Elucidating the Bacterial and Host Mechanisms Governing B. burgdorferi-Related Type I Interferon Responses

项目总结 伯氏杆菌(Bb)是莱姆病的病媒传播病原体，可引起强烈的炎症反应 在逃避和调节宿主免疫以建立多系统感染的同时做出反应。Bb隔离了 36kb线性质粒(Lp36)的存在或序列不同具有不同的传播和 引起莱姆相关的病理，这与宿主炎症和I型的不同诱导有关 干扰素(IFN-I)反应。已经提出了多种先天免疫受体来触发干扰素-I。 然而，控制BB依赖的干扰素-I产生的确切宿主途径，干扰素-I的动力学 感染组织中的反应，以及调节干扰素-I诱导的BB因子仍不清楚。 正在进行的研究已经确定了细胞内cGAS-sting DNA传感通路在 控制BB感染过程中干扰素-I的诱导。因此，这一提议将检验cGAS-sting的假设 和伯氏杆菌lp36基因控制干扰素-I反应的形状感染动力学和莱姆相关 免疫病理学。目标1将利用各种人类和小鼠基因敲除和基因敲除细胞来 BB感染过程中依赖cGAS和刺痛的干扰素-I的产生特征。其次，高分辨率 显微镜和cGAS免疫沉淀-深度测序方法将用于定义细胞内 CGAS感知BB的利基，并识别激活cGAS的细菌和/或宿主DNA物种。至 定义活体、野生型、cGAS和刺痛缺陷小鼠将被生物发光BB感染的角色 用活体成像系统(IVIS)对硼负荷进行时空评估。最后，目标1将使用一个 新型干扰素-I绿色荧光蛋白报告鼠品系研究干扰素的体内动力学和细胞来源 一个35天的疗程。目的2阐明BB36kb线形质粒(Lp36)及其编码基因的作用 Lp36的bbk35-bbk50区域在宿主干扰素-I诱导中的作用。Bb完全缺失Lp36或bbk35-bbk50区 由于bb缺少bbk35-bbk50亚区，将评估其附着、被吸收和/或触发的能力 原代小鼠和人巨噬细胞及成纤维细胞产生的干扰素-I。此外，IVIS的活体成像将是 用于确定所有lp36突变体和相关补体菌株的传播表型。这个 随着时间的推移，将检测突变菌株在小鼠中诱导莱姆相关免疫病理的能力。 这项工作是创新的，因为它将第一次定义BB，一种主要是细胞外的病原体， 通过细胞内cGAS-STING途径诱导干扰素-I。此外，它还将为 候选疏螺旋体基因在感染早期和晚期触发干扰素-I，并将检测其 在莱姆病发病机制中的贡献。从长远来看，这项研究有可能揭示出新的 对活动性感染或持续症状有效的免疫治疗途径。 伯格多菲、莱姆关节炎等。

PROJECT SUMMARY B. burgdorferi (Bb), the vector-borne etiologic agent of Lyme disease, elicits robust inflammatory responses while evading and modulating host immunity to establish a multi-systemic infection. Bb isolates that differ in the presence or sequence of the 36kb linear plasmid (lp36) possess varied ability to disseminate and cause Lyme-related pathology, which has been linked to differential induction of host inflammatory and type I interferon (IFN-I) responses. Multiple innate immune receptors have been proposed to trigger IFN-I during infection; however, the exact host pathways governing Bb-dependent IFN-I production, the kinetics of IFN-I responses in infected tissues, and the Bb factors modulating IFN-I induction remain unknown. Ongoing studies have identified a novel role for the intracellular cGAS-STING DNA sensing pathway in controlling IFN-I induction during Bb infection. This proposal will therefore test the hypothesis that cGAS-STING and B. burgdorferi lp36 genes control the IFN-I response to shape infection kinetics and Lyme-related immunopathology. Aim 1 will employ a variety of human and mouse knockout and knockdown cells to characterize cGAS- and STING-dependent IFN-I production over the course of Bb infection. Next, high resolution microscopy and a cGAS immunoprecipitation-deep sequencing approach will be utilized to define the intracellular niches where cGAS senses Bb and to identify the bacterial and/or host DNA species that activate cGAS. To define roles in vivo, wildtype, cGAS-, and STING-deficient mice will be infected with bioluminescent Bb for spatiotemporal evaluation of borrelial load by an In Vivo Imaging System (IVIS). Finally, Aim 1 will employ a novel, IFN-I GFP reporter mouse strain to characterize the in vivo kinetics and cellular sources of interferon over a 35 day time course. Aim 2 will elucidate the role of Bb 36kb linear plasmid (lp36) and genes encoded in the bbk35-bbk50 region of lp36 in host IFN-I induction. Bb lacking lp36 entirely or the bbk35-bbk50 region, as well as Bb lacking subregions of bbk35-bbk50, will be assessed for their ability to adhere, be taken up, and/or trigger IFN-I by primary murine and human macrophages and fibroblasts. Furthermore, in vivo imaging by IVIS will be used to determine the dissemination phenotypes of all lp36 mutants and associated complement strains. The ability of mutant strains to induce Lyme-related immunopathology in mice will be examined over time. This work is innovative as it will be the first to define how Bb, a predominately extracellular pathogen, induces IFN-I through the intracellular cGAS-STING pathway. Moreover, it will characterize novel roles for candidate borrelial genes in triggering IFN-I during early and late phases of infection and will examine their contribution to Lyme disease pathogenesis. In the long term, this research has the potential to reveal new immunotherapeutic avenues that may be effective against active infection or persistent symptoms of B. burgdorferi, such as Lyme arthritis.