Treponomics: enhanced tools for genetic manipulation in spirochetes

描述（由申请人提供）：微生物基因组学时代的主要挑战之一是通过实验建立基因组数据与基因功能、生理学、行为和致病性之间的联系。齿垢密螺旋体是牙龈粘膜组织的一种条件致病菌，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与T. pallidum、T.齿垢属于 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，具有很高的兴趣。这是由于Td和T之间的生理相似性。pallidum和T.如果没有体外培养系统，苍白球遗传学研究仍然是不可行的。该项目将开发和展示可培养的密螺旋体属螺旋体的遗传操作方法的重大进展，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，特别是不可培养的螺旋体问题提供机会。我们提出了一个T。齿状突特异性适应的反选择诱变加上敲除的限制性修饰系统的关键特征，以促进T.具有（1）大大增加的吸收和掺入异源DNA的能力，和（2）可能的突变类型的扩展范围的齿垢菌株。基于我们小组主要进行的“概念验证”研究，这项工作的结果将有助于T。denticola，并建立了T.齿垢作为其他密螺旋体感染因子分子分析的模式生物，特别是T.苍白球

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.