Natural competence and genetic manipulation of Treponema pallidum

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.苍白球），由于该病原体不能在体外繁殖的事实，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。

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.