Natural competence and genetic manipulation of Treponema pallidum

梅毒螺旋体的自然能力和遗传操作

• 批准号: 8173514
• 负责人: Lorenzo Giacani
• 金额: $ 7.21万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8173514
• 关键词: ATP phosphohydrolase; Ablation; Adhesions; Antibiotics; Bacillus subtilis; Bacteria; Belief; Binding Proteins; Bypass; Cell Culture System; Cells; China; Chloramphenicol Resistance; Chromosomes; Competence; Congenital Syphilis; Cytoplasm; DNA; DNA receptor; Developing Countries; Disease; Doctor of Philosophy; Elements; Enzymes; Funding; Gene Expression; Genes; Genetic; Genetic Materials; Genetic Recombination; Genetic Transformation; Genetic Variation; Genome; Globus Pallidus; Growth; Harvest; Immune; In Vitro; Incidence; Infection; Intake; Knowledge; Location; Mediating; Messenger RNA; Modification; Multiprotein Complexes; Neisseria gonorrhoeae; Oryctolagus cuniculus; Pathogenesis; Prokaryotic Cells; Protocols documentation; Recombinants; Refractory; Research Personnel; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; Scanning; Sequence Homologs; Single-Stranded DNA; Spontaneous abortion; Suspension substance; Suspensions; Syphilis; Testing; Time; Translating; Treponema pallidum; United States; Vaccines; Virulence Factors; Western Europe; base; comparative genomics; ds-DNA; genetic manipulation; homologous recombination; in vivo; innovation; mortality; pathogen; prevent; research study; stillbirth; tool; uptake; vaccine candidate

DESCRIPTION (provided by applicant): The incidence of primary and secondary syphilis has nearly doubled in the United States since 2000, and has increased dramatically in Western Europe and China during that time. In developing countries every year at least half a million babies are born with congenital syphilis, and as many cases of stillbirth and miscarriage are caused by this disease. The improvement of our understanding of the pathogenesis of syphilis is a key element to identify new potential vaccine candidates against the disease, but such progress is greatly limited by the current assumption that the agent of syphilis, Treponema pallidum subsp. pallidum (T. pallidum), cannot be genetically manipulated, due to the fact that this pathogen cannot be propagated in vitro. In vitro propagation in fact is regarded as an obligate step for genetic manipulation of a pathogen because it easily provides billions of exponentially growing bacteria able to withstand the elevated mortality of DNA transfer protocols. Most importantly, in vitro propagation allows selection of recombinant strains. Comparative genomics suggests however that, similarly to other pathogens, T. pallidum might be naturally competent for transformation, namely the ability to express enzymes for the intake of exogenous genetic material and its integration into the genome via homologous recombination. Natural competence is widely present among prokaryotes as an effective means to increase genetic diversity but, for T. pallidum it could as well represent an alternative way to obtain genetically altered T. pallidum strains without the need of in vitro propagation. In this application I therefore propose to investigate if T. pallidum is naturally competent for transformation and if this feature can be used to induce targeted integration of a selection marker into the T. pallidum genome. If successful, these experiments will provide an innovative protocol to alter T. pallidum's genome, perform selective gene ablation, and greatly advance our understanding of the function and location of putative virulence factors in relation to the establishment of the infection. PUBLIC HEALTH RELEVANCE: These studies will challenge the assumption that the agent of syphilis, Treponema pallidum (T. pallidum) is impossible to genetically manipulate. Comparative genomics suggests in fact that this pathogen might be naturally competent for transformation, namely able to intake outside DNA and integrate it into its genome if it is similar enough to the bacterium's own DNA. This application will investigate T. pallidum's natural competence and the possibility to use it to develop a genetic modification protocol that will facilitate the identification of new targets for developing effective vaccines to prevent syphilis.

描述（由申请人提供）：自2000年以来，一期和二期梅毒的发病率在美国几乎翻了一番，在西欧和中国也急剧增加。在发展中国家，每年至少有50万婴儿出生时患有先天性梅毒，同样多的死胎和流产病例也是由这种疾病引起的。提高我们对梅毒发病机制的认识是确定新的潜在疫苗候选人的关键因素，但目前的假设大大限制了这种进展，即梅毒螺旋体（Treponema pallidum subsp.苍白球（T. pallidum），由于该病原体不能在体外繁殖的事实，因此不能进行遗传操作。事实上，体外繁殖被认为是病原体遗传操作的必要步骤，因为它容易提供数十亿个指数生长的细菌，这些细菌能够承受DNA转移方案的高死亡率。最重要的是，体外繁殖允许选择重组菌株。然而，比较基因组学表明，与其他病原体类似，T。苍白球可能天然地具有转化能力，即表达用于摄入外源遗传物质的酶并通过同源重组将其整合到基因组中的能力。天然感受态广泛存在于原核生物中，是增加遗传多样性的有效手段。pallidum，它也可以代表一种获得遗传改变的T. pallidum菌株，而不需要体外繁殖。因此，在本申请中，我提议研究T.苍白球是天然的转化能力，并且如果该特征可用于诱导选择标记靶向整合到T.苍白球基因组如果成功，这些实验将提供一个创新的协议，以改变T。pallidum的基因组，进行选择性基因切除，并大大推进我们的理解的功能和位置的推定毒力因子的建立感染。 公共卫生相关性：这些研究将挑战梅毒病原假设，梅毒螺旋体（T。pallidum）是不可能遗传操纵的。事实上，比较基因组学表明，这种病原体可能天生就具有转化能力，即能够吸收外部DNA并将其整合到其基因组中，如果它与细菌自身的DNA足够相似。本申请将调查T. pallidum的天然能力，并有可能利用它来开发一种基因改造方案，这将有助于确定新的目标，开发有效的疫苗，以防止梅毒。