Development of Novel Genetic Tools for Metabolic Selection in Yersinia Pestis

鼠疫耶尔森菌代谢选择的新型遗传工具的开发

• 批准号: 7846463
• 负责人: DEBORAH M ANDERSON
• 金额: $ 1.62万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-19 至 2009-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7846463
• 关键词: Animals; Antibiotic Resistance; Antibiotic Therapy; Bacteria; Categories; Cell Culture Techniques; Collection; Copper; Deletion Mutation; Development; Diaminopimelic Acid; Engineering; Escherichia coli; Genetic; Genetic Recombination; Goals; Gram-Positive Bacteria; Human; Infection; Ions; Laboratories; Licensing; Mercury; Metabolic; Metabolic Pathway; Mutation; Nutrient; Operon; Pathogenesis; Pathway interactions; Phenotype; Plague; Plague Vaccine; Plasmids; Pneumonic Plague; Prophylactic treatment; Recombinants; Reporter; Research; Resistance; System; Yersinia; Yersinia pestis; Yersinia pestis V antigen; auxotrophy; base; divalent metal; improved; in vivo; mouse model; mutant; novel; null mutation; overexpression; pathogen; plasmid DNA; protein expression; suicide vector; tool

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.

描述（由申请人提供）：耶尔森氏菌是一种精选药物，能够引起高致命的，高度传染性的肺炎瘟疫。目前，尚无用于瘟疫的许可疫苗，迫切需要依靠当前可用的抗生素进行治疗和暴露后预防。多种抗生素耐药性鼠疫的最新出现强调了最大程度地减少使用抗生素耐药性盒作为研究目的的重要性，以最大程度地利用人类鼠疫的治疗选择。然而，这样做的是，人们损害了耶尔森氏念珠菌中遗传学的潜力，因为目前可用的研究工具可以在遗传上操纵细菌，均涉及在一个或多个步骤中引入抗生素耐药性。我们试图通过创建新型质粒来研究耶尔森氏念珠菌的发病机理的遗传工具的可用性，这些新质粒涉及基于代谢途径而不是抗生素耐药性的选择。已经研究了革兰氏阴性和革兰氏阳性细菌的质粒对二价金属离子的抗性，例如汞或铜。此外，可以利用养分的营养丰富营养（例如二氨基二酰酸生物合成途径）作为在体内和实验室培养基中选择质粒DNA的一种手段。将探索这些途径对质粒和自杀媒介等遗传工具的实用性。然后，这些工具将被用来在耶尔森氏菌中创建特定的突变，并在细胞培养和动物感染的背景下进行研究。