Treponomics: enhanced tools for genetic manipulation in spirochetes

描述(申请人提供)：微生物基因组学时代的主要挑战之一是在实验上建立基因组数据与基因功能、生理、行为和致病性之间的联系。齿密螺旋体是牙龈粘膜组织的一种机会性病原体，为研究微生物-宿主相互作用、微生物生理学和分子进化提供了一系列靶点。虽然口腔螺旋体的致病行为和疾病关联明显不同于梅毒螺旋体，但齿状螺旋体是 作为研究包括梅毒在内的螺旋体疾病的生理学、生物合成途径和微生物-宿主相互作用的模型，受到高度关注。这既是由于TD和梅毒螺旋体在生理上的相似性，也是因为如果没有体外培养系统，梅毒螺旋体的遗传学研究仍然是不可行的。该项目将开发和展示可培养螺旋体的遗传操作方法方面的重大进展，这将“启动”密螺旋体遗传研究，从而为合理解决新的生物学问题提供机会，特别是在不可培养的螺旋体中。我们建议将反选择突变与限制性内切酶修饰系统的敲除相结合的反选择突变的特异性适应作为关键特征，以便于构建具有(1)极大地增强了吸收和整合异源DNA的能力，以及(2)扩大了可能的突变类型的范围。在我们小组主要进行的“概念验证”研究的基础上，这项工作的结果将有助于对齿状毛滴虫进行深入的分子分析，并建立齿状毛滴虫作为模式生物用于其他梅毒螺旋体感染病原体，特别是梅毒螺旋体的分子分析。

DESCRIPTION (provided by applicant): One of the major challenges in the era of microbial genomics is to experimentally establish connections between genome data and gene function, physiology, behavior and pathogenicity. Treponema denticola, an opportunistic pathogen of gingival mucosal tissue, offers a range of targets for research into microbe-host interactions, microbial physiology and molecular evolution. While pathogenic behaviors and disease associations of oral spirochetes are clearly distinct from those of T. pallidum, T. denticola is of high interest as a model for studying physiology, biosynthetic pathways and microbe-host interactions in spirochetal diseases including syphilis. This is due both to physiological similarities between Td and T. pallidum and to the fact that T. pallidum genetic studies remain impracticable without an in vitro culture system. This project will develop and demonstrate significant advances in methods for genetic manipulation of cultivable spirochetes of the genus Treponema that will "jump-start" Treponema genetic research, thus providing opportunities to rationally address novel biological questions, particularly in uncultivable spirochetes. We propose a T. denticola-specific adaptation of counter selection mutagenesis coupled with knockouts of restriction-modification systems as key features to facilitate construction of T. denticola strains with (1) greatly increased ability to take up and incorporate heterologous DNA's, and (2) an expanded range of possible mutant types. Building on "proof of concept" studies primarily conducted by our group, results of this work will both facilitate in-depth molecular analysis of T. denticola and establish the utility of T. denticola as a model organism for molecular analysis of agents of other treponemal infections, particularly T. pallidum.