Genetic Manipulation of Unreaplasma spp. Infecting Humans using Synthetic Genomic

描述(申请人提供)：解脲支原体和微小解脲支原体是性传播的人类病原体。感染很普遍。多达40%-80%的女性是携带者。虽然大多数感染是无症状的，但它们会在一些患者中引起侵袭性疾病。尽管它们与多种疾病有关，但它们对社会的最大影响是造成不良妊娠结局和新生儿感染，导致新生儿死亡的风险更高。几乎所有极低出生体重的新生儿(小于2.5公斤或5.5磅)都有呼吸道解脲支原体感染。对抗生素产生抗药性的解脲支原体感染越来越常见，治疗解脲支原体的最佳药物氟喹诺酮类和四环素类药物不推荐孕妇或儿童使用。由于缺乏修改这些细菌DNA的手段，了解解脲支原体生物学和发病机制的努力受到了阻碍。在许多已被充分研究的病原体中，修饰生物基因组的能力一直是了解宿主-病原体相互作用和致病因素的先决条件。开发基因工具来创造设计的突变解脲支原体，将使科学家有能力识别导致解脲支原体致病的因素，以及如何更好地预防解脲支原体感染引起的疾病。最近，我们的J·克雷格·文特尔研究所的研究小组用化学合成的基因组创造了一个细菌细胞。为了达到这一合成细胞里程碑，我们为合成基因组学这门新科学开发了基础工具。其中一种叫做“基因组移植”的工具使我们能够从一种名为支原体的细菌中提取基因组，并将其植入另一种细菌中。用另一种工具，我们在酵母细胞内克隆了相同的丝状支原体基因组，它静静地停在那里。一旦进入酵母细胞，我们就可以使用酵母生物学家使用的成熟的遗传工具，通过删除或添加基因来修改基因组。这两种方法结合在一起，使我们能够制造出一种改变的丝状支原体菌株。这种新的霉菌不可能用任何现有的技术制造出来。该项目的目标是应用这种合成基因组学技术来开发基因工具，以创造改良的解脲支原体菌株，这将有助于了解解脲支原体的致病性。我们的具体目标是首先调整我们的基因组移植方法，将解脲支原体基因组安装到微小解脲支原体或异型解脲支原体的受体细胞中。第二个目标是在酵母细胞中克隆同样的解脲支原体基因组，这个目标在实现第一个目标之前用处不大。这两个目标的结果是能够改变解脲支原体基因的含量和表达，从而验证解脲支原体发病机制的假说，并开发新的治疗方法。除了这些鲜为人知的病原体的直接进展外，可能还会有其他更重要的后果。目前，我们只知道如何移植一种细菌的基因组。学习如何移植解脲支原体基因组可能会导致我们能够为许多其他物种做到这一点，包括具有尚未实现的制药或工业潜力的细菌，而我们对这些细菌没有遗传工具。

DESCRIPTION (provided by applicant): Ureaplasma urealyticum and Ureaplasma parvum are sexually transmitted human pathogens. Infection is widespread. As many as 40-80% of women are carriers. While most infections are asymptomatic, they cause invasive diseases in some patients. Although they have been associated with a wide range of diseases, their largest impact on society is as a cause of adverse pregnancy outcomes and infection of newborns, leading to higher risk of newborn mortality. Virtually all very-low-birth-weight newborns (less than 2.5 kg or 5.5 lbs) have respiratory ureaplasma infections. Ureaplasma infections resistant to antibiotics are increasingly common and the best drugs for ureaplasma treatment, fluoroquinolones and tetracyclines, are not recommended for pregnant women or children. Efforts to understand ureaplasma biology and pathogenesis have been inhibited by the lack of means to modify the DNA of these bacteria. The capacity to modify organisms' genomes has been a prerequisite in understanding host-pathogen interaction and factors involved in pathogenicity in many well studied pathogens. Development of genetic tools for the creation of designed mutant ureaplasmas will give scientists the power to identify factors making ureaplasmas pathogenic and how to better prevent disease due to ureaplasma infection. Recently our J. Craig Venter Institute research team created a bacterial cell with a chemically synthesized genome. To reach this synthetic cell milestone we developed foundation tools for the new science of Synthetic Genomics. One of those tools called "genome transplantation" enabled us to take a genome out of one species of bacteria called Mycoplasma mycoides, and install it into another species of bacteria. With another tool, we cloned the same M. mycoides genome inside a yeast cell, where it sits quietly parked. Once inside that yeast cell we can use well developed genetic tools used by yeast biologists to modify that genome by deleting or adding genes. These two methods in concert allowed us to make an altered M. mycoides strain. This new M. mycoides could not have been made using any existing technology. The goal of this project is to apply this Synthetic Genomics technology to develop genetic tools for creation of modified ureaplasma strains that will aid in the understanding of ureaplasma pathogenicity. Our specific aims are first to adapt our genome transplantation method for installation of a U. urealyticum genome into a U. parvum or U. diversum recipient cell. The second aim, which is of little use until aim one is achieved, is to clone that same ureaplasma genome in a yeast cell. The result of the two aims is the capacity to modify ureaplasma gene content and expression so that we can test hypotheses about ureaplasma pathogenesis and develop new therapies. Beyond the immediate advances for these poorly understood pathogens, there could be other even more important consequences. Currently we only know how to transplant the genome of one bacterial species. Learning how to transplant ureaplasma genomes could lead to our being able to do this for many other species including bacteria with unrealized pharmaceutical or industrial potential, for which we have no genetic tools.