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: 8556045
• 负责人: PATRICIA A ROSA
• 金额: $ 13.91万
• 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/8556045
• 关键词: Aerobic Bacteria; Animal Model; Animals; Antibiotics; Area; Bacteria; Borrelia burgdorferi; Calculi; Chiroptera; Cloning; Cloning Vectors; Color; Culture Media; DNA; Data; Development; Disease; Engineering; Frequencies; Galactosidase; Gene Silencing; Genes; Genetic; Genetic Techniques; Genetic Transformation; Genome; Goals; Growth; Homologous Gene; Housing; Human; Hydrogen Peroxide; In Vitro; Infection; Intention; Knowledge; Leptospira; Leptospirosis; Life; Lyme Disease; Manuscripts; Maps; Measures; Membrane Proteins; Microbial Genetics; Modeling; Molecular Genetics; Mutagenesis; Order Spirochaetales; Organism; Oxidative Stress; Pathogenicity; Phenotype; Physiological; Play; Procedures; Production; Proteins; Proteome; Proteomics; Publications; Publishing; Reactive Oxygen Species; Recipe; Refractory; Reporting; Research; Role; Shuttle Vectors; Site; Source; Subgroup; System; Techniques; Testing; Virulence; Virulence Factors; Work; biological adaptation to stress; coping; experience; genetic manipulation; improved; interest; member; mutant; neglect; oxidative damage; pathogen; research study; 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 initially focused on L. biflexa to master the microbial and genetic techniques needed to manipulate this genus, with the intention to transfer this expertise to the more refractory pathogenic strains. Targeted gene inactivation, shuttle vector transformation, and transposon mutagenesis have all been successfully used in L. biflexa. To date, no shuttle vector system exists for pathogenic species and there are only two published reports of targeted gene inactivation in L. interrogans. 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 or targeted deletion mutants. Our focus has therefore concentrated on increasing and improving the molecular genetic tools available to manipulate leptospires, relying on our 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. Our current work in L. biflexa is now focused on more fully developing it as a model organism for the pathogenic leptospires. Since L. biflexa has a better transformation frequency than other species we plan to optimize new techniques in this organism. However, as a model organism, key information is lacking in this system, specifically regarding what proteins are physiologically important or highly expressed during in vitro cultivation. Therefore, our approach has been to first optimize culture conditions and transformation techniques, followed by proteome mapping and directed mutagenesis against specific targets of interest. In FY2011, Mr. Hunter Stone and Dr. Amit Sarkar optimized the growth conditions for culturing and selecting for Leptospira spp. This included adapting a recipe for EMJH medium that allows Leptospira growth when made from basic ingredients. Previous attempts to make our own medium had failed but by testing various components, we identified the Fe++ and antibiotic concentrations as too high to support sustained Leptospira growth. By testing various concentrations of different media components, we were able to optimize the recipe. We obtained a variety of saprophytic and pathogenic strains from different labs and found they were able to grow in both commercially purchased medium and in our in-house medium. Finally, Hunter modified an existing shuttle vector to simplify genetic manipulations by adding a multiple cloning site region and the -galactosidase gene for color screens. With culture conditions optimized and the construction of a more amenable cloning vector, we proceeded in 2012 to use the saprophyte L. biflexa to perform genetic transformation procedures including shuttle vector transformation and targeted gene inactivation. Using allelic exchange techniques, we engineered deletion mutants in the batABD locus, genes that encode proteins proposed to play a role in protecting some bacteria from oxidative stress. We compared the wild-type strain and deletion mutants under various oxidative stress conditions and found that the data do not support a protective role for the Leptospira Bat proteins in directly coping with oxidative stress, as previously proposed. Further, we demonstrated that L. biflexa is relatively sensitive to reactive oxygen species such as H2O2, suggesting that this spirochete lacks a strong, protective defense against oxidative damage despite being a strict aerobe. These results are described in a manuscript submitted for publication. Currently, we are developing a global proteomic map of in vitro cultivated L. biflexa to identify highly expressed proteins from membrane and soluble cellular fractions. Highly expressed proteins allow us to identify targets that may play important physiological roles and also use as tagged proteins for various expression studies. We have also begun to experiment directly in the pathogenic strain L. interrogans, studying homologs that have been shown in other organisms to target and degrade foreign DNA. This system may help explain why transformation frequencies are much lower in pathogenic strains where this system is present, versus free-living strains that lack these homologs. Now having mastered the techniques to cultivate and manipulate Leptospira spp. we have completed one project studying the oxidative stress response of the model organism L. biflexa and continued to develop our basic knowledge of this organism by mapping its proteome. Carrying these techniques and knowledge on to the pathogenic strains should help to expand the genetic tools needed for elucidating mechanisms of infection and pathogenicity.

钩端螺旋体病是一种由钩端螺旋体属成员引起的全球性人畜共患疾病。尽管钩端螺旋体病广泛存在，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 年首次被识别，但该螺旋体的体外生长速度缓慢，且用于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组：腐生菌、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（一种自由生活的非致病性腐生菌）和 L. interrogans（一种病原体）。然而，非致病性双折乳杆菌比致病性询问乳杆菌更容易培养，也更容易进行基因操作。因此，我们最初将重点放在 L. biflexa 上，以掌握操纵该属所需的微生物和遗传技术，旨在将这种专业知识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均已成功应用于 L. biflexa。迄今为止，尚不存在针对致病物种的穿梭载体系统，并且仅发表了两份关于问号钩体中靶向基因失活的报告。转座子诱变可应用于问号钩端螺旋体，但其作用效率如此之低，以至于不能用于任何广泛的应用，例如营养缺陷型筛选或特征标记诱变。 问号钩端螺旋体穿梭载体的缺乏阻碍了互补，从而限制了对转座子或靶向缺失突变体的任何结果表型的解释。 因此，我们的重点集中在增加和改进可用于操纵钩端螺旋体的分子遗传工具，依靠我们为另一种螺旋体——伯氏疏螺旋体（莱姆病的病原体）开发遗传系统的经验。该项目的长期目标是利用改进的工具和技术来了解问号钩端螺旋体的感染和致病机制，并加速制定钩端螺旋体病的预防措施。 我们目前对双曲拉氏菌的工作重点是更充分地将其开发为致病性钩端螺旋体的模式生物。由于 L. biflexa 比其他物种具有更好的转化频率，我们计划优化该生物体的新技术。然而，作为模式生物，该系统缺乏关键信息，特别是关于哪些蛋白质在体外培养过程中具有生理重要性或高度表达的信息。因此，我们的方法是首先优化培养条件和转化技术，然后针对特定目标靶点进行蛋白质组图谱和定向诱变。 2011财年，Hunter Stone先生和Amit Sarkar博士优化了钩端螺旋体的培养和选择生长条件。其中包括调整 EMJH 培养基的配方，使由基本成分制成的钩端螺旋体能够生长。之前尝试制作我们自己的培养基失败了，但通过测试各种成分，我们发现 Fe++ 和抗生素浓度过高，无法支持钩端螺旋体的持续生长。通过测试不同培养基成分的不同浓度，我们能够优化配方。我们从不同的实验室获得了多种腐生和致病菌株，发现它们能够在商业购买的培养基和我们的内部培养基中生长。最后，亨特修改了现有的穿梭载体，通过添加多克隆位点区域和用于彩色筛选的β半乳糖苷酶基因来简化遗传操作。 随着培养条件的优化和更适合的克隆载体的构建，我们于 2012 年开始使用腐生菌 L. biflexa 进行遗传转化程序，包括穿梭载体转化和靶向基因失活。利用等位基因交换技术，我们在 batABD 基因座中设计了缺失突变体，这些基因编码的蛋白质有望在保护某些细菌免受氧化应激方面发挥作用。我们比较了各种氧化应激条件下的野生型菌株和缺失突变体，发现数据并不支持钩端螺旋体蝙蝠蛋白在直接应对氧化应激方面的保护作用，正如之前提出的那样。此外，我们证明 L. biflexa 对 H2O2 等活性氧相对敏感，表明这种螺旋体尽管是严格需氧菌，但缺乏针对氧化损伤的强大保护性防御。这些结果在提交出版的手稿中进行了描述。 目前，我们正在开发体外培养的 L. biflexa 的全球蛋白质组图谱，以鉴定膜和可溶性细胞组分中高表达的蛋白质。高表达的蛋白质使我们能够识别可能发挥重要生理作用的靶标，并可用作各种表达研究的标记蛋白质。 我们还开始直接在致病菌株问号钩端螺旋体上进行实验，研究已在其他生物体中显示出能够靶向和降解外源 DNA 的同源物。该系统可能有助于解释为什么存在该系统的致病菌株的转化频率比缺乏这些同源物的自由生活菌株低得多。 现在已经掌握了钩端螺旋体的培养和操纵技术。我们已经完成了一个研究模式生物 L. biflexa 氧化应激反应的项目，并通过绘制其蛋白质组图谱继续发展我们对该生物体的基础知识。将这些技术和知识应用于致病菌株应该有助于扩展阐明感染和致病机制所需的遗传工具。