Real-time Imaging Analysis of Vector-borne Lyme Borreliosis Pathogenesis & Persis

描述(申请人提供)：莱姆病，由于感染硬蜱传播的伯氏疏螺旋体(BB)，是北美最常见的媒介传播疾病，是一个重大的公共卫生问题。虽然通常可以接受抗生素治疗，但BB感染之后可能会出现长期的临床症状，特别是如果治疗延迟的话。最近对小鼠的研究表明，在某些情况下，在抗生素治疗后，螺旋体可以在结缔组织中持续较长时间。解决抗生素治疗后螺旋体持久性的临床问题的核心是了解BB是如何逃避正常的宿主防御的，这些防御应该补充抗生素的效果。我们在体外对BB与吞噬细胞相互作用的初步发现表明，BB外表膜蛋白(OSP)具有高度的流动性，这一特征与螺旋体的运动性一起，使其即使在调理后也能逃脱吞噬细胞的几乎全部吞噬。我们进一步展示了使用多光子成像来获得时间分辨图像的可行性，该图像显示了小鼠体内的扁虱摄食和Bb运动。这项应用将使用体外成像技术和体内实时多光子显微镜来进一步评估BB OSP移动性和螺旋体移动性对哺乳动物宿主内吞噬细胞逃避和BB持久性的贡献。利用一组在小鼠体内具有不同存活率的突变体/转化体BB，我们将通过共聚焦、电子和视频显微镜评估其优势表达的OSP的膜流动性以及BB突变体逃避人类单核细胞和小鼠巨噬细胞吞噬的能力。然后，我们将使用实时多光子成像的活体麻醉小鼠，以检查SPC的要求，在硬虱传播的BB感染和传播从皮肤接种部位，以及SPC缺陷对螺旋体运动的影响在体内。最后，我们将使用多光子显微镜来检查BB运动模式随时间的变化，因为它适合在小鼠体内持续存在，以及在感染的急性和慢性阶段使用抗生素时对BB的影响。这些研究将深入了解膜蛋白流动性和螺旋体运动在体内BB逃避免疫破坏中的作用，并深入了解在抗生素治疗莱姆病后BB可能如何持续。此外，我们的结果还将提供一个强大的、特征明确的系统，用于未来实时研究特定的基因操作对蜱媒、螺旋体和/或哺乳动物传播的BB感染的影响。 公共卫生意义：莱姆病是北美最常见的病媒传播疾病，由壁虱传播的伯氏疏螺旋体(BB)感染引起。BB在哺乳动物体内感染、传播和持续存在的机制尚不清楚。这一应用将使用包括多光子显微镜在内的最先进的成像方法来研究BB和BB运动模式的外膜特征，这些外膜可能使其能够逃脱体内免疫和抗生素介导的破坏。

DESCRIPTION (provided by applicant): Lyme disease, due to infection with the Ixodes tick-borne spirochete Borrelia burgdorferi (Bb) is the most common vector-borne disease in North America and a significant public health concern. Although usually amenable to antibiotic therapy, Bb infection can be followed by prolonged clinical symptoms, especially if treatment is delayed. Recent studies in mice reveal that under some circumstances, spirochetes can persist in connective tissue for extended periods after antibiotic treatment. Central to resolving the clinical issue of spirochete persistence after antibiotic therapy is an understanding of how Bb evades the normal host defenses that should complement antibiotic effects. Our preliminary findings of Bb interactions with phagocytes in vitro suggest that Bb outer surface membrane proteins (Osps) are highly mobile and that this feature, along with spirochete motility, facilitate its escape from near total engulfment by phagocytes, even after opsonization. We further show the feasibility of using multiphoton imaging to obtain time-resolved images of tick feeding and Bb movements within the murine host. This application will use in vitro imaging techniques and in vivo real-time multiphoton microscopy to further evaluate the contribution of Bb Osp mobility and spirochete motility to phagocyte evasion and Bb persistence within the mammalian host. Using a panel of mutant/transformant Bb with differential survival in mice, we will evaluate through confocal, electron and videomicroscopy the membrane mobility of their dominantly expressed Osp and the capacity of the Bb variants to escape engulfment by human monocytes and murine macrophages. We will then use real-time multiphoton imaging of live anesthesized mice to examine the requirement for OspC in tick-transmitted Bb infection and dissemination from the skin inoculation site, and effects of OspC deficiency on spirochete motility in vivo. Finally, we will use multiphoton microscopy to examine the changes in Bb motility patterns over time as it adapts to persist within mice, and the effects of antibiotics on Bb when administered during acute and chronic phases of infection. These studies will provide insight into the role of membrane protein mobility and spirochete motility in Bb evasion of immune destruction in vivo, and insight into how Bb may persist after antibiotic therapy for Lyme disease. Moreover, our results will also provide a powerful, well-characterized system for future real-time studies of the effects of specific genetic manipulations of the tick vector, the spirochete, and/or the mammal on tick-borne Bb infection. PUBLIC HEALTH RELEVANCE: Lyme disease, due to infection with the tick-transmitted spirochete Borrelia burgdorferi (Bb), is the most common vector-borne disease in North America. The mechanisms whereby Bb infects, disseminates and persists in mammals are poorly understood. This application will use state-of-the-art imaging approaches, including multiphoton microscopy, to study features of the outer membrane of Bb and Bb motility patterns that may allow it to escape immune and antibiotic-mediated destruction in vivo.