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: 8745399
• 负责人: PATRICIA A ROSA
• 金额: $ 92.99万
• 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/8745399
• 关键词: Address; Antigenic Variation; Bacteria; Base Sequence; Biological Assay; Borrelia; Borrelia burgdorferi; Chromosomes; Collaborations; Cues; Data; Development; Elements; Environment; Flagella; Florida; Future; Gene Expression; Gene Proteins; Genes; Genetic; Genome; Genomics; Goals; Growth; Immune; Immunodeficient Mouse; In Vitro; Individual; Infection; Laboratories; Life Cycle Stages; Lipids; Lipoproteins; Lyme Disease; Mammals; Membrane; Membrane Proteins; Methods; Modeling; Molecular Genetics; Mus; Nature; Order Spirochaetales; OspA protein; OspC protein; Other Genetics; Pattern; Phenotype; Plasmids; Play; Postdoctoral Fellow; Process; Production; Proliferating; Proteins; Relative (related person); Replicon; Reporting; Research Design; Research Personnel; Role; Signal Transduction; Site; Staging; Structure; Sultan; System; Technology; Temperature; Testing; Ticks; Time; Universities; Variant; Virulence; Work; attenuation; base; cell motility; design; feeding; fitness; genetic element; graduate student; in vivo; insight; interest; mutant; outer surface lipoprotein; pathogen; pathogenic bacteria; periplasm; response; tool; transmission process; vector

Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature through an infectious cycle that alternates between various species of small mammals and a tick vector. Like many bacterial pathogens, B. burgdorferi must adapt to a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi has an unusual segmented genome that includes a large number of linear and circular plasmids. Increasing evidence indicates that plasmid-encoded genes are critical for successful adaptation by B. burgdorferi to the different environments that the spirochete encounters during its infectious cycle. A major focus of this project is to determine how and why the Lyme disease spirochete maintains such a unique genomic structure, and the specific contributions of individual plasmids and genes at each stage of the infectious cycle. In FY2013, Dr. Dan Dulebohn, a post-doctoral fellow, investigated the role of a 28 kilobase (kb) linear plasmid, designated lp28-3, in the B. burgdorferi infectious cycle (1). Analysis of the nucleotide sequence of lp28-3 indicates that this plasmid is highly conserved among B. burgdorferi strains and that the predicted functions of some lp28-3 encoded gene products are consistent with a role in the infectious cycle. Dan characterized lp28-3 deficient strains obtained by two independent methods and analyzed these B. burgdorferi variants during in vitro growth and the entire mouse-tick infectious cycle. Dan found that lp28-3 does not carry any genes that are strictly required for B. burgdorferi infection of a mouse or tick, and that spirochetes lacking lp28-3 can cause a disseminated and persistent infection. However, these lp28-3-minus spirochetes were at a selective dis-advantage relative to wild type spirochetes when both were co-injected into a mouse, and this attenuation was reflected in the relative proportion of lp28-3 deficient to wild type spirochetes acquired by feeding ticks. These data demonstrate that lp28-3-encoded genes, although not essential, contribute to the fitness of B. burgdorferi during infection of the mammalian host (1). This study demonstrates the utility of using a co-infection model to analyze B. burgdorferi mutants for which there is no obvious phenotype when assayed individually (1). This strategy facilitates determining the strict requirement for a genetic element during infection, while also providing a quantitative assessment of the contribution to the fitness of Borrelia in vivo. This approach could be especially useful for studying other genetic elements of B. burgdorferi that would appear to be important, but have been shown to be dispensable in an experimental infectious cycle. Our data suggest that although many B. burgdorferi genes are not strictly required for completion of an experimental mouse-tick-mouse infectious cycle, they can make substantial contributions in the competitive environment of a mixed infection, which is frequently encountered in a natural infectious cycle. As described above, Borrelia burgdorferi alternates between ticks and mammals, requiring variable gene expression and protein production to adapt to these diverse niches. These adaptations include switching between the major outer surface lipoproteins OspA, OspC, and VlsE at different stages of the infectious cycle. Although this pattern of lipoprotein succession has been described, the functions of these outer surface proteins remain undefined. Previous work by other investigators suggested that several B. burgdorferi lipoproteins, including OspA and VlsE, could substitute for OspC at the initial stage of mouse infection, when OspC is transiently but absolutely required. In FY2013, Dr. Kit Tilly and Aaron Bestor assessed whether complementation with the vlsE or ospA gene could restore infectivity to an ospC mutant, and found that neither gene product effectively compensated for the absence of OspC during early infection (2). In contrast, Kit and Aaron determined that OspC production was required by B. burgdorferi for persistent infection of an immunodeficient mouse when the vlsE gene was absent. Together, these results indicate that OspC can substitute for VlsE on B. burgdorferi when antigenic variation is unnecessary, but that these two abundant outer surface lipoproteins are optimized for their related but specific roles during early and persistent mammalian infection by B. burgdorferi. A simple model to explain these and other results is that these major B. burgdorferi outer surface lipoproteins serve a common basic function at different stages of the bacterial mouse-tick life cycle. In this model, similar roles are played by OspC during the initial stage of mammalian infection, by VlsE during persistent infection, and by OspA during the tick stage of the life cycle. The seemingly non-specific nature of that function may be protection against some aspect of mammalian and tick immune defenses, or structural stabilization of the outer membrane. The FY2013 study described above was designed to further test the model that OspC and VlsE play similar roles at different stages of mammalian infection (2). Because it appears that OspC and VlsE are not fully interchangeable, we propose that these two proteins require both appropriate context and appropriate timing to be fully functional. By context, we encompass the roles of other proteins produced at the same time and also variations in membrane composition and arrangement, which are influenced by lipid availability and temperature. In fiscal year 2013 we reported the culminations of two long-standing collaborations with the laboratories of Dr. Md Motaleb at East Carolina University and Dr. Mollie Jewett at the University of Central Florida (3, 4). Dr. Syed Sultan, a post-doctoral fellow in Dr. Motalebs lab, completed a genetic study investigating the requirement for motility in the mouse-tick infectious cycle of B. burgdorferi (3). This study demonstrated that motility driven by the spirochete's periplasmic flagella is required for optimal survival in and transmission between the tick vector and the mammalian host. Tish Choudry Ellis, a graduate student in Dr. Jewetts lab, reported the development and application of an In Vivo Expression Technology system (IVET) for identification of B. burgdorferi genes that are expressed during infection of the mammalian host (4). Using this approach, they have identified a number of genes that are expressed during active infection, including a gene that appears to contribute to the ability of the spirochete to persist in an immune-competent host. Our contributions to both of these studies were discrete but significant at their inceptions.

伯氏疏螺旋体（Borrelia burgdorferi）是莱姆病的病原体，通过在各种小型哺乳动物和蜱媒介之间交替的传染周期在自然界中得以维持。与许多细菌病原体一样，伯氏疏螺旋体必须适应一系列不断变化的环境条件，才能成功生存、增殖并在宿主之间传播。伯氏疏螺旋体具有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，质粒编码基因对于伯氏疏螺旋体成功适应螺旋体在感染周期中遇到的不同环境至关重要。该项目的一个主要重点是确定莱姆病螺旋体如何以及为何保持如此独特的基因组结构，以及单个质粒和基因在感染周期每个阶段的具体贡献。 2013 财年，博士后 Dan Dulebohn 博士研究了 28 kilobase (kb) 线性质粒（命名为 lp28-3）在伯氏疏螺旋体感染周期中的作用 (1)。对lp28-3的核苷酸序列的分析表明，该质粒在伯氏疏螺旋体菌株中高度保守，并且一些lp28-3编码的基因产物的预测功能与在感染周期中的作用一致。 Dan 对通过两种独立方法获得的 lp28-3 缺陷菌株进行了表征，并分析了这些伯氏疏螺旋体变体在体外生长和整个小鼠蜱感染周期中的情况。 Dan 发现 lp28-3 不携带任何小鼠或蜱感染伯氏疏螺旋体所必需的基因，并且缺乏 lp28-3 的螺旋体会导致播散性和持续性感染。然而，当将这些lp28-3-缺失螺旋体共同注射到小鼠体内时，这些lp28-3-缺失螺旋体相对于野生型螺旋体处于选择性劣势，并且这种减弱反映在通过喂养蜱获得的lp28-3缺陷型螺旋体与野生型螺旋体的相对比例上。这些数据表明，lp28-3 编码的基因虽然不是必需的，但有助于伯氏疏螺旋体在哺乳动物宿主感染期间的适应度 (1)。 本研究证明了使用共感染模型来分析伯氏疏螺旋体突变体的实用性，这些突变体在单独检测时没有明显的表型 (1)。该策略有助于确定感染过程中对遗传元件的严格要求，同时还提供对疏螺旋体体内适应性的贡献的定量评估。这种方法对于研究伯氏疏螺旋体的其他遗传元件特别有用，这些元件似乎很重要，但已被证明在实验感染周期中是可有可无的。我们的数据表明，尽管许多伯氏疏螺旋体基因并不是完成实验性小鼠-蜱-小鼠感染周期所必需的，但它们可以在自然感染周期中经常遇到的混合感染的竞争环境中做出重大贡献。 如上所述，伯氏疏螺旋体在蜱和哺乳动物之间交替，需要可变的基因表达和蛋白质生产来适应这些不同的生态位。 这些适应包括在感染周期的不同阶段在主要外表面脂蛋白 OspA、OspC 和 VlsE 之间进行转换。 尽管已经描述了这种脂蛋白连续模式，但这些外表面蛋白的功能仍不清楚。其他研究人员之前的工作表明，几种伯氏疏螺旋体脂蛋白，包括 OspA 和 VlsE，可以在小鼠感染的初始阶段替代 OspC，此时 OspC 是暂时但绝对需要的。 2013 财年，Kit Tilly 博士和 Aaron Bestor 博士评估了与 vlsE 或 ospA 基因的互补是否可以恢复 ospC 突变体的感染性，并发现这两种基因产物都无法有效补偿早期感染期间 OspC 的缺失 (2)。 相反，Kit 和 Aaron 确定，当 vlsE 基因缺失时，伯氏疏螺旋体需要产生 OspC 来持续感染免疫缺陷小鼠。总之，这些结果表明，当不需要抗原变异时，OspC 可以替代伯氏疏螺旋体上的 VlsE，但这两种丰富的外表面脂蛋白针对其在伯氏疏螺旋体早期和持续哺乳动物感染期间的相关但特定的作用进行了优化。 解释这些和其他结果的一个简单模型是，这些主要的伯氏疏螺旋体外表面脂蛋白在细菌小鼠蜱生命周期的不同阶段发挥着共同的基本功能。 在该模型中，OspC 在哺乳动物感染的初始阶段、VlsE 在持续感染期间以及 OspA 在生命周期的蜱阶段发挥着类似的作用。 该功能看似非特异性的性质可能是针对哺乳动物和蜱免疫防御的某些方面的保护，或外膜的结构稳定。 上述 2013 财年研究旨在进一步测试 OspC 和 VlsE 在哺乳动物感染的不同阶段发挥相似作用的模型 (2)。 由于 OspC 和 VlsE 似乎不能完全互换，因此我们认为这两种蛋白需要适当的背景和适当的时机才能充分发挥功能。 根据上下文，我们涵盖了同时产生的其他蛋白质的作用，以及膜组成和排列的变化，这些变化受到脂质可用性和温度的影响。 2013 财年，我们报告了与东卡罗来纳大学 Md Motaleb 博士和中佛罗里达大学 Mollie Jewett 博士实验室的两项长期合作的成果 (3, 4)。 Motalebs 博士实验室的博士后研究员 Syed Sultan 博士完成了一项基因研究，调查了伯氏疏螺旋体小鼠蜱感染周期中运动性的要求 (3)。这项研究表明，螺旋体周质鞭毛驱动的运动对于蜱载体和哺乳动物宿主之间的最佳生存和传播是必需的。 Jewetts 博士实验室的研究生 Tish Choudry Ellis 报告了体内表达技术系统 (IVET) 的开发和应用，用于鉴定在哺乳动物宿主感染期间表达的伯氏疏螺旋体基因 (4)。 利用这种方法，他们鉴定了许多在主动感染期间表达的基因，其中包括一个似乎有助于螺旋体在免疫能力正常的宿主中持续存在的能力的基因。 我们对这两项研究的贡献是离散的，但在它们开始时意义重大。