Development of Novel Genetic Tools for Metabolic Selection in Yersinia Pestis

描述(由申请人提供)：鼠疫耶尔森氏菌是一种A类精选制剂，能够引起高度致命、高度传染性的肺鼠疫。目前，没有获得许可的鼠疫疫苗，这使得人们非常需要依赖现有的抗生素进行治疗和暴露后的预防。最近出现的多重抗药性鼠疫杆菌突出表明，为了最大限度地提供人类鼠疫的治疗选择，最大限度地减少出于研究目的而使用抗药性盒带的重要性。然而，这样做会损害鼠疫耶尔森氏菌的遗传学潜力，因为目前可用的对细菌进行基因操作的研究工具都涉及在一个或多个步骤中引入抗生素耐药性。我们试图通过创造新的质粒来提高研究鼠疫耶尔森氏菌致病机理的遗传工具的可用性，这涉及到基于代谢途径而不是抗生素耐药性的选择。在革兰氏阴性和革兰氏阳性细菌中，已经研究了质粒携带的对二价金属离子，例如汞或铜的耐药性，并具有良好的特性。此外，营养营养缺陷症，如二氨基戊二酸的生物合成途径，可作为在体内和实验室介质中选择质粒DNA的一种手段。将探索这些途径作为基因工具的用途，如质粒和自杀载体。然后，这些工具将被用来在鼠疫耶尔森氏菌中产生特定的突变，并在细胞培养和动物感染的背景下进行研究。

DESCRIPTION (provided by applicant): Yersinia pestis is a Category A Select Agent capable of causing highly fatal, highly contagious pneumonic plague. At present, there is no licensed vaccine for plague, creating an enormous need to rely on currently available antibiotics for treatment and post-exposure prophylaxis. Recent emergence of multi-antibiotic resistant Y. pestis underscores the importance of minimizing the use of antibiotic resistance cassettes for research purposes in order to maximize the availability of treatment options for human plague. However in so doing, one compromises the potential for genetics in Yersinia pestis as currently available research tools to genetically manipulate the bacteria all involve the introduction of antibiotic resistance at one or more steps. We seek to improve the availability of genetic tools to study the pathogenesis of Yersinia pestis through the creation of novel plasmids which involve selection based on metabolic pathways rather than antibiotic resistance. Plasmid borne resistance to divalent metal ions, for example mercury or copper, has been studied in both Gram negative and Gram positive bacteria and is well characterized. In addition, nutrient auxotrophy, such as the diaminopimelic acid biosynthetic pathway, can be exploited as a means to select plasmid DNA both in vivo and in laboratory media. The utility of these pathways for genetic tools such as plasmids and suicide vectors will be explored. These tools will then be employed to create specific mutations in Yersinia pestis and studied in the context of cell culture and animal infections.