Investigation of FadA adhesin from Fusobacterium nucleatum

描述（由申请人提供）：具核梭杆菌是一种与各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，是羊膜内感染中最普遍的物种之一，导致早产。F.具核质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部位定殖的机制。以往的研究表明，F. nucleatum可以通过造血传递转移到妊娠小鼠胎盘，随后激活局部胎盘炎症反应，导致不良妊娠结局。F.对小鼠胎盘内皮细胞的侵袭作用。在体内也观察到有核质。到目前为止，只有一种粘附素，FadA（梭杆菌粘附素A），已被确定为所需的组织培养和动物模型中的宿主细胞的细菌结合和入侵。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非分泌形式（pre-FadA）和由111个氨基酸残基组成的成熟分泌形式（mFadA）。mFadA的晶体结构显示由8- aa环连接的两个反平行α-螺旋。mFadA的晶体结构表明通过新的“亮氨酸链”基序以头-尾模式寡聚化。细丝形成和与宿主细胞的结合需要pre-FadA和mFadA两者。已经提出了FadA粘附素模型，其中pre-FadA锚定在内膜中，并且pre-FadA顶部的mFadA链突出穿过外膜。我们推测受体结合位点可能位于环区，仅在丝的尖端完全暴露。此外，几个FadA细丝可以捆绑在一起以形成环簇，这可能是结合所需的。本研究的一个重点是测试FadA粘附素模型。使用酵母双杂交系统，已经鉴定出几种推定的受体与FadA相互作用。因此，本研究的第二个重点是验证假定的受体和FadA之间的相互作用，并调查宿主对FadA的反应。我们的具体目标是：目标一。进一步对F.核质本研究提出了五个子目标：（i）研究fadA在不同条件下的表达;（ii）研究FadA在F. nucleatum，（iii）调查参与的环区在宿主细胞的结合，（iv）调查的作用，信号肽在FadA复合物的形成，和（v）调查可能的辅助分子在F。与FadA相关的核质。Aim II. FadA和宿主细胞相互作用的研究。提出了两个次级目标：（i）继续鉴定和表征FadA受体，和（ii）研究FadA对细胞过程的作用。通过这项研究，我们希望确定（i）抑制F的潜在治疗靶点。（ii）受FadA影响的宿主组分和途径，其将促进相应细胞过程的调节和靶向药物递送。 公共卫生相关性：在这项竞争性更新申请中，我们建议继续深入研究口腔细菌具核梭杆菌中与牙周病和早产相关的新型FadA粘附素。FadA在宿主细菌定植和引起早产中起重要作用。对FadA结构-功能的深入研究将有助于开发抑制细菌在宿主体内定植和降低早产发生率的治疗方法。鉴定受FadA影响的宿主组分和途径将促进调节特定细胞过程和靶向药物递送的治疗开发。

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.