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

多种伯氏疏螺旋体因子共同逃避补体介导的防御

• 批准号: 9187413
• 负责人: Jenifer L Coburn
• 金额: $ 62.92万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9187413
• 关键词: Antigen-Antibody Complex; Bacteremia; Bacteria; Bacterial Adhesins; Bacterial Attachment Site; Binding; Binding Proteins; Bite; Blood; Blood Circulation; Borrelia burgdorferi; Carbohydrates; Chronic; Complement; Complement 3 Convertase; Complement 3b; Complement 4b; Complement Activation; Complement Degradation; Complement Factor H; Complex; Cytolysis; Data; Development; Disease; Distant; Environment; Event; Extracellular Matrix; Genes; Immune response; In Vitro; Infection; Inflammation; Injury; Investigation; Joints; Knowledge; Lead; Lectin; Life Cycle Stages; Lyme Disease; Mannose Binding Lectin; Mannose-Binding Lectins; Mediating; Membrane Proteins; Methods; Modeling; Molecular; Mus; Order Spirochaetales; Organism; OspC protein; Pathogenesis; Pathway interactions; Phenotype; Play; Proteins; Proteolysis; Recombinant Proteins; Resistance; Role; Serum; Site; Surface; Testing; Therapeutic Intervention; Ticks; Tissues; Variant; Vector-transmitted infectious disease; Virulence Factors; Work; base; complement 4b-binding protein; complement pathway; defined contribution; enzootic; fitness; genetic regulatory protein; genetic variant; genome-wide; in vitro activity; in vivo; insight; killings; microbial; mutant; novel; pathogen; prevent; protein degradation; public health relevance; resistance factors

 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. .

 描述（由申请人提供）：莱姆病，由螺旋体伯氏疏螺旋体（Bb）引起，是美国最常见的病媒传播疾病。补体抵抗，使血流存活，可能是螺旋体传播到远处的必要条件。 补体激活的关键步骤是形成C3转化酶C4 b2 a或C3 bBb，这是导致炎症、调理作用和病原体裂解的复合物。由于补体激活引起的潜在组织损伤需要通过结合补体蛋白并促进其降解的血清补体调节蛋白（CRP）进行严格控制。为了避免补体介导的杀伤，病原体通常产生结合CRP的表面蛋白，这是一种被认为有助于菌血症和组织定植的现象。然而，对于Bb来说，这一点缺乏严格的证明。Bb表面蛋白BBK 32和DbpA是被充分研究的Bb细胞外基质（ECM）粘附素，并且我们发现OspC也作为ECM结合粘附素起作用。我们的新发现形成了这一建议的基础是，所有三种粘附素也促进体外血清存活和体内血流存活。DbpA和BBK 32都与C4 BP结合，预测其阻断C4 b2 a的形成。产生Bb的DbpAI 156 A是一种DbpA点突变体，缺乏C4 BP-但不结合ECM，在血液和关节定植中延迟，表明DbpA介导早期菌血症。OspC与C4 b结合，预测阻断C4 b2 a的形成，并且OspC的等位基因变体显示出与体外血清抗性活性和体内早期菌血症相关的不同C4 b结合活性。这是第一次证明Bb产生补体逃避因子，干扰经典和凝集素，并首次证明任何补体抗性因子在哺乳动物宿主中Bb传播中起重要作用。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逃避补体的机制并鉴定新的血清抗性因子，我们将1）通过评估C4 BP结合缺陷型BBK 32突变体的血清抗性和血流存活来确定BBK 32介导的C4 BP结合对血清抗性和菌血症的贡献; 2）通过测试C4 b-结合活性来确定C4 b-结合活性是否是OspC介导的血清抗性和早期菌血症所需的。结合缺陷型OspC突变体以丧失促进这些表型的能力; 3）表征DbpA，BBK 32和OspC通过检查DbpA-或BBK 32-C4 BP复合物是否促进C4 b的蛋白水解并抑制细菌裂解以及C4 b-或BBK 32-C4 BP复合物是否促进C4 b的蛋白水解并抑制细菌裂解来阻断补体激活。OspC复合物抑制C4 b2 a的形成;和4）进行基于Tn-Seq的大规模筛选以鉴定编码促进血清抗性的因子的Bb基因。这项工作将对潜在的治疗干预和理解B的基本机制产生重大影响。burgdorferi发病机制和生活史。.