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

描述（由申请方提供）：莱姆病，由于感染硬蜱传播的螺旋体伯氏疏螺旋体（Bb），是北美最常见的病媒传播疾病，是一个重大的公共卫生问题。虽然通常适合抗生素治疗，但Bb感染后可能会出现长期的临床症状，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，螺旋体可以在抗生素治疗后在结缔组织中持续很长一段时间。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Bb如何逃避应补充抗生素作用的正常宿主防御。我们的初步研究结果表明，Bb与吞噬细胞在体外的相互作用表明，Bb外表面膜蛋白（Osps）是高度移动的，这一功能，沿着与螺旋体运动，促进其逃避几乎完全吞噬吞噬细胞，即使在调理作用。我们进一步表明，使用多光子成像的可行性，以获得时间分辨的图像蜱喂养和Bb的运动在小鼠宿主。本申请将使用体外成像技术和体内实时多光子显微镜进一步评估Bb Osp流动性和螺旋体运动性对吞噬细胞逃避和Bb在哺乳动物宿主中的持久性的贡献。使用一组在小鼠中具有差异存活的突变体/β-Bb，我们将通过共聚焦、电子和视频显微镜评估其显性表达的Osp的膜流动性和Bb变体逃避人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麻醉小鼠检查蜱传播的Bb感染和传播的皮肤接种部位的OspC的要求，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，因为它适应持续在小鼠体内，以及抗生素对Bb的影响时，在急性和慢性感染阶段。这些研究将提供深入了解的作用，膜蛋白的流动性和螺旋体的运动在Bb逃避体内免疫破坏，并深入了解如何Bb可能持续抗生素治疗莱姆病。此外，我们的研究结果也将提供一个强大的，良好的表征系统，为未来的实时研究的影响，特定的遗传操作的蜱虫载体，螺旋体，和/或蜱传Bb感染的哺乳动物。 公共卫生相关性：莱姆病，由于感染蜱传播的螺旋体伯氏疏螺旋体（Bb），是北美最常见的病媒传播疾病。Bb在哺乳动物中感染、传播和持续存在的机制知之甚少。本申请将使用最先进的成像方法，包括多光子显微镜，以研究Bb和Bb运动模式的外膜的特征，这些特征可能使其逃避体内免疫和免疫介导的破坏。

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.