CRASP-mediated Serum Resistance by Borrelia burgdorferi

 描述(申请人提供)：莱姆病，由伯氏疏螺旋体引起，是美国最常见的媒介传播疾病。伯氏疏螺旋体可以感染扁虱叮咬部位的皮肤，并通过血液传播到不同的组织，表明该细菌 可以抵御血液系统的防御。补体系统是一种血液防御系统，通过三种不同的途径激活。该系统激活的一个关键步骤是形成C3转换酶，C4b2a或C3bBb，它催化导致炎症、调理和病原体溶解的事件。补体激活后潜在的宿主损害需要血清补体调节剂的严格调节，补体调节剂与补体成分结合并促进其降解。例如，补体调节因子H(FH)和C4BP分别调节C3bBb和C4b2a的形成。我们最近发现，伯氏杆菌产生DbpA，这是一种表面蛋白，通过与硫酸皮肤素和核心蛋白结合来促进组织定植，也通过与C4BP结合来促进菌血症。一株产生DbpA-I156A的伯氏杆菌菌株表现出菌血症和关节定植的延迟，DbpA-I156A是C4BP结合缺陷的点突变。此外，伯氏杆菌还产生CRASP(补体调节因子获取表面蛋白)，与补体调节因子结合。其中CSPA、CspZ与FH和FH样蛋白(FHL)结合，ErpP、ErpC和ErpA与FH和补体因子H相关蛋白(CFHR)结合。虽然已有几个CRASP在体外被证明有助于血清抵抗，但没有一个明确地被证明与哺乳动物感染有关。最近，我们发现一株携带erpA：：TN插入的伯氏杆菌在感染早期的组织定植方面存在缺陷。这些结果表明，DbpA和ErpA分别调节C4b2a和C3bBb，促进菌血症和组织定植。DbpA和ErpA抑制这两类C3转换酶的形成的能力可能意味着伯氏杆菌对宿主血液防御的协同攻击。为了验证这一假设，我们将追求以下目标。(1)检测FH结合在菌血症和定植中的作用。我们将测试一个靶向的、非极性的erpA缺失突变体是否在FH/CFHR结合、体外补体激活以及小鼠模型中的菌血症和组织定植方面显示缺陷。如果突变体显示出缺陷，我们将测试ErpA与FH的结合是否对这些功能是必不可少的，以及其他CRASP是否可以在功能上补充这些缺陷。(2)。测试伯氏杆菌的C4BP和FH结合是否提供了促进菌血症和定植的非多余作用。为了确定一个戏剧性的传播缺陷是否需要失去C4BP和FH结合活性，我们将在伯氏杆菌DbpA-I156A突变体的菌株背景中突变erpA。这些双突变菌株将在小鼠模型中进行严重菌血症和定植缺陷的测试。

 DESCRIPTION (provided by applicant): Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most common vector-borne disease in U.S. B. burgdorferi can infect the skin at the site of the tick bite and spread via blood to diverse tissues, indicating that the bacterium can withstand bloodstream defenses. The complement system is a bloodstream defense that is activated via three different pathways. A critical step of the activation of this system is the formation of C3 convertases, C4b2a or C3bBb, which catalyze events resulting in inflammation, opsonization and pathogen lysis. Potential host damage following complement activation necessitates tight regulation by serum complement regulators that bind to complement components and promote their degradation. For example, the complement regulator factor H (FH) and C4BP modulates formation of C3bBb and C4b2a, respectively. We recently showed that B. burgdorferi produces DbpA, a surface protein that promotes tissue colonization by binding to dermatan sulfate and decorin, also fosters bacteremia by binding to C4BP. A B. burgdorferi strain producing DbpA-I156A, a point mutant deficient in C4BP binding, showed a delay in bacteremia and joint colonization. In addition, B. burgdorferi also produces CRASPs (Complement Regulator Acquiring Surface Proteins) that bind to complement regulators. The paralogs CspA, CspZ bind to FH and FH-like protein (FHL), and the paralogs ErpP, ErpC, and ErpA bind to FH and complement factor H-related protein (CFHR). Although several CRASPs have been demonstrated to contribute to serum resistance in vitro, none have been definitely shown to contribute to mammalian infection. Recently, we found that a B. burgdorferi strain harboring an erpA::Tn insertion displayed a defect in tissue colonization at early stages of infection. These results suggest that DbpA and ErpA modulate C4b2a and C3bBb, respectively, to promote bacteremia and tissue colonization. The ability of DbpA and ErpA to inhibit the formation of both classes of C3 convertases may signify a coordinated attack on host bloodstream defenses by B. burgdorferi. To test this hypothesis, the following aims will be pursued. (1) Test the role of FH-binding in bacteremia and colonization. We will test if a targeted, non-polar erpA deletion mutant display defects in FH/CFHR binding, complement activation in vitro, and bacteremia and tissue colonization in the murine model. If the mutant displays defects, we will test if FH binding by ErpA is essential for these functions, and whether other CRASPs can functionally complement the defects. (2). Test if C4BP- and FH-binding by B. burgdorferi provide non-redundant roles to promote bacteremia and colonization. To determine if a dramatic dissemination defect requires loss of both C4BP- and FH-binding activities, we will mutate erpA in the strain background of the B. burgdorferi DbpA-I156A mutant. These double mutant strains will be tested for the severe bacteremia and colonization defect in the murine model.