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。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，我们的目标是通过定向突变技术对感兴趣的基因进行定向突变，并构建膜相关蛋白和可溶性蛋白的蛋白质组图谱，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。 于二零一三财政年度，我们继续使用saparte L。biflexa对螺旋体中保守的基因家族进行靶向基因失活。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对活性氧物质如过氧化氢和超氧化物相对敏感，这表明该螺旋体尽管是严格的需氧菌，但缺乏对氧化损伤的强保护性防御。这些结果描述于2013财年发表的手稿中（Stewart等人，BMC Microbiol. 2012，12：29）。 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来鉴定来自膜级分和可溶级分的高表达蛋白。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。这项工作正在与NIAID持续性病毒性疾病实验室的James卡罗尔博士进行内部合作。 我们还开始直接用致病菌株L.研究在其他生物体中已经显示出的靶向和降解外源DNA的基因的同源物。这个系统可能有助于解释为什么这些基因存在的致病菌株的转化频率要低得多，而缺乏这些同源物的自由生活菌株。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. 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 target genes of interest for directed mutagenesis and to construct a proteomic map of both membrane-associated proteins and soluble proteins.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. We proceeded in FY2013 to use the saprophyte L. biflexa to perform targeted gene inactivation against a gene family conserved among spirochetes. 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 hydrogen peroxide and superoxide, suggesting that this spirochete lacks a strong, protective defense against oxidative damage despite being a strict aerobe. These results are described in a manuscript published in FY2013 (Stewart et al. BMC Microbiol. 2012, 12:29). Currently, we are developing a global proteomic map of in vitro cultivated L. biflexa to identify highly expressed proteins from membrane- and soluble-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. This work is being completed with an internal collaboration with Dr. James Carroll in the Laboratory of Persistent Viral Diseases, NIAID. We have also begun to experiment directly with the pathogenic strain L. interrogans, studying homologs of genes 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 these genes are 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.