Investigation of FadA adhesin from Fusobacterium nucleatum

描述（由申请人提供）：核梭杆菌是一种革兰氏阴性厌氧菌，与各种形式的牙周病有关。它也与身体其他部位的感染有关，是羊膜内感染中最常见的物种之一，导致早产。F. nucleatum结合并侵入宿主上皮细胞和内皮细胞，这种机制允许在不同的宿主位置定植。先前的研究表明，具核梭菌可以通过血液传播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。在体内也观察到核仁梭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，只有一种黏附素FadA（梭杆菌黏附素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）和111个氨基酸的成熟分泌形式（mFadA）。mFadA的晶体结构为由8- aa环连接的两个反平行α螺旋。mFadA的晶体结构表明，通过一种新的“亮氨酸链”基序，mFadA以从头到尾的模式进行寡聚。丝的形成和与宿主细胞的结合需要pre-FadA和mFadA。提出了一种FadA黏附素模型，其中pre-FadA固定在内膜中，而在pre-FadA之上的mFadA链突出穿过外膜。我们假设受体结合位点可能位于环区，仅完全暴露在丝的尖端。此外，几个FadA细丝可以束在一起形成环簇，这可能是结合所必需的。本研究的一个重点是测试FadA粘附素模型。利用酵母-双杂交系统，已经确定了几种可能与FadA相互作用的受体。因此，本研究的第二个重点是验证假定的受体与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：目的1 .进一步表征核梭菌的FadA粘附素。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在核仁梭菌中的空间排列，（iii）研究环区参与宿主细胞结合，（iv）研究信号肽在fadA复合物形成中的作用，以及(v)研究核仁梭菌中可能与fadA相关的辅助分子。目的二世。FadA与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，这些成分和途径将促进各自细胞过程和靶向药物传递的调节。

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.