Molecular Genetic Basis of the Infectious Cycle of Borrelia burgdorferi

伯氏疏螺旋体感染周期的分子遗传学基础

• 批准号: 10014097
• 负责人: PATRICIA A ROSA
• 金额: $ 86.85万
• 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/10014097
• 关键词: Address; Affect; Amino Acid Motifs; Arthropod Vectors; Bacteria; Binding; Binding Proteins; Black-legged Tick; Borrelia; Borrelia burgdorferi; Cell Membrane Permeability; Cells; Cellular Structures; Chromosomes; Code; Collaborations; Complement; Cues; DNA; DNA Binding; DNA Restriction-Modification Enzymes; DNA Sequence; Data; Development; Diagnosis; Diffuse; Distant; Dyes; Elements; Engineering; Enterobacteriaceae; Environment; Europe; Flow Cytometry; Fluorescence; Fluorescence Microscopy; Future; Gene Expression; Gene Expression Regulation; Gene-Modified; Genes; Genetic; Genetic Screening; Genetic study; Genome; Genomics; Goals; Imagery; Individual; Infection; Investigation; Kentucky; Label; Laboratories; Location; Lyme Disease; Maintenance; Mammals; Martens; Mediating; Membrane; Membrane Proteins; Metabolism; Modification; Molecular; Molecular Biology; Molecular Genetics; Mus; Nature; Order Spirochaetales; Organism; Phylogenetic Analysis; Physiology; Plasmids; Postdoctoral Fellow; Process; Proteins; RNA-Binding Proteins; Rodent; Role; Shuttle Vectors; Signal Transduction; Site; Stains; Structure; Superoxide Dismutase; System; Techniques; Testing; Tick-Borne Diseases; Ticks; Time; Transcript; Translations; United States; Universities; Variant; Virulence; Work; Zoonoses; design; differential expression; epigenetic regulation; gang; genetic analysis; genetic approach; genetic manipulation; graduate student; human pathogen; in vivo; insight; interest; medical schools; member; novel; pathogen; pathogenic bacteria; prevent; promoter; protein folding; response; sabbatical; sample fixation; soft drink; tool; transcriptome sequencing; transmission process; vector tick; vector-borne pathogen

Lyme disease is the most common tick-borne illness in the United States and Europe. It is caused by Borrelia burgdorferi, a bacterial pathogen that is maintained in nature in a zoonotic cycle between various species of small mammals and an ixodid tick vector. A hallmark of the Lyme disease spirochete is its unusual segmented genome, which includes a large number of linear and circular plasmids. Increasing evidence indicates that plasmid-encoded functions are critical for successful adaptation to the different environments that B. burgdorferi encounters during its infectious cycle. We have developed genetic tools to investigate basic aspects of the unusual genomic organization, cellular structure and metabolism of B. burgdorferi. We have extended this investigation to an in vivo setting with an experimental system that closely mimics the natural arthropod vector/rodent host infectious cycle. Through an understanding of the basic molecular biology of the organism, we hope to gain insight into the infectious strategy utilized by this significant vector-borne pathogen and thereby facilitate efforts to prevent, diagnose and treat Lyme disease. In FY2019 we described the novel application to spirochetes of the FlAsH technique for fluorescently labeling specific proteins in living cells (1). This technique entails engineering a tetracysteine motif in the coding sequence of a protein of interest and staining with a membrane-permeable biarsenical dye that binds to this motif. The small increase in protein size conferred by the 6 amino acid motif minimizes potential negative effects that can occur when targets are fused to larger fluorogenic proteins, such as steric hindrance, improper protein folding, incorrect cellular location, or inappropriate oligomerization. This technique worked well for labeling both inner and outer spirochetal membrane proteins, indicating that the dye is freely diffusible across the intact outer membrane of living spirochetes without the need for fixation or permeabilization. Labeled spirochetes were quantitatively detected by fluorescence microscopy and flow cytometry. We found that concatenating two copies of the tetracysteine motif increased the intensity and duration of fluorescence. Genes encoding tetracysteine-tagged proteins were expressed from endogenous loci or in trans from shuttle vectors, indicating that single or multicopy expression sites are effective options. We successfully applied this technique to the distantly related spirochetes B. burgdorferi, a zoonotic human pathogen, and L. biflexa, a free-living saprophyte, suggesting that the FlAsH system can be applied broadly across all spirochete species. Mouse-tick infection studies of tetracysteine-tagged B. burgdorferi demonstrated that the tetracysteine motif was stably maintained in vivo and did not adversely affect infectivity in either the arthropod vector or murine host. Finally, biarsenical-bound proteins could be followed over several days, indicating that the FlAsH dye approach can be used in time-course studies of live cells (1). Genetic manipulation of B. burgdorferi is currently extremely inefficient, requiring microgram quantities of DNA, yet yielding only a few transformants. This severely limits the application of effective genetic screens to the Lyme disease spirochete. Endogenous plasmid-encoded restriction/modification (R/M) systems constitute part of the barrier to stable introduction of foreign DNA in B. burgdorferi. In FY2019, we continued a long-standing collaboration with Dr. Gang Fang at Mt. Sinai School of Medicine, NY. We have identified the DNA sequence motifs recognized by R/M systems of the widely used B. burgdorferi type strain B31 and extended this analysis to prototypic B. garinii and B. afzelii strains, which are agents of Lyme borreliosis in Europe. Armed with this information, Dr. Jenny Wachter, a postdoctoral fellow in MGS, has designed shuttle vectors and selectable markers that lack these R/M sites. Additionally, in collaboration with Dr. Craig Martens and Stacy Ricklefs of the RTB at RML, Jenny has completed RNA-seq analysis of strains containing or lacking R/M genes and found limited evidence for epigenetic regulation of gene expression in B. burgdorferi. Ongoing studies will assess the utility of sequence-optimized constructs for efficient transformation of B. burgdorferi and explore their potential to expand genetic studies in the Lyme disease spirochete. In FY2019 we assisted Christina Savage, William Arnold and Brandon Jutras, current and former graduate students in Dr. Brian Stevenson's lab at the University of Kentucky, with an investigation of the role of the BpuR DNA- and RNA-binding protein of B. burgdorferi in the modulation of spirochete physiology (2). Our contribution to this study comprised an analysis of bpuR transcript levels in the spirochete during acquisition, persistence and transmission of B. burgdorferi by the tick vector. In FY2019, Dr. Rosa completed a sabbatical in the laboratory of Dr. Christine Jacobs-Wagner at Yale University. During FY2019, Dr. Rosa contributed to a Jacobs-Wagner lab study that resulted in the development of a number of new molecular tools for B. burgdorferi, including several new fluorescent proteins, promotors of varying strength for modulated levels of gene expression, and novel selectable markers. (3) Dr. Rosa also assisted members of the Jacobs-Wagner lab in an investigation of how Borrelia replicates and segregates its highly segmented genome, as required for the maintenance and survival of B. burgdorferi in nature and ultimately central to transmission of Lyme disease. Finally, Dr. Rosa took advantage of the expertise of the Jacobs-Wagner lab to fluorescently tag and computationally analyze the cellular locations of a recently described set of bacterial cytoskeletal elements termed bactofilins. This work was done in conjunction with an ongoing genetic analysis of the role of bactofilins in B. burgdorferi, conducted by Valentina Carracoi in Dr. Rosas lab at RML. Various aspects of the genome localization and bactofilin projects continue as a collaborative effort between the Jacobs-Wagner and Rosa. 1. Hillman, C., Stewart, P.E., Strnad, M., Stone, H., Starr, T., Carmody, A., Evans, T.J., Carracoi, V., Wachter, J., and Rosa, P.A., Visualization of spirochetes by labeling membrane proteins with fluorescent biarsenical dyes. Front. Cell. Infect. Microbiol., in press 2019. 2. Jutras, B., Savage, C., Arnold, W., Lethbridge, K. Carroll, D., Tilly, K., Bestor, A., Zhu, H., Seshu, J., Zueckert, W., Stewart, P., Rosa, P., Brissette, C., and Stevenson, B. ,The Lyme disease spirochete's BpuR DNA- / RNA-binding protein is differentially expressed during the mammal-tick infectious cycle and affects translation of the SodA superoxide dismutase. Mol.Microbiol., in press 2019. 3. Takacs, C.N., Kloos, Z.A., Scott, M., Rosa, P.A., and Jacobs-Wagner, C., Fluorescent proteins, promoters and selectable markers for applications in the Lyme disease spirochete Borrelia burgdorferi. Appl. Environ. Microbiol. 84:e01824-18, 2018

莱姆病是美国和欧洲最常见的蜱传疾病。它是由伯氏疏螺旋体引起的，这是一种细菌病原体，在自然界中以各种小型哺乳动物和蜱虫媒介之间的人畜共患病循环存在。莱姆病螺旋体的一个标志是其不寻常的分段基因组，其中包括大量线性和环状质粒。越来越多的证据表明，质粒编码的功能对于成功适应伯氏疏螺旋体在感染周期中遇到的不同环境至关重要。我们开发了遗传工具来研究伯氏疏螺旋体不寻常的基因组组织、细胞结构和代谢的基本方面。我们将这项研究扩展到了体内环境，使用了一个密切模仿自然节肢动物载体/啮齿动物宿主感染周期的实验系统。通过了解生物体的基本分子生物学，我们希望深入了解这种重要的媒介传播病原体所利用的感染策略，从而促进莱姆病的预防、诊断和治疗。 在 2019 财年，我们描述了 FlaAsH 技术在螺旋体中的新应用，用于荧光标记活细胞中的特定蛋白质 (1)。该技术需要在感兴趣的蛋白质的编码序列中设计一个四半胱氨酸基序，并用与该基序结合的膜渗透性双砷染料进行染色。 6 个氨基酸基序赋予蛋白质大小的小幅增加，最大限度地减少了当靶标与较大荧光蛋白融合时可能发生的潜在负面影响，例如空间位阻、不正确的蛋白质折叠、不正确的细胞位置或不适当的寡聚化。该技术对于标记内螺旋体膜蛋白和外螺旋体膜蛋白效果良好，表明染料可以自由扩散到活螺旋体的完整外膜上，而不需要固定或透化。通过荧光显微镜和流式细胞术定量检测标记的螺旋体。我们发现连接两个拷贝的四半胱氨酸基序增加了荧光的强度和持续时间。编码四半胱氨酸标签蛋白的基因从内源基因座表达或从穿梭载体反式表达，表明单拷贝或多拷贝表达位点是有效的选择。 我们成功地将这项技术应用于远缘螺旋体伯氏疏螺旋体（一种人畜共患的人类病原体）和双曲螺旋体（一种自由生活的腐生菌），这表明 FlAsH 系统可以广泛应用于所有螺旋体物种。四半胱氨酸标记的伯氏疏螺旋体的小鼠蜱感染研究表明，四半胱氨酸基序在体内稳定维持，并且不会对节肢动物载体或鼠宿主的感染性产生不利影响。最后，可以对双砷结合蛋白进行几天的跟踪，这表明 FlAsH 染料方法可用于活细胞的时程研究 (1)。 目前，伯氏疏螺旋体的基因操作效率极低，需要微克量的 DNA，但只能产生少量转化体。这严重限制了对莱姆病螺旋体进行有效遗传筛选的应用。内源质粒编码的限制/修饰 (R/M) 系统构成了外源 DNA 稳定引入伯氏疏螺旋体的部分障碍。 2019 财年，我们继续与纽约西奈山医学院的 Gang Fang 博士进行长期合作。我们已经鉴定了广泛使用的伯氏疏螺旋体型菌株 B31 的 R/M 系统所识别的 DNA 序列基序，并将该分析扩展到原型伯氏疏螺旋体菌株和欧洲莱姆疏螺旋体病的病原体 B. garinii 和 B. afzelii 菌株。有了这些信息，MGS 的博士后 Jenny Wachter 博士设计了缺乏这些 R/M 位点的穿梭载体和选择性标记。此外，Jenny 与 RML RTB 的 Craig Martens 博士和 Stacy Ricklefs 合作，完成了对含有或缺乏 R/M 基因的菌株的 RNA-seq 分析，并发现了伯氏疏螺旋体基因表达表观遗传调控的有限证据。正在进行的研究将评估序列优化构建体在有效转化伯氏疏螺旋体方面的效用，并探索其扩大莱姆病螺旋体遗传学研究的潜力。 2019 财年，我们协助肯塔基大学 Brian Stevenson 博士实验室的现任和前任研究生 Christina Savage、William Arnold 和 Brandon Jutras 调查了伯氏疏螺旋体的 BpuR DNA 和 RNA 结合蛋白在调节螺旋体生理学中的作用 (2)。我们对这项研究的贡献包括分析蜱虫媒介在伯氏疏螺旋体的获取、持续和传播过程中螺旋体中的 bpuR 转录水平。 2019 财年，Rosa 博士在耶鲁大学 Christine Jacobs-Wagner 博士的实验室完成了休假。 2019 财年，Rosa 博士为 Jacobs-Wagner 实验室研究做出了贡献，该研究开发了许多针对伯氏疏螺旋体的新分子工具，包括几种新的荧光蛋白、用于调节基因表达水平的不同强度的启动子以及新型选择性标记。 (3) Rosa 博士还协助 Jacobs-Wagner 实验室的成员研究伯氏疏螺旋体如何复制和分离其高度分段的基因组，这是伯氏疏螺旋体在自然界中维持和生存所必需的，并且最终是莱姆病传播的核心。最后，Rosa 博士利用 Jacobs-Wagner 实验室的专业知识，对最近描述的一组细菌细胞骨架元件（称为 bactofilins）的细胞位置进行荧光标记和计算分析。这项工作是与 RML Rosas 博士实验室的 Valentina Carracoi 正在进行的对伯氏疏螺旋体中杆菌素作用的遗传分析结合完成的。基因组定位和杆菌素项目的各个方面继续作为雅各布斯-瓦格纳和罗莎之间的合作努力。 1. Hillman, C.、Stewart, P.E.、Strnad, M.、Stone, H.、Starr, T.、Carmody, A.、Evans, T.J.、Carracoi, V.、Wachter, J. 和 Rosa, P.A.，通过用荧光双砷染料标记膜蛋白来可视化螺旋体。正面。细胞。感染。微生物学，2019 年出版。 2. Jutras, B., Savage, C., Arnold, W., Lethbridge, K. Carroll, D., Tilly, K., Bestor, A., Zhu, H., Seshu, J., Zueckert, W., Stewart, P., Rosa, P., Brissette, C., and Stevenson, B.，莱姆病螺旋体的 BpuR DNA-/RNA 结合蛋白差异表达 在哺乳动物蜱感染周期中并影响 SodA 超氧化物歧化酶的翻译。 Mol.Microbiol.，2019 年出版。 3. Takacs, C.N.、Kloos, Z.A.、Scott, M.、Rosa, P.A. 和 Jacobs-Wagner, C.，荧光蛋白、启动子和选择标记在莱姆病螺旋体伯氏疏螺旋体中的应用。应用。环境。微生物。 84:e01824-18, 2018