Cultivation and Genetic Manipulation of Free-Living and Pathogenic Leptospires

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年首次被发现，但缓慢的体外生长速度和有限的基因工具操作这种螺旋体的基因组，阻碍了毒力因子的识别和疫苗的开发。 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双歧乳杆菌(一种自由生活的非致病腐生植物)和问号乳杆菌(一种病原体)。然而，与致病性问号钩端螺旋体相比，非致病性双向钩端螺旋体更容易培养，更容易受到遗传操作的影响。因此，我们最初将重点放在双挠性乳杆菌上，以掌握操纵该属所需的微生物和基因技术，目的是将这一专业知识转移到更具耐药性的致病菌株上。靶向基因失活、穿梭载体转化和转座子诱变都已在双歧杆菌中得到成功应用。到目前为止，还没有针对致病物种的穿梭载体系统，只有两份已发表的关于问号钩端螺旋体靶向基因失活的报告。转座子诱变可以应用于问号钩端螺旋体，但它的作用效率很低，不能用于任何广泛的应用，如营养缺陷性筛选或标记突变。缺乏问号钩端螺旋体的穿梭载体阻碍了互补，从而限制了对转座子或靶向缺失突变体的任何表型的解释。 因此，我们的重点是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-伯氏疏螺旋体开发遗传系统的经验，伯氏疏螺旋体是莱姆病的病原体。由于双挠性乳杆菌比其他物种具有更高的转化频率，我们计划优化这种生物的新技术。然而，作为一个模式生物，在这个系统中缺乏关键信息，特别是关于在体外培养过程中哪些蛋白质具有重要的生理意义或高表达。因此，我们的方法是针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问号钩端螺旋体感染和致病的机制，并加速开发预防钩端螺旋体病的措施。 我们在2013财年继续使用腐生的双曲霉对螺旋体中保守的基因家族进行靶向基因失活。利用等位基因交换技术，我们在BatABD基因座设计了缺失突变体，该基因编码蛋白质，被认为在保护一些细菌免受氧化应激的作用。我们比较了野生型菌株和缺失突变体在不同的氧化应激条件下，发现数据不支持钩端螺旋体BAT蛋白在直接应对氧化应激中的保护作用，如先前提出的那样。此外，我们还证明了双挠性乳杆菌对过氧化氢和超氧化物等活性氧物种相对敏感，这表明这种螺旋体尽管是严格的需氧菌，但缺乏对氧化损伤的强大保护防御。这些结果在2013财年发表的一篇手稿中进行了描述(Stewart等人。BMC微生物。2012，12：29)。 目前，我们正在开发体外培养的双歧杆菌的全球蛋白质组学图谱，以从膜和可溶部分中鉴定高表达的蛋白质。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点，并将其用作各种表达研究的标记蛋白。这项工作是在NIAID持久性病毒疾病实验室与James Carroll博士的内部合作下完成的。 我们还开始直接用致病菌株问号乳杆菌进行实验，研究其他生物中已显示的基因的同源物，以靶向和降解外来DNA。这一系统可能有助于解释为什么存在这些基因的致病菌株的转化频率比没有这些同源基因的自由生活菌株低得多。现在已经掌握了培养和操作钩端螺旋体的技术。我们已经完成了一个研究模式生物L.biflea的氧化应激反应的项目，并通过绘制其蛋白质组图来继续发展我们对该生物的基本知识。将这些技术和知识带到致病菌株上，应该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.