Investigation of FadA adhesin from Fusobacterium nucleatum

描述(申请人提供)：核梭杆菌是一种革兰氏阴性厌氧菌，与各种形式的牙周疾病有关。它还与身体其他部位的感染有关，是导致早产的羊水内感染最常见的物种之一。核藻结合并入侵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植的机制。以往的研究表明，核杆菌可以通过血源性传播转移到妊娠小鼠的胎盘，随后激活局部的胎盘炎性反应，导致不良妊娠结局。在体内也观察到核假单胞菌侵袭小鼠胎盘内皮细胞。到目前为止，在组织培养和动物模型中，只有一种粘附素FADA(对于梭杆菌粘附素A)被鉴定为与宿主细胞的细菌结合和入侵所必需的。FADA是一种独特的粘附素，由两种形式组成：由129个氨基酸残基组成的完整非分泌型(前FADA)和由111个氨基酸残基组成的成熟分泌型(MFadA)。MFadA的晶体结构显示有两个由8-AA环连接的反平行α-螺旋。MFadA的晶体结构表明，通过一个新的“亮氨酸链”基序，以头到尾的模式进行齐聚。细丝的形成和与宿主细胞的结合都需要前FADA和mFadA。提出了一种FADA粘附素模型，将Pre-FADA锚定在内膜上，在Pre-FADA的顶部通过外膜伸出mFadA链。我们假设受体结合部位可能位于环区，仅在细丝顶端完全暴露。此外，几个FADA细丝可以捆绑在一起形成一簇环，这可能是结合所需的。这项拟议研究的重点之一是测试FADA粘附素模型。利用酵母双杂交系统，已经确定了几个可能的受体与FADA相互作用。因此，本研究的第二个重点是验证可能的受体与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：目标1.进一步表征核藻中的FADA粘附素。提出了五个子目标：(1)研究不同条件下FADA的表达；(2)研究FADA在核盘藻中的空间排列；(3)研究环区在宿主细胞结合中的作用；(4)研究信号肽在FADA复合体形成中的作用；(5)研究核盘藻中可能与FADA有关的辅助分子。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)继续鉴定和鉴定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)抑制核镰刀菌在宿主中定植的潜在治疗靶点，以及(Ii)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输送。 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来自口腔细菌核梭杆菌的新型FADA粘附素，它与牙周病和早产有关。FADA在细菌在宿主体内的定植和导致早产方面起着重要作用。继续分析FADA的结构和功能将有助于治疗的发展，以抑制细菌在宿主中的定植，减少早产的发生率。识别受FADA影响的宿主成分和途径将有助于调控特定细胞过程和靶向药物输送的治疗发展。

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.