Exploring New Virulence Factors of the Oral Spirochete Treponema denticola

描述（由申请人提供）：螺旋体齿垢密螺旋体（Td）与严重和难治性牙周疾病密切相关。作为“红色复合体”细菌的一员，Td主要生活在龈下菌斑的顶端表面，与龈下菌斑直接接触。 上皮因此，Td处于直接遭遇巨大宿主免疫攻击的最前沿，例如，补体杀伤和吞噬作用。在囊袋中，多形核白细胞（PMNs）是通过吞噬作用杀死病原体来保护宿主的主要免疫细胞。此外，龈沟液（GCF）主要由血清组成，含有补体系统，这是宿主防御的关键成分。以前的报道表明，Td对补体杀伤具有抗性，并且能够调节PMNs，并且能够在口腔植物群中茁壮成长。然而，涉及的分子机制仍然难以捉摸。本申请的中心假设是Td的新毒力因子TDE 0362（Tmac）具有双重功能：其C-末端（C362）是切割对于PMN活化和补体杀伤必需的免疫因子的蛋白酶;其N-末端（N362）含有细菌免疫球蛋白（Big）样结构域，其通过分子模拟机制阻断PMN活化。总的来说，Tmac保护Td免受吞噬作用和补体杀伤，从而增强其存活和感染的建立。为了验证这一假设，本研究的具体目的是：（1）确定Tmac是否被切割和分泌，以及切割的机制;（2）研究Tmac对人IgG的内肽酶活性;（3）阐明Tmac在PMN活化中的作用及其机制;（4）研究Tmac在Td致病性中的作用。该项目的完成将推进我们目前对Td致病性的理解，特别是先天免疫逃避的机制，这可能会导致新的牙周病治疗干预措施。

DESCRIPTION (provided by applicant): The spirochete Treponema denticola (Td) is strongly associated with severe and refractory periodontal conditions. As a member of the 'red-complex' bacteria, Td primarily lives on the apical surface of subgingival plaque in direct contact with the epithelium. Thus Td is at the forefront that directly encounters enormous host immune attacks, e.g., complement killing and phagocytosis. In the pocket, polymorphoneclear leukocytes (PMNs) are the major immune cells that protect the host by killing pathogens via phagocytosis. In addition, the gingival crevice fluid (GCF), which is mainly composed of serum, contains the complement system, a critical component in host defense. Previous reports suggest that Td is resistant to the complement killing, and is able to modulate PMNs and can thrive in the oral flora. However, the molecular mechanisms involved remain elusive. The central hypothesis of this application is that TDE0362 (Tmac), a new virulence factor of Td, has dual functions: its C-terminus (C362) is a protease that cleaves immune factors that are essential for the activation of PMNs and the complement killing; its N-terminus (N362) contains a bacterial immunoglobulin (Big)-like domain which blocks PMN activation via a molecular mimicry mechanism. Collectively, Tmac protects Td from the phagocytosis and the complement killing, and consequently enhances its survival and establishment of infection. To test this hypothesis, this application wil focus on the following Specific Aims: (1) To determine if Tmac is cleaved and secreted, and the mechanism involved in the cleavage; (2) To study the endopeptidase activity of Tmac on human IgG; (3) To elucidate the role of Tmac in PMN activation and its mechanism involved; and (4) To investigate the role of Tmac in the pathogenicity of Td in vitro and in vivo. Completion of this project will advance our current understanding of the pathogenicity of Td, in particular, the mechanisms involved in the innate immune evasion, which could potentially lead to new therapeutic interventions against periodontal diseases.