 表达，和3）在相对简单的细胞培养模型中的宿主响应生物标志物产生。数据集2和 3将与杜克小组生成的结果进行比较，以识别和描述生成的响应 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 细菌毒力决定簇和内皮细胞反应之间的关系。目标1将评估人内皮细胞 不同钩端螺旋体分离株的细胞反应、侵袭动力学和体内组织嗜性。内皮AJ 感染后，将比较临床和 属于高毒力和低毒力致病物种的环境分离物，以及嗜热链球菌 种，在接种后的不同时间。上清将用于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以鉴定变化 在宿主和细菌基因表达中。致病性、内皮细胞 损伤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，这可以告知改进的诊断结合杜克集团获得的结果。 目标2将确定已知和候选L的角色。问号粘附蛋白粘附，和 内皮细胞层的侵袭。一些感兴趣的候选粘附素包括富含亮氨酸的- Pasteur小组鉴定的重复序列（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。我们的工作将建立基因组内容，蛋白质功能， 和内皮反应在钩端螺旋体病内皮损伤中的作用。

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.