The roles of Lyme spirochete adhesins in hematogenous dissemination

通过媒介扁虱叮咬传播后，莱姆病螺旋体，主要是美国的伯氏杆菌(B.burgdorferi)， 建立局部皮肤感染，然后扩散到多个组织。乙型脑炎的慢性感染通常与 患有关节炎。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，克服了确定其在BB生物学中的作用的挑战。 我们的方法包括分析生化活性和选择性地产生目标突变体。 单一黏附活性缺陷及多个小鼠感染模型中突变体的分析。为了获得 对活体内BB传播过程中可能发生的相互作用的详细机械洞察，如下 静脉接种我们使用活体显微镜来表征血管附着和移行。 在皮肤和关节近端组织中。这些研究表明，粘附素BBK32和VlsE几乎可以解释所有 在接种几分钟后发生的暂时性BB-内皮结合，称为“相遇”相互作用。一个 一组独特的粘附素，DbpB/A，OspC和P66，介导了侵袭血管外所需的接触 24小时后关节间隙(Hr)，称为“轮回”相互作用。使用具有相同基因的菌株集 在我们展示的多种短期和长期感染模型中，获得或失去特定的黏附活性 六种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的定植。而轮回迁移的粘附素并不能促进“相遇” 相互作用，我们发现内皮细胞的黏附能力随着感染的进展而增强 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。在人力资源中，“内皮” 激活“允许与BBK32和VlsE无关的(”问候“)交互。约24小时后“内皮” 增强“的发生，反映在关节血管系统支持BB转位的能力上。虽然两者都是 Ospc和p66起转移性粘附素的作用，只有p66，一种整合素结合的粘附素，改变 在培养的内皮细胞中，转录也是增强的，即促进快速迁移所必需的 属于第二个BB菌株。通过外源处理产生几种细胞因子来模拟小鼠的激活 但只有在肿瘤坏死因子-α、单核细胞趋化蛋白-1或白介素10的作用下才能增强。这些发现揭示了之前 未识别的步骤对BB传播至关重要，并提供了一种区分每个粘附素在 不同的感染阶段：相遇、问候、加重、迁徙和定植。在目标1中，我们将 确定有助于问候互动的已知粘附素；为了更好地确定粘附素列表的优先顺序，我们建议 全基因组筛选也可能识别新的粘附素。在目标2中，我们将阐明已知(和，如果 适用的、新颖的)粘附素在内皮增强和移行中的作用。我们使用严格的基因 感染模型的分析--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。

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.