Invasion Dynamics

入侵动力学

• 批准号: 10643292
• 负责人: Jenifer L Coburn
• 金额: $ 31.85万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-16 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10643292
• 关键词: Adherence; Adherens Junction; Adhesions; Affinity; Animal Model; Bacteria; Bacterial Adhesins; Bacterial Genes; Binding; Biological Markers; Blood Circulation; Blood specimen; Cell Communication; Cell Culture Techniques; Cells; Clinical; Collaborations; Data; Data Set; Development; Diagnostic; Disease; Endothelial Cells; Endothelium; Environment; Family; Gene Expression; Genome; Genomics; Hematogenous; Hemorrhage; Human; Immune response; Infection; Inflammatory; Invaded; Knock-in; Leptospira; Leptospira interrogans; Leptospirosis; Mediating; Modeling; Multiple Organ Failure; Pathogenicity; Patients; Phenotype; Production; Proteins; Reaction; Receptor Cell; Role; Sampling; Severities; Statistical Data Interpretation; Testing; Vascular Permeabilities; Virulence; Virulence Factors; Wisconsin; Work; Zoonoses; cadherin 5; cytokine; experimental study; gain of function; human disease; improved; in vivo; insight; interest; knock-down; leucine-rich repeat protein; medical schools; migration; mouse model; mutant; novel; pathogen; potential biomarker; protein function; receptor; response; response biomarker; tissue tropism; transcriptome sequencing

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.

摘要：项目3：威斯康星州医学院 钩端螺旋病是全球最广泛的人畜共患病，从轻度疾病到严重程度各不相同 致命的出血性疾病，具有多器官衰竭。由于内皮损害和血管增加 渗透性是钩端螺旋体病的重要特征 介导对人内皮细胞培养的附着，鉴定内皮细胞受体的鉴定 细菌结合，以及钩端螺旋 - 内皮细胞相互作用对完整性的后果 内皮层。致病性L.探讨有效地横穿内皮层（跨内皮迁移）， 虽然非致病L. biflexa却没有。内皮细胞 - 细胞粘附连接的主要贡献者 （AJS）和障碍功能，VE-钙粘蛋白，是L. erentogans的受体，而L. erentogans会导致破坏 AJS，而L. biflexa则没有。我们确定了两种与纯化的VE-钙粘蛋白结合的L.探究蛋白 高亲和力。我们将与巴斯德和加州大学洛杉矶分校组合作评估现有的表型 转座子突变体克隆，以及细胞培养中的敲入（功能增益）和敲门突变体 动物模型以识别和更好地表征有助于侵袭内皮的钩端螺旋体蛋白 障碍。在这些实验中，我们还将生成样品以分析1）细菌和2）宿主基因 表达和3）在相对简单的细胞培养模型中宿主反应生物标志物的产生。数据集2和 3将与杜克小组生成的那些识别和表征产生的响应的人进行比较。 在人类感染期间。我们的假设是钩端螺旋体病中的内皮损害是由协作引起的 在细菌毒力决定因素和内皮细胞反应之间。 AIM 1将评估人类内皮 细胞对各种钩端螺旋体分离株的细胞反应，侵袭动力学和体内组织的偏移症。内皮AJ 在感染临床和 属于高病毒和低侵入性致病物种的环境分离株和腐生物种 物种，在接种后的不同时间。上清液将用于量化细胞因子和其他潜力 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识别变化 在宿主和细菌基因表达中。致病性，内皮性之间关联的统计分析 损坏，宿主反应将识别潜在的生物标志物作为患者血液中钩端螺旋体病的指标 样本可以为改进的诊断提供，并结合杜克小组获得的结果。 AIM 2将确定已知和候选乳杆菌粘附蛋白在遵守和 内皮细胞层的入侵。一些感兴趣的候选粘合剂包括一个富含亮氨酸的家族 重复（LRR）蛋白质由巴斯德组鉴定出来，尤其是LRR蛋白在 高致病性的钩端螺旋体基因组。我们的工作将建立基因组含量，蛋白质功能， 和钩端螺旋体病中内皮损伤的内皮反应。