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类特定病原体，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，因此需要依靠现有的抗生素进行治疗和接触后预防。最近出现的多重抗生素耐药鼠疫耶尔森氏菌突出表明，为了最大限度地提供人类鼠疫治疗方案，必须尽量减少抗生素耐药磁带用于研究目的。然而，在这样做的过程中，人们牺牲了鼠疫耶尔森氏菌遗传学的潜力，因为目前可用的研究工具来遗传操纵细菌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遗传工具的可用性，以研究鼠疫耶尔森氏菌的发病机制，这些质粒涉及基于代谢途径而不是抗生素耐药性的选择。质粒对二价金属离子（如汞或铜）的抗性已经在革兰氏阴性和革兰氏阳性细菌中进行了研究，并得到了很好的表征。此外，营养缺陷，如二氨基苯甲酸生物合成途径，可以作为在体内和实验室培养基中选择质粒DNA的手段。将探讨这些途径对遗传工具（如质粒和自杀载体）的效用。这些工具随后将用于在鼠疫耶尔森氏菌中产生特定突变，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.