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钩端螺旋体属的几种致病螺旋体引起的是全世界最广泛的人畜共患病。钩端螺旋体属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的、免疫功能正常的宿主动物中的播散性感染，但该属还包括腐生的、非致病的物种。水库宿主不会发展出严重的疾病，但钩端螺旋体与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（血清型）和其他未知因素。人类疾病范围包括轻度自限性疾病至伴有肾衰竭和肝衰竭的急性危及生命的感染功能障碍、广泛的内皮损伤、通透性增加和出血。内皮屏障的破坏可能会促进传播并有助于出血性疾病的表现。我们重点研究了之间的相互作用内皮细胞表面受体和钩端螺旋体体外，鉴定内皮细胞表面询问钩体的受体，并鉴定出候选询问钩体粘附素（表示为 LIC11574 和 LIC13411) 与 VE-钙粘蛋白（主要内皮屏障决定簇）结合。我们假设多种粘附素与宿主跨膜受体的粘附有助于破坏内皮屏障和细菌的迁移。在本提案中，我们将采取多种方法来确定先前的角色确定了细菌迁移中的粘附素、VE-钙粘蛋白的破坏以及跨内皮电阻（TEER）。我们将使用 Tn 突变体，并获得功能产生 VE-钙粘蛋白粘附素的腐生菌株的衍生物，以评估潜力 7 个粘附素的作用。创新在于使用两种转基因钩端螺旋体菌株我们的内皮细胞培养模型。在目标 1 中，我们将表征问号钩体的表型编码已知或候选粘附素的基因中的转座子突变体用于粘附和跨内皮层的迁移。在目标 2 中，我们将类似地描述函数增益的特征产生结合 VE-钙粘蛋白的问号钩体粘附素的腐生菌株。我们提出的工作将构成解决生物学基本问题的重要一步钩端螺旋体将病原体与病原体分开的功能决定因素是什么？非病原体。拟议的工作还将采取措施解决长期存在的问题该领域的争议：内皮损伤是细菌与细菌相互作用的结果吗？内皮或宿主反应，增加影响。