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

描述（由申请人提供）：莱姆病，由蜱传螺旋体伯氏疏螺旋体（Bb）感染引起，是北美最常见的媒介传播疾病，也是一个重要的公共卫生问题。虽然通常可以接受抗生素治疗，但Bb感染后可出现延长的临床症状，特别是如果治疗延迟。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存在较长时间。解决抗生素治疗后螺旋体持续存在的临床问题的核心是了解螺旋体如何逃避正常的宿主防御，这应该补充抗生素的作用。我们在体外对Bb与吞噬细胞相互作用的初步研究结果表明，Bb外表面膜蛋白（Osps）具有高度的流动性，这一特征与螺旋体的运动性一起，有助于其从吞噬细胞的几乎完全吞噬中逃脱，即使在活化后也是如此。我们进一步展示了使用多光子成像获得鼠宿主内蜱进食和Bb运动的时间分辨图像的可行性。该应用程序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流动性和螺旋体运动对哺乳动物宿主内吞噬细胞逃避和Bb持久性的贡献。利用小鼠中具有不同存活率的突变/转化Bb，我们将通过共聚焦、电子和视频显微镜来评估其主要表达的Osp的膜迁移性以及Bb变体逃避人类单核细胞和小鼠巨噬细胞吞噬的能力。然后，我们将使用活体麻醉小鼠的实时多光子成像来检查蜱传播的Bb感染和皮肤接种部位传播对OspC的需求，以及OspC缺乏对体内螺旋体运动的影响。最后，我们将使用多光子显微镜检查Bb运动模式随时间的变化，因为它适应在小鼠体内持续存在，以及在感染的急性和慢性阶段给予抗生素对Bb的影响。这些研究将深入了解膜蛋白迁移和螺旋体运动在Bb逃避体内免疫破坏中的作用，并深入了解莱姆病抗生素治疗后Bb如何持续存在。此外，我们的研究结果也将为未来实时研究蜱虫媒介、螺旋体和/或哺乳动物的特定遗传操作对蜱传Bb感染的影响提供一个强大的、具有良好特征的系统。

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.