Cultivation and Genetic Manipulation of Free-Living and Pathogenic Leptospires

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，我们首先关注L。biflexa掌握操纵该属所需的微生物和遗传技术，目的是将这种专门知识转移到更难治的致病菌株。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。迄今为止，还没有针对致病菌的穿梭载体系统，也只有两篇关于L.质问者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.问号阻碍互补作用，因此限制了转座子或靶向缺失突变体的任何所得表型的解释。 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的经验。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。 我们目前在L. biflexa目前正致力于更全面地将其开发为致病性钩端螺旋体的模式生物。自L. biflexa比其他物种有更好的转化频率，我们计划优化这种生物的新技术。然而，作为一种模式生物，该系统中缺乏关键信息，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。因此，我们的方法是首先优化培养条件和转化技术，然后对感兴趣的特定靶标进行蛋白质组作图和定向诱变。 在2011财年，Hunter Stone先生和Amit Sarkar博士优化了钩端螺旋体培养和选择的生长条件。这包括调整EMJH培养基的配方，当由基本成分制成时，该培养基允许钩端螺旋体生长。以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，我们确定Fe++和抗生素浓度太高，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各种浓度，我们能够优化配方。我们从不同的实验室获得了各种致病性和致病性菌株，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，以简化遗传操作，通过添加多克隆位点区域和半乳糖苷酶基因的彩色屏幕。 随着培养条件的优化和更适合克隆载体的构建，我们在2012年开始使用sapliteL. biflexa进行遗传转化程序，包括穿梭载体转化和靶向基因失活。使用等位基因交换技术，我们设计了batABD基因座的缺失突变体，这些基因编码的蛋白质在保护某些细菌免受氧化应激中发挥作用。我们在各种氧化应激条件下比较了野生型菌株和缺失突变体，发现数据不支持蝙蝠钩端螺旋体蛋白在直接应对氧化应激中的保护作用，如先前提出的。进一步证明了L. biflexa对活性氧如H2O2相对敏感，这表明尽管是严格的需氧菌，但这种螺旋体缺乏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，我们正在开发一个全球蛋白质组图谱，在体外培养L。biflexa来鉴定来自膜和可溶性细胞级分的高度表达的蛋白质。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点，并用作各种表达研究的标记蛋白。 我们还开始直接在致病菌株L中进行实验。研究在其他生物体中显示的靶向和降解外源DNA的同源物。这个系统可能有助于解释为什么转化频率低得多的致病菌株，该系统是本，与自由生活的菌株，缺乏这些同源物。 目前已掌握钩端螺旋体的培养和操作技术。我们已经完成了一个研究模式生物L的氧化应激反应的项目。biflexa，并继续通过绘制其蛋白质组来发展我们对这种生物体的基本知识。将这些技术和知识应用于致病菌株应有助于扩大阐明感染和致病机制所需的遗传工具。

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.