Investigation of FadA adhesin from Fusobacterium nucleatum

具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，是体内最流行的物种之一， 羊膜感染导致早产F.有核物质结合并侵入宿主上皮和内皮细胞 细胞，一种允许在不同宿主位点定殖的机制。以往的研究表明，F. nucleatum可以通过血行传递转移到孕鼠胎盘， 局部胎盘炎症反应的激活，导致不良妊娠结局。入侵 培养小鼠胎盘内皮细胞，用F.在体内也观察到有核质。到目前为止，只有一种粘附素， FadA（Fusobacterium adhesin A）已被鉴定为细菌结合和侵入细菌所需。 在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（aa）残基组成的完整的非分泌形式（pre-FadA），以及成熟的分泌形式 （mFadA）为111 aa。mFadA的晶体结构揭示了由8-羟基-N-甲基连接的两个反平行的α-螺旋。 aa循环。mFadA的晶体结构表明通过新的“亮氨酸”以头-尾模式寡聚化。 链”母题。细丝形成和与宿主细胞的结合需要pre-FadA和mFadA两者。A FadA粘附素 已经提出了一种模型，其中pre-FadA锚定在内膜中，而mFadA链在膜的顶部。 pre-FadA通过外膜突出。我们推测受体结合位点可能是 位于环区域中，仅在灯丝的尖端处完全暴露。此外，一些FadA 细丝可以捆扎在一起，形成一簇环，这可能是装订所需要的。其中一个焦点是 本研究旨在验证FadA粘附素模型。使用酵母双杂交系统，几个假定的 已经鉴定出受体与FadA相互作用。因此，本研究的第二个重点是验证 本发明的目的在于研究推定受体与FadA之间的相互作用，并研究宿主对FadA的反应。我们 具体目标是：目标一。进一步对F.核质五个次级目标是 建议：（i）调查fadA在不同条件下的表达，（ii）调查空间排列 FadA在F.核质，（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，和（v）研究可能的辅助作用， 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）潜在的 用于抑制F.在宿主中的核质定殖，和（ii）宿主组分和途径 受FadA影响，这将有助于调节各自的细胞过程和靶向药物递送。

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.