Virulence Mechanisms of Multifunctional Borrelial Proteins

项目摘要 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 来自该组的众所周知的病原体是狭义伯氏疏螺旋体，其引起超过300，000例 莱姆病在美国每年。B。garinii和B. afzelii，属于B.狭义伯氏疏螺旋 Lato复合体是欧洲和亚洲莱姆病的主要病原体。疏螺旋体螺旋体也是 古老的人类疾病回归热的病原体，以及一种新认识的传染病 这种病叫做宫本疏螺旋体病。莱姆病相关、复发性发热相关和B。宫本井 螺旋体有不同的生命周期，它们的感染伴随着不同的临床表现。 然而，已知这些病原体中的每一种都编码多功能表面表达脂蛋白， 与脊椎动物宿主分子相互作用。在这些蛋白质中，有一小部分免疫调节剂， 特异性地靶向并抑制称为补体系统的先天免疫的主要分支。我们有 最近报道了两条独立的证据线，支持这一假设，即这些途径之一， 被称为经典途径，在控制B方面很重要。伯氏菌感染首先，我们已经证明， 缺乏经典途径模式识别分子C1 q的小鼠， 对B敏感。伯氏感染其次，我们已经证明脂蛋白B。burgdorferi BBK 32是一个 经典途径起始蛋白酶C1 r的高亲和力抑制剂。 在本项目的目的1中，我们试图了解BBK 32广义蛋白在以下条件下的C1 r抑制活性： 分子水平。在目标2中，我们将确定三种免疫调节剂的免疫调节作用和毒力贡献。 BBK 32直向同源物称为FbpA、Fbp B和FbpC，其在回归热和B中独特地发现。 宫本氏螺旋体。在目的3中，我们将描述C1 r抑制在疏螺旋体发病机制中的作用， 疾病的体内模型。为了实现这一目标，我们提出了一个多学科的战略，采用x射线 晶体学、生物物理学方法和补充功能测定，以确定关键的“热点”残基 对BBK 32的作用，产生了其有效的抗C1 r活性。这些数据将为bbk 32突变体的设计提供信息， 将用于小鼠感染性研究，以在氨基酸水平上将BBK 32的结构特征与 体内表型。平行研究将使用回归热fbp基因的遗传缺失突变体- 相关螺旋体B。turiclavirus和B.赫姆斯。这些研究将与以下实验模型配对： 使用C1 r-/-小鼠研究莱姆病和回归热疏螺旋体病，以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。通过解决医学上如何 重要的疏螺旋体螺旋体识别和逃避宿主免疫，这里提出的研究表明， 对细菌致病机理领域产生了广泛而重大的影响。

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.