Virulence Mechanisms of Multifunctional Borrelial Proteins

项目总结 疏螺旋体属的螺旋体是几种流行的媒介传播疾病的原因。最多的 这一组的著名病原体是伯氏疏螺旋体，它导致30多万例 莱姆病每年在美国流行。B.garinii和B.afzelii，属于伯氏杆菌 拉托复合体是欧洲和亚洲莱姆病的主要病原体。螺旋体螺旋体也是 古代人类疾病复发热的病原体，以及一种新发现的传染病 这种情况被称为宫本疏螺旋体病。与莱姆相关的、与复发性发热相关的和宫本杆菌 螺旋体具有不同的生命周期，其感染伴随着不同的临床表现。 然而，这些病原体中的每一种都已知编码多功能表面表达的脂蛋白，这些脂蛋白 与脊椎动物的宿主分子相互作用。在这些蛋白质中，有一小部分免疫调节剂 特别是针对和灭活先天免疫的主要手臂，称为补体系统。我们有 最近报道了两条独立的证据，支持这样一种假设，即其中一条途径， 被称为经典途径，在控制伯氏杆菌感染方面很重要。首先，我们已经证明了 缺乏经典途径的模式识别分子C1q的小鼠明显更多 易受伯氏杆菌感染的。其次，我们已经证明了伯氏杆菌BBK32是一种 经典途径启动酶C1r的高亲和力抑制物。 在本项目的目标1中，我们试图了解BBK32正义蛋白的C1R抑制活性 分子水平。在目标2中，我们将确定三种病毒的免疫调节作用和毒力贡献。 BBK32同源基因被称为FbpA、FbpB和FbpC，它们是在复发热和B中唯一发现的。 宫本氏螺旋体。在目标3中，我们将描述C1r抑制在斑疹伤寒发病中的作用。 疾病的活体模型。为了实现这一目标，我们提出了一种使用x射线的多学科策略。 结晶学、生物物理方法和补充功能分析，以确定关键的热点残基 在BBK32上，使其具有强大的抗C1r活性。这些数据将为bbk32突变体的设计提供信息 将用于小鼠感染性研究，将BBK32在氨基酸水平的结构特征与 活体表型。平行研究将使用复发热病的FBP基因的遗传缺失突变。 伴生螺旋体B.turicatae和B.hermsii。这些研究将与实验模型配对 利用C1R-/-小鼠更好地了解经典途径的作用 启动蛋白酶控制疏螺旋体感染。通过解决基本问题，即如何在医学上 重要的疏螺旋体识别和逃避宿主免疫，这里提出的研究将有一个 对细菌发病机制领域影响广泛而重大。

PROJECT SUMMARY Spirochetes of the Borrelia genus are the cause of several prevalent vector-borne diseases. The most well-known pathogen from this group is Borrelia burgdorferi sensu stricto, which causes over 300,000 cases of Lyme disease in the United States each year. B. garinii and B. afzelii, which belong to the B. burgdorferi sensu lato complex, are the primary agent of Lyme disease in Europe and Asia. Borrelia spirochetes are also the etiological agent of the ancient human disease relapsing fever, as well as a newly recognized infectious condition called Borrelia miyamotoi disease. Lyme-associated, relapsing fever-associated, and B. miyamotoi spirochetes have differing lifecycles and their infections are accompanied by distinct clinical presentations. However, each of these pathogens are known to encode multifunctional surface-expressed lipoproteins that interact with vertebrate host molecules. Among these proteins are a small arsenal of immunomodulators that specifically target and inactivate a primary arm of innate immunity known as the complement system. We have recently reported two independent lines of evidence that support the hypothesis that one of these pathways, known as the classical pathway, is important in controlling B. burgdorferi infections. First, we have shown that mice deficient in the pattern recognition molecule of the classical pathway, C1q, are significantly more susceptible to B. burgdorferi infection. Secondly, we have shown that the lipoprotein B. burgdorferi BBK32 is a high-affinity inhibitor of the initiating protease of the classical pathway, C1r. In Aim 1 of this project we seek to understand the C1r inhibitory activity of BBK32 sensu lato proteins at the molecular level. In Aim 2 we will determine the immunomodulatory roles and virulence contribution of three BBK32 orthologues known as FbpA, FbpB, and FbpC which are found uniquely in relapsing fever and B. miyamotoi spirochetes. In Aim 3 we will delineate the role of C1r inhibition in borrelial pathogenesis using in vivo models of disease. To achieve this, we propose a multi-disciplinary strategy that employs x-ray crystallography, biophysical approaches, and complement functional assays to pinpoint key ‘hot-spot’ residues on BBK32 that give rise to its potent anti-C1r activity. These data will inform the design of bbk32 mutants which will be used in mouse infectivity studies to connect structural features of BBK32, at the amino-acid level, to an in vivo phenotype. Parallel studies will use genetic deletion mutants of fbp genes from the relapsing fever- associated spirochetes B. turicatae and B. hermsii. These studies will be paired with experimental models of Lyme and relapsing fever borrelioses using C1r-/- mice to better understand the role of the classical pathway initiating protease in the control of borrelial infections. By addressing fundamental questions of how medically important Borrelia spirochetes recognize and evade host immunity, the studies proposed here stand to have a broad and significant impact on the field of bacterial pathogenesis.