Genetic Approaches to Evaluation of the Roles of Leptospira interrogans Adhesins in Endothelial Interactions

评估问号钩端螺旋体粘附素在内皮相互作用中作用的遗传学方法

基本信息

批准号：
10612825
负责人：
Jenifer L Coburn
金额：
$ 19.5万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-22 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10612825
关键词：
Acute Adhesions Affect Animals Bacteria Bacterial Adhesins Bacterial Proteins Binding Blood Vessels Blood capillaries Body Fluids Case Fatality Rates Cell Culture Techniques Cell Surface Receptors Cells Clinical Complex Development Disease E-Cadherin Electrical Resistance Endothelial Cells Endothelium Environment Environmental Pollution Epithelium Evaluation Future Genes Hemorrhage Human Immune response Immunocompetent In Vitro Infection Intercellular Junctions Invaded Investigation Kidney Failure Laboratories Leptospira Leptospira interrogans Leptospirosis Life Liver Dysfunction Lung Mediating Membrane Modeling Mucous Membrane Mutation Order Spirochaetales Outcome Participant Pathogenicity Permeability Phenotype Proteins Proximal Kidney Tubules Role Serology Skin Source Stains Testing Therapeutic Tissues Urine Variant Virulence Weil&apos s Disease Work Zoonoses cadherin 5 candidate identification chronic infection gain of function genetic approach innovation macromolecule migration mutant pathogen receptor urinary

项目摘要

Abstract Leptospirosis is caused by several pathogenic species of spirochetes of the genus Leptospira and is the most widespread zoonosis worldwide. Pathogens in the genus Leptospira are maintained in zoonotic cycles, demanding that they be able to establish persistent, disseminated infection in immunocompetent host animals, but the genus also includes saprophytic, non-pathogenic species. Reservoir hosts do not develop severe disease, but the outcome of the Leptospira-host interaction depends on the host species, the Leptospira species, serological variant (serovar), and additional unknown factors. Disease in humans ranges from mild self-limited illness to acute life-threatening infection with kidney failure, and liver dysfunction, and widespread endothelial damage, increased permeability, and hemorrhage. Disruption of endothelial barriers is likely to facilitate dissemination and to contribute to hemorrhagic disease manifestations. We have focused on investigation of interactions between endothelial cell surface receptors and Leptospira in vitro, identified endothelial cell-surface receptors for L. interrogans, and identified candidate L. interrogans adhesins (denoted LIC11574 and LIC13411) that bind to VE-cadherin, the primary endothelial barrier determinant. We hypothesize that adhesion to host transmembrane receptors by multiple adhesins contributes to disruption of endothelial barriers and transmigration of the bacteria. In this proposal, we will take multiple approaches to determining the roles of previously identified adhesins in bacterial transmigration, disruption of VE-cadherin, and changes in transendothelial electrical resistance (TEER). We will employ Tn mutants, and gain of function derivatives of a saprophytic strain that produce VE-cadherin adhesins, to evaluate the potential roles of 7 adhesins. Innovation lies in use of both genetically modified Leptospira strain sets in our endothelial cell culture model. In Aim 1 we will characterize the phenotypes of L. interrogans transposon mutants in genes encoding known or candidate adhesins for adhesion to and transmigration across endothelial layers. In Aim 2 we will similarly characterize gain of function saprophytic strains producing L. interrogans adhesins that bind VE-cadherin. Our proposed work will constitute a major step toward resolving the biologically fundamental question of what are the functional determinants of Leptospira that separate the pathogens from the non-pathogens. The proposed work will also take steps toward resolving a long-standing controversy in the field: Is endothelial damage the result of bacterial interactions with the endothelium or to the host response, increasing impact.

抽象的钩端螺旋体病是由几种病原体螺旋体引起的，并且是全球最广泛的人畜共患病。钩端螺旋体中的病原体是维持在人畜共患循环中，要求他们能够建立持久性，免疫能力的宿主动物中传播感染，但该属也包括腐生，非致病物种。水库寄主不会患上严重疾病，而是钩端螺旋体宿主相互作用的结果取决于宿主物种，钩端螺旋体物种，血清学变异（血清）和其他未知因素。人类疾病范围轻度的自限性疾病，因肾衰竭而急性威胁生命的感染和肝脏功能障碍和广泛的内皮损伤，渗透率增加和出血。内皮障碍的破坏可能有助于传播并做出贡献出血性疾病表现。我们专注于研究之间的相互作用内皮细胞表面受体和钩端螺旋体在体外，鉴定出内皮细胞表面 L. discemogans的受体，并确定了候选L. descentogans粘附素（表示 LIC11574和LIC13411）与主要内皮屏障决定因素结合的VE-Cadherin。我们假设通过多种粘附素的宿主跨膜受体的粘附有助于干扰细菌的内皮屏障和移民。在此提案中，我们将采用多种方法来确定以前的作用鉴定在细菌透射术，VE-钙粘蛋白的破坏以及变化中的粘附素跨内皮电阻（TEER）。我们将采用TN突变体并获得功能产生VE-钙粘着蛋白粘附蛋白的腐生菌株的衍生物，以评估潜力 7种粘附素的作用。创新在于使用两个基因修饰的钩端螺旋体菌株我们的内皮细胞培养模型。在AIM 1中，我们将表征L. ventergans的表型编码已知或候选粘附素的基因中的转座子突变体，以粘附到和跨内皮层的迁移。在AIM 2中，我们将类似地表征功能增长产生结合VE-cadherin的杂物蛋白的腐生菌株。我们拟议的工作将构成解决生物学基本的重要一步分离病原体的钩端螺旋体功能决定因素是什么问题非公主。拟议的工作还将采取措施解决长期存在的该领域的争议：是内皮损害与细菌相互作用与内皮或对宿主反应，增加影响。