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。虽然这种疾病很普遍，估计每年有59,000人死亡，但钩端螺旋体病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在100多年前首次被发现，但缓慢的体外生长速度和有限的遗传工具可用于操纵这种螺旋体的基因组，阻碍了毒力因素的识别和疫苗的开发。 钩端螺旋体大致可分为两类：自由生活的腐生生物和传染性病原体。最广泛使用和研究的物种是双歧杆菌(一种非传染性腐生植物)和问号钩端螺旋体(一种病原体)。然而，与致病性问号钩端螺旋体相比，非致病性双歧杆菌更容易培养，更容易受到遗传操作的影响。因此，我们把注意力集中在双弯乳杆菌上，将其作为研究保守基因和了解该属整体的模型，开发新的技术，并作为异源寄主表达病原菌特异性基因，以确定其功能。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，问号钩端螺旋体靶向基因失活的报道还很少。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。由于双挠性乳杆菌的转化频率比其他物种高，因此我们努力优化该生物体的新技术。 2019年1月，钩端螺旋体病项目首席研究员菲利普·斯图尔特博士被详细介绍到NIAID RML病毒学实验室的马歇尔·布鲁姆斯博士媒介传播病毒科，并于2019年3月被任命为职员科学家。在2019财年，由于预计Stewarts博士将过渡到LV并离开分子遗传学部门，因此没有开展钩端螺旋体病项目的额外工作。钩端螺旋体项目的试剂和菌株与合作者共享或陪同斯图尔特博士去LV。

Leptospirosis is a global, zoonotic disease caused by members of the genus Leptospira. Although the disease is widespread with an estimated mortality rate of 59,000 people annually, leptospirosis is considered a neglected and understudied disease. The causative agent of Leptospirosis was first identified over 100 years ago, but the slow in vitro growth rate and limited genetic tools available to manipulate the genome of this spirochete have hampered the identification of virulence factors and development of a vaccine. Leptospires can be broadly divided into two groups: free-living saprophytes and infectious pathogens. The most widely used and studied species are L. biflexa (a non-infectious saprophyte) and L. interrogans (a pathogen). However, the nonpathogenic L. biflexa is more easily cultivated and more amenable to genetic manipulation than the pathogenic L. interrogans. Therefore, we focused our attention on L. biflexa as a model to characterize conserved genes and to understand the genus as a whole, to develop new techniques, and as a heterologous host to express pathogen-specific genes in order to characterize their function. Targeted gene inactivation, shuttle vector transformation, and transposon mutagenesis have all been successfully used in L. biflexa. To date, there are few published reports of targeted gene inactivations in L. interrogans. Transposon mutagenesis can be applied to L. interrogans but it functions at such low efficiency that it cannot be utilized for any broad applications, such as auxotrophic screens or signature tagged mutagenesis. Since L. biflexa has a better transformation frequency than other species, we endeavored to optimize new techniques in this organism. In January 2019, Dr. Philip Stewart, Lead Investigator of the Leptospirosis project, was detailed to Dr. Marshall Blooms Biology of Vector Borne-Viruses Section in the Laboratory of Virology, RML, NIAID, where he was appointed Staff Scientist in March 2019. In FY2019, no additional work was undertaken on the Leptospirosis project in anticipation of Dr. Stewarts transition to LV and departure from the Molecular Genetics Section. Reagents and strains for the Leptospira project were shared with collaborators or accompanied Dr. Stewart to LV.