Treponomics: enhanced tools for genetic manipulation in spirochetes

密螺旋体组学：螺旋体基因操作的增强工具

• 批准号: 8719805
• 负责人: J CHRISTOPHER FENNO
• 金额: $ 19.43万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-10 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8719805
• 关键词: Address; Anaerobic Bacteria; Animal Model; Behavior; Biological; Cellular Structures; Chromosomes; Computer Simulation; Coupled; DNA; DNA Restriction-Modification Enzymes; Data; Development; Disease; Disease Association; Electroporation; Essential Genes; Future; Gene Mutation; Genes; Genetic; Genetic Research; Genetic Transformation; Genome; Genomics; Gingiva; Human; In Vitro; Knock-out; Laboratories; Libraries; Mediating; Membrane Proteins; Methods; Microbe; Microbial Physiology; Microbiology; Molecular; Molecular Analysis; Molecular Evolution; Molecular Genetics; Mucous Membrane; Mutagenesis; Mutation; Order Spirochaetales; PAWR protein; Parents; Pathogenesis; Pathogenicity; Pathway interactions; Physiological; Physiology; Prokaryotic Cells; Protocols documentation; Qualifying; Recovery; Research; Signal Transduction; Study models; Syphilis; System; Targeted Research; Treponema; Treponema denticola; Treponema pallidum; Treponemal Infections; Work; base; cell motility; design; experience; gene function; genetic manipulation; genome sequencing; improved; interest; microbial; microbial community; microbial genome; mutant; novel; oral spirochetes; pathogen; public health relevance; tool; uptake

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.

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实验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。 treponema denticola是牙龈粘膜组织的机会性病原体，为研究微生物相互作用，微生物生理学和分子进化提供了一系列目标。虽然口腔螺旋体的致病行为和疾病关联显然与pallidum的疾病明显不同，但T. denticola是 作为研究生理学，生物合成途径和微生物 - 宿主相互作用的模型，包括梅毒在内的模型。这既是由于TD和T. pallidum之间的生理相似性，也是由于没有体外培养系统的情况下，pallidum遗传研究仍然不可行。该项目将发展并证明在遗传操纵treponema属的可培养螺旋体的方法方面取得了重大进展，该方法将“启动” treponema遗传研究，从而为理性地解决新的生物学问题提供了机会，尤其是在不可培养的螺旋体中。我们提出了反选择诱变的牙霉菌特异性适应，加上限制性修饰系统的敲除，作为促进牙霉菌菌株的构建的关键特征，具有（1）大大提高了吸收和融合异源性DNA的能力，以及（2）扩展的可能突变类型的范围。在我们小组主要进行的“概念证明”研究的基础上，这项工作的结果既可以促进对牙霉菌的深入分子分析，并确立T. denticola作为模型生物体的实用性，用于对其他曲中感染的药物的分子分析，尤其是T. pallidum。