Investigation of FadA adhesin from Fusobacterium nucleatum

具核梭杆菌 FadA 粘附素的研究

• 批准号: 7736929
• 负责人: Yiping Han
• 金额: $ 35.05万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7736929
• 关键词: Abscess; Address; Affect; Amino Acids; Anaerobic Bacteria; Animal Model; Apoptosis; Attention; Award; Bacteria; Bacterial Adhesins; Binding; Binding Sites; Biological Process; Body part; Cell Adhesion; Cell Adhesion Molecules; Cell Communication; Cell physiology; Cell-Cell Adhesion; Cells; Complex; Diagnosis; Discipline of obstetrics; Disease; Drug Delivery Systems; Endothelial Cells; Epithelial; Filament; Focal Adhesions; Foundations; Fusobacteria; Fusobacterium; Fusobacterium nucleatum; Goals; Growth; Head; Hematogenous; Immune response; In Vitro; Incidence; Infection; Inflammatory Response; Invaded; Investigation; Leucine; Light; Mediating; Membrane; Membrane Proteins; Modeling; Mus; Neoplasm Metastasis; Oral; Oral cavity; Organism; Pathogenesis; Pathway interactions; Pattern; Peptide Signal Sequences; Peptides; Periodontal Diseases; Placenta; Play; Pregnancy Outcome; Premature Birth; Proteins; Receptor Cell; Research; Resolution; Role; Screening procedure; Site; Structure; Structure-Activity Relationship; System; Tail; Testing; Tissues; Uncertainty; Virulent; Wound Healing; Yeasts; alpha helix; angiogenesis; biological adaptation to stress; cancer cell; cell motility; frontier; in vitro testing; in vivo; innovation; intraamniotic infection; microorganism; monomer; mutant; novel; oral bacteria; oral biology; pathogen; pregnant; public health relevance; receptor; receptor binding; therapeutic development; therapeutic target; tissue culture; transmission process; yeast two hybrid system

DESCRIPTION (provided by applicant): Fusobacterium nucleatum is a Gram-negative anaerobe implicated in various forms of periodontal diseases. It is also associated with infections in other parts of the body and is one of the most prevalent species in intra- amniotic infection, causing preterm birth. F. nucleatum binds to and invades host epithelial and endothelial cells, a mechanism allowing colonization at different host sites. Previous studies have shown that F. nucleatum can translocate to the pregnant mouse placenta via haematogenous transmission, followed by activation of localized placental inflammatory responses, leading to adverse pregnancy outcomes. Invasion of mouse placental endothelial cells by F. nucleatum has also been observed in vivo. So far, only one adhesin, FadA (for Fusobacterium adhesin A), has been identified to be required for bacterial binding and invasion of host cells in both tissue-culture and animal models. FadA is a unique adhesin consisting of two forms: the intact non-secreted form (pre-FadA) composed of 129 amino-acid (aa) residues, and the mature secreted form (mFadA) of 111 aa. The crystal structure of mFadA reveals two anti-parallel alpha-helices connected by an 8- aa loop. The crystal structure of mFadA suggests oligomerization in a head-to-tail pattern via a novel "leucine chain" motif. Filament formation and binding to host cells require both pre-FadA and mFadA. A FadA adhesin model has been proposed, with pre-FadA anchored in the inner membrane and a chain of mFadA on top of pre-FadA protruding through the outer membrane. We hypothesize that the receptor-binding site may be located in the loop region, only fully exposed at the tip of the filament. Furthermore, several of the FadA filaments may bundle together to form a cluster of loops, which may be required for binding. One focus of this proposed study is to test the FadA adhesin model. Using a yeast-two-hybrid system, several putative receptors have been identified to interact with FadA. Thus, a second focus of this study is to validate the interactions between the putative receptors and FadA and to investigate the host responses to FadA. Our specific aims are: Aim I. Further characterization of the FadA adhesin in F. nucleatum. Five sub-aims are proposed: (i) investigating fadA expression under different conditions, (ii) investigating the spatial arrangement of FadA in F. nucleatum, (iii) investigating the involvement of the loop region in host-cell binding, (iv) investigating the role of the signal peptide in FadA complex formation, and (v) investigating possible accessory molecules in F. nucleatum associated with FadA. Aim II. Investigation of FadA and host cell interactions. Two sub-aims are proposed: (i) continued identification and characterization of the FadA receptor, and (ii) investigating the effect of FadA on cellular processes. From this study, we hope to identify (i) potential therapeutic targets for inhibiting F. nucleatum colonization in the host, and (ii) host components and pathways affected by FadA which will facilitate modulation of respective cellular processes and targeted drug delivery. PUBLIC HEALTH RELEVANCE: In this competitive renewal application, we propose to continue in-depth investigation of the novel FadA adhesin from oral bacterium Fusobacterium nucleatum, which is associated with periodontal disease and preterm birth. FadA plays an important role in the bacterial colonization in the host and in causing preterm birth. Continued structure-function analysis of FadA will facilitate therapeutic development to inhibit the bacterial colonization in the host and to reduce the incidence of preterm birth. Identification of host components and pathways affected by FadA will facilitate therapeutic development of modulate specific cellular processes and targeted drug delivery.

描述（由申请人提供）：具核梭杆菌是一种与多种形式的牙周病有关的革兰氏阴性厌氧菌。它还与身体其他部位的感染有关，是羊膜内感染最常见的物种之一，可导致早产。具核梭菌结合并侵入宿主上皮细胞和内皮细胞，这是一种允许在不同宿主位点定植的机制。既往研究表明，具核梭菌可通过血行传播转移至妊娠小鼠胎盘，激活局部胎盘炎症反应，导致不良妊娠结局。在体内也观察到具核梭杆菌对小鼠胎盘内皮细胞的侵袭。到目前为止，在组织培养和动物模型中，只有一种粘附素 FadA（梭杆菌粘附素 A）被确定为细菌结合和入侵宿主细胞所必需的。 FadA 是一种独特的粘附素，由两种形式组成：由 129 个氨基酸 (aa) 残基组成的完整非分泌形式 (pre-FadA)，以及由 111 个氨基酸 (aa) 组成的成熟分泌形式 (mFadA)。 mFadA 的晶体结构揭示了两个由 8-aa 环连接的反平行 α 螺旋。 mFadA 的晶体结构表明通过新颖的“亮氨酸链”基序以头尾相连的方式寡聚化。丝状形成和与宿主细胞的结合需要 pre-FadA 和 mFadA。已经提出了 FadA 粘附素模型，其中 pre-FadA 锚定在内膜中，而 mFadA 链位于 pre-FadA 顶部，穿过外膜突出。我们假设受体结合位点可能位于环区域，仅在丝的尖端完全暴露。此外，一些 FadA 丝可能捆绑在一起形成一簇环，这可能是结合所必需的。这项研究的重点之一是测试 FadA 粘附素模型。使用酵母双杂交系统，已鉴定出几种与 FadA 相互作用的推定受体。因此，本研究的第二个重点是验证假定受体和 FadA 之间的相互作用，并研究宿主对 FadA 的反应。我们的具体目标是： 目标 I. 具核梭杆菌中 FadA 粘附素的进一步表征。提出了五个子目标：（i）研究不同条件下的 fadA 表达，（ii）研究 F. nucleatum 中 FadA 的空间排列，（iii）研究环区域在宿主细胞结合中的参与，（iv）研究信号肽在 FadA 复合物形成中的作用，以及 (v) 研究 F. nucleatum 中与 FadA 相关的可能辅助分子。目标二。 FadA 和宿主细胞相互作用的研究。提出了两个子目标：(i) 继续鉴定和表征 FadA 受体，以及 (ii) 研究 FadA 对细胞过程的影响。通过这项研究，我们希望确定（i）抑制具核梭杆菌在宿主体内定植的潜在治疗靶点，以及（ii）受 FadA 影响的宿主成分和途径，这将有助于调节各自的细胞过程和靶向药物递送。 公共健康相关性：在本次竞争性更新申请中，我们建议继续深入研究来自口腔细菌具核梭杆菌的新型 FadA 粘附素，该粘附素与牙周病和早产有关。 FadA 在宿主体内的细菌定植和引起早产方面发挥着重要作用。 FadA 的持续结构功能分析将促进治疗方法的开发，以抑制宿主体内的细菌定植并降低早产发生率。鉴定受 FadA 影响的宿主成分和途径将有助于调节特定细胞过程和靶向药物递送的治疗开发。