Multiple B. burgdorferi Factors Collaborate to Evade Complement-Mediated Defenses

 描述(申请人提供)：莱姆病，由伯氏疏螺旋体(BB)引起，是美国最常见的媒介传播疾病。补体抵抗，使血液存活，可能是螺旋体传播到远处部位的关键。 补体激活的一个关键步骤是C3转换酶C4b2a或C3bBb的形成，这是导致炎症、调理和病原体溶解的复合体。补体激活引起的潜在组织损伤需要血清补体调节蛋白(CRPS)的严格控制，补体调节蛋白结合并促进补体蛋白的降解。为了避免补体介导的杀伤，病原体通常会产生结合CRP的表面蛋白，这种现象被认为是导致菌血症和组织定植的原因。然而，对于BB来说，缺乏关于这一点的严格证明。BB表面蛋白BBK32和DbpA是研究较多的BB细胞外基质(ECM)粘附素，我们发现OspC也具有ECM结合粘附素的功能。我们的新发现形成了这一建议的基础，这三种粘附素都可以促进体外血清存活和体内血流存活。DbpA和BBK32都与C4BP结合，预测C4BP可以阻止C4b2a的形成。Bb产生DbpAI156A是一个DbpA点突变，缺乏C4BP结合，但不缺乏ECM结合，在血液和关节定植延迟，提示DbpA介导早期菌血症。OspC与C4b结合，预测可阻断C4b2a的形成，OspC的等位基因变体显示出明显的C4b结合活性，与体外血清耐药活性和体内早期菌血症相关。这是第一次证明BB产生的补体逃避因子干扰经典和凝集素，并首次证明任何补体抵抗因子在哺乳动物宿主中的传播中起着重要作用。然而，我们的数据也表明，其他未知的细菌因素可能在持续感染中发挥作用。为了确定BB逃避补体的机制和识别新的血清耐药因素，我们将1)通过评估C4BP结合缺陷的BBK32突变体对血清抵抗力和血液存活的影响来确定BBK32介导的C4BP结合对血清抵抗和菌血症的贡献；2)通过检测C4b结合缺陷的OspC突变体是否失去促进这些表型的能力来确定C4b结合活性是否是OspC介导的血清抵抗和早期菌血症所必需的；3)通过检测DbpA-或BBK32-C4BP复合体是否促进C4b的蛋白分解和抑制细菌裂解以及C4b-OspC复合体是否抑制C4b2a的形成来表征DbpA、BBK32和OspC阻断补体激活的机制；4)进行基于TN-Seq的大规模筛查，以确定编码促进血清抵抗的因子的BB基因。这项工作将对潜在的治疗干预措施和了解伯氏杆菌致病和生活史的基本机制产生重大影响。。

 DESCRIPTION (provided by applicant): Lyme disease, caused by the spirochete Borrelia burgdorferi (Bb), is the most common vector-borne disease in U.S. Complement resistance, enabling bloodstream survival, is likely essential for spirochetes to disseminate to distant sites. A critical step in complement activation is the formation of C3 convertases C4b2a or C3bBb, complexes that lead to inflammation, opsonization and pathogen lysis. Potential tissue injury due to complement activation necessitates stringent control by serum complement regulatory proteins (CRPs) that bind to and promote degradation of complement proteins. To avoid complement-mediated killing, pathogens often produce surface proteins that bind CRPs, a phenomenon thought to contribute to bacteremia and colonization of tissues. For Bb, however, a rigorous demonstration of this is lacking. Bb surface proteins BBK32 and DbpA are well-studied Bb extracellular matrix (ECM) adhesins, and we found that OspC also functions as an ECM-binding adhesin. Our novel findings forming the basis for this proposal are that all three adhesins also promote serum survival in vitro and bloodstream survival in vivo. DbpA and BBK32 both bind to C4BP, which is predicted to block the formation of C4b2a. Bb producing DbpAI156A, a DbpA point mutant deficient for C4BP- but not ECM-binding, was delayed in blood and joint colonization, suggesting that DbpA mediates early stage bacteremia. OspC bound to C4b, predicted to block the formation of C4b2a, and allelic variants of OspC displayed distinct C4b-binding activities that correlated with serum resistance activity in vitro and early-stage bacteremia in vivo. These are the first demonstrations that Bb produces complement evasion factors that interfere with both the classical and lectin, and the first to demonstrate tha any complement resistance factor plays an important role in Bb dissemination in the mammalian host. However, our data also suggest that other, unknown bacterial factors may play a role in persistent infection. To determine mechanisms of complement evasion by Bb and to identify novel serum resistance factors, we will 1) Define the contribution of BBK32-mediated C4BP binding to serum resistance and bacteremia by evaluating a C4BP-binding deficient BBK32 mutant for serum resistance and bloodstream survival; 2) Determine whether C4b-binding activity is required for OspC-mediated serum resistance and early bacteremia by testing C4b-binding deficient OspC mutants for loss of ability to promote these phenotypes; 3) Characterize mechanisms by which DbpA, BBK32 and OspC block complement activation by examining whether DbpA- or BBK32- C4BP complexes promote proteolysis of C4b and inhibit bacterial lysis and whether the C4b-OspC complex inhibits the formation of C4b2a; and 4) Perform a Tn-Seq -based large-scale screen to identify Bb genes encoding factors that promote serum resistance. This work will have significant impact on potential therapeutic interventions and understanding fundamental mechanisms of B. burgdorferi pathogenesis and life cycle. .