Cultivation and Genetic Manipulation of Free-Living and Pathogenic Leptospires

自由生活和致病性钩端螺旋体的培养和基因操作

• 批准号: 8336349
• 负责人: PATRICIA A ROSA
• 金额: $ 25.56万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8336349
• 关键词: Animals; Area; Borrelia burgdorferi; Cloning; Culture Media; Development; Disease; Gene Silencing; Genetic; Genetic Techniques; Genome; Goals; Growth; Human; In Vitro; Infection; Leptospira; Leptospirosis; Life; Lyme Disease; Measures; Microbial Genetics; Modeling; Molecular Genetics; Mutagenesis; Order Spirochaetales; Pathogenicity; Phenotype; Production; Publishing; Refractory; Reporting; Research; Shuttle Vectors; Source; Subgroup; System; Techniques; Virulence; Virulence Factors; experience; genetic manipulation; improved; member; mutant; neglect; pathogen; tool; vaccine development

Leptospirosis is a global, zoonotic disease caused by members of the genus Leptospira. Although widespread and sometimes fatal, leptospirosis is considered a neglected and understudied disease. The causative agent of Leptospirosis was first identified in 1916 but the slow in vitro growth rate and limited genetic tools with which to manipulate the genome of this spirochete have hampered the identification of virulence factors and development of a vaccine. Leptospires can be divided into three subgroups: saprophytes, pathogens, and a middle group of unknown pathogenicity. The most widely used and studied species are L. biflexa (a free-living, non-pathogenic saprophyte) and L. interrogans (a pathogen). However, the non-pathogenic L. biflexa is more easily cultivated and more amenable to genetic manipulation than the pathogenic L. interrogans. Therefore, we have decided to initially focus on L. biflexa in order to develop the microbial and genetic expertise we plan to transfer to the more refractory pathogenic strains. Targeted gene inactivation, shuttle vector transformation, and transposon mutagenesis have all been successfully used in L. biflexa. Only one report of targeted gene inactivation in L. interrogans has been published and no successful shuttle vector system currently exists for the pathogen. Transposon mutagenesis can be applied to L. interrogans but it functions at such a low efficiency that it cannot be utilized for any broad applications, such as auxotrophic screens or signature tagged mutagenesis. The lack of a shuttle vector for L. interrogans hinders complementation and thus limits interpretation of any resulting phenotypes of transposon mutants. We have therefore decided to focus on increasing and improving the molecular genetic tools available to manipulate leptospires, as we have experience in developing a genetic system for another spirochete, Borrelia burgdorferi, the causative agent of Lyme disease. The long-term objective of this project is to use the improved tools and techniques to understand the mechanisms of infection and pathogenecity of L. interrogans and accelerate the development of preventative measures against Leptospirosis.

钩端螺旋体病是由钩端螺旋体属成员引起的全球性人畜共患疾病。虽然钩端螺旋体病广泛传播，有时甚至致命，但它被认为是一种被忽视和研究不足的疾病。钩端螺旋体病的病原体于1916年首次被鉴定，但体外生长速度慢和操纵该螺旋体基因组的遗传工具有限阻碍了毒力因子的鉴定和疫苗的开发。 钩端螺旋体可分为三个亚群：寄生虫、病原体和致病性未知的中间组。最广泛使用和研究的物种是L. biflexa和L.问号（病原体）。而非致病性L. biflexa比致病L.更容易培养，更适合遗传操作。质问者因此，我们决定首先关注L。biflexa为了开发微生物和遗传学专门知识，我们计划将其转移到更难治的致病菌株上。靶向基因失活、穿梭载体转化和转座子诱变都已成功地应用于L. biflexa。只有一个报告的目的基因失活的L。已经发表了Interrogans，目前还没有针对该病原体的成功穿梭载体系统。转座子诱变可应用于L.但是它的功能效率很低，以致于不能用于任何广泛的应用，例如营养缺陷型筛选或标记突变。 由于缺乏穿梭载体，L.问号阻碍了互补作用，因此限制了转座子突变体的任何所得表型的解释。 因此，我们决定把重点放在增加和改进可用于操纵钩端螺旋体的分子遗传工具上，因为我们在开发另一种螺旋体莱姆病病原体伯氏疏螺旋体的遗传系统方面有经验。本项目的长期目标是利用改进的工具和技术来了解L.并加快制定钩端螺旋体病的预防措施。