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

• 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
• 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' s Disease; Work; Zoonoses; cadherin 5; candidate identification; chronic infection; gain of function; genetic approach; innovation; macromolecule; migration; mutant; pathogen; receptor; urinary

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.

摘要 钩端螺旋体病是由钩端螺旋体属的几种致病性螺旋体引起的 是世界范围内传播最广的人畜共患病。钩端螺旋体属的病原体是 维持在人畜共患病的循环中，要求它们能够建立持久的， 在免疫活性宿主动物中的播散性感染，但该属还包括 无害的非致病性物种。水库主机不发展严重的疾病，但 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， 血清学变体（血清型）和其他未知因素。人类的疾病范围从 轻度自限性疾病至急性危及生命的感染伴肾衰竭和肝衰竭 功能障碍和广泛的内皮损伤、增加的渗透性和出血。 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。我们专注于研究 内皮细胞表面受体和体外钩端螺旋体，鉴定内皮细胞表面 受体L.问号，并确定了候选人L。问号粘附素（表示为 LIC 11574和LIC 13411），其结合主要内皮屏障决定簇VE-钙粘蛋白。 我们推测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以前的角色， 在细菌迁移中鉴定的粘附素，VE-钙粘蛋白的破坏， 跨内皮电阻（TEER）。我们将使用Tn突变体， 产生VE-钙粘蛋白粘附素的嗜酸性菌株的衍生物，以评估其潜在的 7种粘附素的作用。创新在于使用两种转基因钩端螺旋体菌株集， 我们的内皮细胞培养模型在目标1中，我们将描述L.问号 编码已知或候选粘附素的基因中的转座子突变体， 穿过内皮层。在目标2中，我们将类似地表征函数的增益 产生L.结合VE-钙粘蛋白的问号肌粘附素。 我们提出的工作将构成解决生物学基础的重要一步。 问题是什么是钩端螺旋体的功能决定因素，区分病原体， 非病原体。拟议的工作还将采取措施解决长期存在的 该领域的争议：内皮损伤是否是细菌与内皮细胞相互作用的结果？ 内皮细胞或对宿主的反应，增加影响。