Variable-Protein Surface Display in Treponema denticola

描述(由申请人提供)：齿状毛滴虫是构成龈下菌斑的复杂多菌生物膜的常见成分。它是一种“晚期殖民者”，并被认为在牙周病的发生发展中起病因学作用。T.denticola ATCC 35405在其2.8Mb的环状基因组中含有一个完整的产生多样性的逆转录元件(DGR)。DGRs是一种新发现的逆转录元件家族，可在靶基因中产生多样性。它们通过模板依赖、逆转录酶介导的机制发挥作用，该机制在蛋白质编码序列中的特定位置引入核苷酸替换。可变残基显示在专门的C型凝集素支架的配体结合口袋中，该支架平衡了蛋白质多样性和结构稳定性，为潜在的配体-受体相互作用创造了大量的受体。除了DGR连锁的靶标外，齿状毛滴虫基因组还包括六个额外的靶标基因，所有这些基因都被预测为编码表面暴露的脂蛋白。我们估计了7个目标蛋白中每一个的1024个独特的多肽序列的潜在谱系，远远超过了迄今为止描述的任何自然发生的蛋白质多样化系统的估计能力。该R21的目标是评估齿状毛滴虫已经进化出一种基于DGR的系统来在其细胞表面显示大量可变的蛋白质谱的可能性。我们的具体目标是：1)检验DGR能够指导预测的靶基因的定点突变的假设；2)检验多样化的靶基因编码表面暴露的脂蛋白的假设。DGRs代表了一种新发现的产生蛋白质多样性的机制，它们具有赋予宿主强大的选择性优势的潜力。对于T.denticola，我们认为新的结合相互作用的加速进化可以增加它所在的高度动态的微生物群落中的有机体适合度。了解该物种中的DGR系统不仅将揭示细菌进化的新机制，而且还将为理解促进参与在健康和疾病中发挥作用的微生物群落的适应提供新的范式。 公共卫生相关性：齿密螺旋体是龈下菌斑的常见成分，并被认为与牙周病的发病机制有关。我们的研究将有助于了解这种口腔细菌的定植和毒力机制。

DESCRIPTION (provided by applicant): T. denticola is a frequent component of the complex polymicrobial biofilms that constitute subgingival plaque. It is a "late colonizer" and is proposed to play an etiologic role in the development of periodontal disease. T. denticola ATCC 35405 contains a complete diversity-generating retroelement (DGR) within its 2.8 Mb circular genome. DGRs are a newly discovered family of retroelements that generate diversity in target genes. They function through a template-dependent, reverse trancriptase-mediated mechanism that introduces nucleotide substitutions at specified sites in protein-coding sequences. Variable residues are displayed in the ligand- binding pocket of a specialized C-type lectin scaffold which balances protein diversity with structural stability, creating vast repertoires of receptors for potential ligand-receptor interactions. In addition to a DGR-linked target, the T. denticola genome includes six additional target genes, all of which are predicted to encode surface-exposed lipoproteins. We estimate potential repertoires of 1024 unique polypeptide sequences for each of seven target proteins, far exceeding the estimated capabilities of any naturally occurring protein diversification system described to date. The goal of this R21 is to evaluate the possibility that T. denticola has evolved a DGR-based system to display massively variable protein repertoires on its cell surface. Our specific aims are to: 1) test the hypothesis that the T. denticola DGR is capable of directing site-specific mutagenesis of predicted target genes, and 2) test the hypothesis that diversified target genes encode surface-exposed lipoproteins. DGRs represent a newly discovered mechanism for generating protein diversity and they have the potential to confer powerful selective advantages to their hosts. For T. denticola, we imagine that the accelerated evolution of novel binding interactions could increase the organisms fitness in the highly dynamic microbial community in which it resides. Understanding the DGR system in this species will not only reveal new mechanisms for bacterial evolution, but also provide new paradigms for understanding adaptations that facilitate participation in microbial consortia that play roles in health and disease. PUBLIC HEALTH RELEVANCE: Treponema denticola is a common component of subgingival plaque and is proposed to contribute to the pathogenesis of periodontal disease. Our studies will contribute to an understanding of colonization and virulence mechanisms by this oral bacterium.