Mechanisms of Complement Evasion by Treponema denticola

描述（申请人提供）：牙周病是中年人最常见的感染性疾病。密螺旋体是一种口腔细菌，与牙周炎的发展密切相关。牙周袋是牙齿和牙龈之间的感染部位，它浸泡在含有抗菌肽、免疫球蛋白和丰富的补体蛋白的沟液中。补体是一种先天免疫系统，可以杀死革兰氏阴性细菌，标记病原体进行破坏，并调节局部炎症。齿状体可以通过产生FhbB和牙本质素来逃避补体介导的破坏。FhbB与宿主负补体调节因子H结合，对人血清中细菌的存活至关重要。牙釉质能够分裂中枢补体成分C3和C3b，牙釉质可能导致局部免疫反应失调，导致炎症相关的组织损伤，这是牙周炎的一个标志。本项目旨在对多种临床分离的牙齿弓形菌fhbB进行测序，并应用重组蛋白表达纯化、表面等离子体共振、免疫印迹和等位基因交换等生化方法，确定蛋白序列变化是否影响fhbB:FH相互作用和保护功能。此外，本研究旨在通过纯化和质谱法鉴定牙lisin产生的因子H片段，该片段仍然与细胞结合。几种生化和细胞培养技术将被应用于确定因子H片段是否保留调控活性，是否可以保护T. denticola免受补体破坏，以及是否可以改变全长FH的宿主细胞结合。更全面地了解牙周感染期间由牙牙霉引起的免疫失调将有助于潜在的未来治疗和疫苗设计。

DESCRIPTION (provided by applicant): Periodontal disease is the most common infectious disease of middle-aged adults. Treponema denticola is an oral, bacterial species that is strongly associated with the development of periodontitis. The periodontal pocket, the site of infection between the tooth and the gums, is bathed in crevicular fluid containing antimicrobial peptides, immunoglobulin and highly abundant complement proteins. Complement is an innate immune system that can kill Gram-negative bacteria, mark pathogens for destruction, and modulate local inflammation. T. denticola can evade complement- mediated destruction by production of FhbB and dentilisin. FhbB binds the host negative complement regulator factor H and is critical to the survival of the bacterium in human serum. Dentilisin is capable of cleaving the central complement components C3 and C3b as well as factor H. T. denticola may cause a dysregulation of the local immune response leading to inflammation-associated tissue damage that is a hallmark of periodontitis. This project aims to sequence fhbB from a variety of clinical isolates of T. denticola then apply biochemical approaches such as recombinant protein expression and purification, surface plasmon resonance, immunoblotting and allelic exchange to determine if protein sequence variation affects the FhbB:FH interaction and protective function. Additionally, this study aims to identify a dentilisin generated factor H fragment, which remains bound to the cell, by purification and mass spectrometry. Several biochemical and cell culture techniques will be applied to determine if the factor H fragment retains regulatory activity, can protect T. denticola from complement destruction, and alter host cell binding of full length FH. A more complete picture of immune dysregulation caused by T. denticola during periodontal infection will aid in potential future therapeutic and vaccine design.