Functional Domains of Borrelia burgdorferi Virulence Factor OspC

描述(申请人提供)：莱姆病是北半球最常见的媒介传播疾病，通过携带伯氏疏螺旋体的扁虱传播给人类。在扁虱饲养过程中突然涌入的营养物质使这种细菌能够迅速重塑其表面，以便有效地建立哺乳动物感染。伯氏杆菌宿主-病原体的界面主要由脂蛋白主导，脂蛋白通过脂类修饰的半胱氨酸被锚定在外膜的外叶中。最近在小鼠感染模型中的研究表明，外表面脂蛋白C(OSPC)的表达对于建立早期感染至关重要。本研究的总体目标是确定这一重要的螺旋体毒力因子的TE结构和功能关系。OspC在细胞表面形成同源二聚体，其三维结构可分为三个不同的结构域：(I)无序的N-末端“系绳”肽，(Ii)α螺旋束“支架”，(Iii)膜-远端螺旋连接环的“穹顶”。然而，目前还不清楚这三个结构域是如何影响脂蛋白在细菌定植和传播中的生物学功能的。OspC与复发性发热疏螺旋体VSP蛋白属于同一蛋白质折叠家族，参与免疫逃避和组织趋向性。事实上，疏螺旋体Vsp1的晶体结构与SpC的差异很小，但最显著的是在螺旋连接环上。因此，我们假设OspC的穹顶残基在感染早期对OspC的特定功能至关重要。此外，我们假设锚链多肽除了提供蛋白质分选信息(见我们发表的工作)外，还通过优化其长度进化出在细菌表面蛋白质组中正确定位毒力因子的位置。我们将在以下两个特定的目标中检验这些假设：1.通过测试VSP蛋白或VSP支架-SPC穹顶嵌合体在体外以及在小鼠感染模型中是否能够在功能上取代SPC在宿主分子的结合中来确定SPC穹顶残基在定植和传播中的作用。最终，功能所需的最小OspC穹顶结构域将被确定。2.通过在体外和竞争小鼠感染实验中测试现有和新产生的OspC锚链长度突变体的功能，确定OspC脂蛋白锚链长度在毒力因子可及性和功能中的作用。这些研究将(I)在我们对螺旋体表面脂蛋白结构-功能的研究方面取得进一步的里程碑，(Ii)显著增加我们对哺乳动物感染伯氏疏螺旋体的早期事件的了解，以及(Iii)产生重要的线索，这些线索最终可能转化为莱姆病的新型干预策略的设计。

DESCRIPTION (provided by applicant): Lyme disease, the most common vector-borne disease in the Northern hemisphere, is transmitted to humans by ticks carrying the spirochete Borrelia burgdorferi. Enabled by a sudden influx of nutrients during tick feeding, the bacterium quickly remodels its surface to allow for efficient establishment of mammalian infection. The B. burgdorferi host-pathogen interface is dominated by lipoproteins that are peripherally anchored in the outer leaflet of the outer membrane via a lipid-modified cysteine. Recent studies in a mouse model of infection have shown that expression of outer surface lipoprotein C (OspC) is crucial for establishment of early infection. The overall objective of this study is to determine te structure function relationships of this important spirochetal virulence factor. OspC forms a homodimer on the surface of the cells, and its three-dimensional structure can be separated into three distinct domains: (i) a disordered N-terminal "tether" peptide, (ii) an alpha helical bundle "scaffold", and (iii) a membrane-distal "dome" of helix-connecting loops. Yet, it remains unclear how these three domains are contributing to the lipoprotein's biological function in bacterial colonization and dissemination. OspC belongs to the same protein fold family as the relapsing fever Borrelia Vsp proteins, which are involved in immune evasion and tissue tropism. In fact, the crystal structure of Borrelia turicatae Vsp1 deviates from OspC only slightly but most significantly in the helix-connecting loops. We therefore hypothesize that OspC's dome residues are crucial for OspC's specific function during early infection. Furthermore, we hypothesize that the tether peptide, in addition to providing protein sorting information (see our published work), evolved to properly position the virulence factor within the bacterium's surface proteome through optimization of its length. We will test these hypotheses in the following two specific aims: 1. T determine the role of OspC dome residues in colonization and dissemination by testing if Vsp proteins or Vsp scaffold-OspC dome chimeras can functionally replace OspC both in binding of host molecules in vitro as well as in in a mouse model of infection. Ultimately, the minimal OspC dome domain required for function will be identified. 2. To determine the role of OspC lipoprotein tether length in virulence factor accessibility and function by testing existing and newly generated OspC tether length mutants for functions in vitro as well as in a competitive mouse infection assay. These studies will (i) achieve further milestones in our investigation of spirochetal surface lipoprotein structure- function, (ii) significantly increase our understanding f the early events during mammalian infection with Borrelia burgdorferi, and (iii) yield important clues that may ultimately translate into the design of novel intervention strategies for Lyme disease.