The roles of Lyme spirochete adhesins in hematogenous dissemination

莱姆病螺旋体粘附素在血行传播中的作用

• 批准号: 10570186
• 负责人: George Chaconas
• 金额: $ 71.46万
• 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: 2022-02-10 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10570186
• 关键词: Adhesives; Animals; Arthritis; Bacteria; Bacterial Adhesins; Binding; Biochemical; Biology; Blood Vessels; Borrelia afzelii; Borrelia burgdorferi; Borrelia garinii; CCL2 gene; Cell Line; Cells; Chemosensitization; Complex; DBL Oncoprotein; Endothelial Cells; Endothelium; Event; Extracellular Matrix; Gene Family; Generations; Genes; Genetic Transcription; Geography; Health system; Hematogenous; Hour; Infection; Infectious Skin Diseases; Integrin Binding; Interleukin-10; Intravenous; Invaded; Investigation; Joints; Laboratories; Left; Ligands; Lyme Disease; Measures; Mediating; Modeling; Monitor; Mus; Nature; Order Spirochaetales; Organism; OspC protein; Phase; Prevention; Principal Investigator; Process; Protein Array; Research Personnel; Role; Skin; TNF gene; Testing; Time; Tissues; Vector-transmitted infectious disease; Visualization; chronic infection; cytokine; decorin binding protein B; genetic analysis; genome wide screen; in vivo; inhibitor; insight; intravital microscopy; meetings; microbial colonization; migration; mouse model; mutant; neutrophil; novel; pathogenic microbe; tick bite; transmission process; vector tick; virtual

Upon transmission by a vector tick bite, Lyme disease spirochetes, primarily B. burgdorferi (Bb) in the US, establish a local skin infection, then disseminate to multiple tissues. Chronic infection by Bb is often associated with arthritis. Our laboratories have identified and/or characterized many Bb cell- or extracellular matrix (ECM)- binding adhesins using multiple approaches, overcoming the challenges of defining their roles in Bb biology. Our approaches include analyses of biochemical activities and generation of targeted mutants selectively defective for a single adhesive activity and analysis of the mutants in multiple murine infection models. To gain detailed mechanistic insight into interactions that may occur during Bb dissemination in vivo, following intravenous inoculation we used intravital microscopy to characterize vascular attachment and transmigration in skin and joint-proximal tissue. These studies revealed that adhesins BBK32 and VlsE account for virtually all of the transient Bb-endothelium binding occurring minutes after inoculation, termed “Meeting” interactions. A distinct set of adhesins, DbpB/A, OspC, and P66, mediate contacts required for invasion into extravascular joint space after 24 hours (hr), termed “Transmigrating” interactions. Using isogenic strain sets that have acquired or lost specific adhesive activities in multiple short-term and long-term infection models we showed roles for five of the six Meeting or Transmigrating adhesins in short-term tissue localization and/or long-term colonization in other murine infection models. While Transmigrating adhesins do not promote “Meeting” interactions, our discovery of enhanced adhesive capacity of the endothelium as infection progresses has helped clarify why different adhesins have roles at different stages of infection. Within hr, “Endothelial Activation” permits BBK32- and VlsE-independent (“Greeting”) interactions. After ~24 hr “Endothelial Potentiation” occurs, reflected by the ability of the joint vasculature to support Bb transmigration. Although both OspC and P66 function as Transmigrating adhesins, only P66, an integrin-binding adhesin that alters transcription in cultured endothelial cells, is also Potentiating, i.e., required to promote the rapid transmigration of a second Bb strain. Activation is mimicked by exogenous treatment of mice with several cytokines produced by infected mice, but potentiation is seen only with TNF-α, MCP-1 or IL-10. These findings reveal previously unrecognized steps that are critical for Bb spread and provide a means to distinguish roles for each adhesin in distinct infection stages: Meeting, Greeting, Potentiating, Transmigrating and Colonizing. In Aim 1 we will identify known adhesins that facilitate Greeting interactions; to better prioritize our adhesin list, we propose a genome-wide screen that may also identify novel adhesins. In Aim 2 we will clarify the roles of known (and, if applicable, novel) adhesins in endothelial Potentiation and Transmigration. Our use of rigorous genetic analyses in infection models from visualization of key interactions in vivo to quantitative analysis of Bb at different stages of infection will result in detailed understanding of a critical facet of Bb biology: dissemination.

通过媒介蜱叮咬传播莱姆病螺旋体，主要是美国的伯氏疏螺旋体 (Bb)， 造成局部皮肤感染，然后播散至多个组织，通常与 Bb 的慢性感染有关。 我们的实验室已经鉴定和/或表征了许多 Bb 细胞或细胞外基质 (ECM)- 使用多种方法结合粘附素，克服了定义其在 Bb 生物学中的作用的挑战。 我们的方法包括生化活性分析和选择性生成目标突变体 单一粘附活性缺陷以及对多种小鼠感染模型中突变体的分析。 对 Bb 在体内传播期间可能发生的相互作用的详细机制了解如下 静脉注射我们使用活体显微镜来表征血管附着和迁移 这些研究表明，粘附素 BBK32 和 VlsE 几乎占所有。 接种后几分钟发生的短暂 Bb-内皮结合，称为“相遇”相互作用 A。 一组不同的粘附素 DbpB/A、OspC 和 P66，介导侵入血管外所需的接触 24 小时 (hr) 后的关节空间，称为“迁移”相互作用，使用具有同基因应变组。 我们在多个短期和长期感染模型中获得或失去了特定的粘附活性 六种相遇或迁移粘附素中的五种在短期组织定位和/或长期组织定位中的作用 在其他小鼠感染模型中定植，而迁移粘附素不会促进“相遇”。 相互作用，我们发现随着感染的进展，内皮的粘附能力增强 有助于阐明为什么不同的粘附素在感染的不同阶段发挥作用，“内皮细胞。 约 24 小时后“内皮细胞的激活”允许 BBK32 和 VlsE 独立的（“问候”）相互作用。 发生“增强”，反映在关节脉管系统支持 Bb 迁移的能力上。 OspC 和 P66 具有迁移粘附素的功能，只有 P66 是一种整合素结合粘附素，可改变 培养的内皮细胞中的转录也是增强的，即促进快速迁移所必需的 通过用产生的几种细胞因子对小鼠进行外源治疗来模拟第二种 Bb 菌株的激活。 受感染的小鼠，但仅在 TNF-α、MCP-1 或 IL-10 下才观察到增强作用。 未识别的步骤对于 Bb 传播至关重要，并提供了区分每种粘附素的作用的方法 不同的感染阶段：相遇、问候、增强、迁移和殖民 在目标 1 中，我们将进行。 确定有助于问候互动的已知粘附素；为了更好地优先考虑我们的粘附素列表，我们提出了一个 全基因组筛选也可能鉴定新的粘附素 在目标 2 中，我们将阐明已知的（并且，如果）的作用。 适用的，新颖的）粘附素在内皮增强和迁移中我们使用严格的遗传。 感染模型分析，从体内关键相互作用的可视化到 Bb 的定量分析 感染的不同阶段将导致对 Bb 生物学的一个关键方面的详细了解：传播。