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

基本信息

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
项目类别：
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

项目摘要

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）：莱姆病是由螺旋体伯氏疏螺旋体 (Bb) 引起的，是美国最常见的媒介传播疾病。补体抗性使血液能够存活，可能对于螺旋体传播到远处至关重要。补体激活的关键步骤是 C3 转化酶 C4b2a 或 C3bBb 的形成，这些复合物会导致炎症、调理作用和病原体裂解。补体激活导致的潜在组织损伤需要通过结合并促进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，病原体通常会产生结合 CRP 的表面蛋白，这种现象被认为会导致菌血症和组织定植。然而，缺乏对此的严格证明。Bb 表面蛋白 BBK32 和 DbpA 是经过充分研究的 Bb 细胞外基质 (ECM) 粘附素，我们发现 OspC 也可作为 ECM 结合粘附素，我们的新发现构成了这一点的基础。提议认为，所有三种粘附素还可促进体外血清存活和体内血流存活，DbpA 和 BBK32 均与 C4BP 结合，预计可阻止 C4BP 的形成。 C4b2a.Bb 产生 DbpAI156A，这是一种 C4BP 结合缺陷的 DbpA 点突变体，在血液和关节定植延迟，表明 DbpA 介导与 C4b 结合的早期菌血症，预计会阻止 C4b2a 的形成，并且OspC 的等位基因变体表现出独特的 C4b 结合活性，与体外血清耐药活性和体内早期菌血症相关。首次证明 Bb 产生干扰经典和凝集素的补体逃避因子，并且首次证明任何补体抗性因子在哺乳动物宿主中的 Bb 传播中发挥重要作用。未知的细菌因素可能在持续感染中发挥作用，为了确定 Bb 的补体逃逸机制并确定新的血清耐药因素，我们将 1) 定义 BBK32 介导的 C4BP 结合对血清耐药的贡献和通过评估 C4BP 结合缺陷的 BBK32 突变体的血清耐药性和血流存活来确定菌血症；2) 通过测试 C4b 结合缺陷的 OspC 突变体促进能力的丧失，确定 OspC 介导的血清耐药性和早期菌血症是否需要 C4b 结合活性；这些表型；3) 通过检查 DbpA- 或BBK32-C4BP复合物促进C4b的蛋白水解并抑制细菌裂解以及C4b-OspC复合物是否抑制C4b2a的形成；以及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措施和了解伯氏疏螺旋体发病机制和生命周期的基本机制产生重大影响。