Investigation of FadA adhesin from Fusobacterium nucleatum

具核梭杆菌 FadA 粘附素的研究

• 批准号: 8461710
• 负责人: Yiping Han
• 金额: $ 23.65万
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8461710
• 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 Cells; 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; 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; cellular targeting; frontier; in vitro testing; in vivo; innovation; intraamniotic infection; microorganism; monomer; mutant; novel; oral bacteria; oral biology; pathogen; pregnant; receptor; receptor binding; screening; therapeutic development; therapeutic target; tissue culture; transmission process; yeast two hybrid system

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.

核梭杆菌是一种革兰氏阴性厌氧菌，与多种牙周病有关。它 也与身体其他部位的感染有关，是体内最常见的物种之一 羊水感染，导致早产。F.核霉与宿主上皮和内皮细胞结合并侵袭 细胞，一种允许在不同宿主位置定居的机制。以往的研究表明，F. 有核细胞可以通过血源性传播转移到怀孕的小鼠胎盘，随后 激活局部胎盘炎性反应，导致不良妊娠结局。入侵 小鼠胎盘内皮细胞也在体内观察到了核假单胞菌。到目前为止，只有一种粘附素， FADA(对于梭杆菌粘附素A)，已被发现是细菌结合和入侵所必需的 组织培养和动物模型中的宿主细胞。FADA是一种独特的粘附素，由两种形式组成： 由129个氨基酸残基组成的完整的非分泌型(前FADA)和成熟的分泌型 (MFadA)为111 aa。MFadA的晶体结构显示两个反平行的α-螺旋由一个8-螺旋相连。 AA循环。MFadA的晶体结构表明，通过一种新的亮氨酸，以从头到尾的方式进行齐聚 链“基序。细丝的形成和与宿主细胞的结合需要前FADA和mFadA。FADA粘附素 已经提出了一个模型，其中前FADA锚定在内膜上，mFadA链锚定在 Pre-fada通过外膜突出。我们推测受体结合部位可能是 位于环状区域，只完全暴露在细丝的顶端。此外，几位时尚人士 细丝可以捆绑在一起形成一簇环，这可能是结合所需的。其中一个焦点就是 建议的研究是对Fada粘附素模型进行测试。使用酵母双杂交系统，几个假定的 已发现与FADA相互作用的受体。因此，本研究的第二个重点是验证 推测的受体与FADA之间的相互作用，并研究寄主对FADA的反应。我们的 具体目标为：目标1.进一步鉴定核盘藻中的FADA粘附素。五个子目标是 建议：(I)研究不同条件下FADA的表达；(Ii)调查空间排列 (Iii)研究环区在宿主细胞结合中的作用，(Iv) 研究信号肽在FADA复合体形成中的作用，以及(V)研究可能的辅助 核盘藻中与FADA相关的分子。目的II.研究FADA与宿主细胞的相互作用。 提出了两个子目标：(I)FADA受体的持续鉴定和鉴定；(Ii) 研究FADA对细胞过程的影响。从这项研究中，我们希望发现(一)潜力 抑制核镰刀菌在寄主中定植的治疗靶点，以及(Ii)寄主成分和途径 受FADA影响，这将有助于调节各自的细胞过程和靶向药物输送。