Natural competence and genetic manipulation of Treponema pallidum

描述(申请人提供)：自2000年以来，一期梅毒和二期梅毒在美国的发病率几乎翻了一番，在此期间，西欧和中国的发病率急剧上升。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，同样多的死产和流产病例是由这种疾病造成的。提高我们对梅毒发病机制的认识是发现新的潜在疫苗候选的关键因素，但这种进展受到目前梅毒病原体梅毒螺旋体亚种的假设的极大限制。由于这种病原体不能在体外繁殖，因此不能在基因上进行操作。事实上，体外繁殖被认为是对病原体进行遗传操作的必备步骤，因为它很容易提供数十亿指数级增长的细菌，能够抵御DNA转移协议的高死亡率。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，梅毒螺旋体可能具有天然的转化能力，即表达摄取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。自然能力在原核生物中广泛存在，是增加遗传多样性的有效手段，但对于梅毒螺旋体来说，它也可能是一种不需要体外繁殖就能获得基因改变的梅毒螺旋体菌株的替代方法。因此，在这项应用中，我建议调查梅毒螺旋体是否具有天然的转化能力，以及这一特征是否可以用于诱导选择标记定向整合到梅毒螺旋体基因组中。如果成功，这些实验将提供一种创新的方案来改变梅毒螺旋体的基因组，进行选择性的基因消融，并极大地促进我们对与感染建立相关的假定毒力因子的功能和位置的理解。 公共卫生相关性：这些研究将挑战梅毒病原体梅毒螺旋体(梅毒螺旋体)不可能通过基因操纵的假设。比较基因组学表明，事实上，这种病原体可能具有天然的转化能力，即能够摄取外部DNA，并将其整合到其基因组中，如果它与细菌自己的DNA足够相似的话。这项应用将调查梅毒螺旋体的天然能力，以及利用它开发基因修改方案的可能性，该方案将有助于识别新的靶点，以开发有效的预防梅毒疫苗。

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.