Molecular Genetics Of Infectious Borrelia Burgdorferi

传染性伯氏疏螺旋体的分子遗传学

• 批准号: 7196704
• 负责人: PATRICIA A ROSA
• 金额: --
• 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/7196704
• 关键词: Borrelia; Lyme disease; Mus musculus; SDS polyacrylamide gel electrophoresis; Spirochaetales; arthropod borne communicable disease; bacteria infection mechanism; bacterial genetics; bacterial proteins; disease vectors; gene complementation; gene expression; gene mutation; genetic manipulation; laboratory mouse; mass spectrometry; microarray technology; microorganism culture; molecular genetics; northern blottings; nucleic acid sequence; pathologic process; plasmids; polymerase chain reaction; ticks; western blottings

Borrelia burgdorferi, the cauative agent of Lyme disease, is maintained in nature through an infectious cycle between wild mammals and ticks. Like many bacterial pathogens, B. burgdorferi must cope with a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi has an unusual genomic structure composed of a linear chromosome and a large number of linear and circular plasmids. Abundant evidence indicates that plasmid-encoded genes are critical for adaptation in the infectious cycle. In contrast to their essential role in vivo, plasmids are often lost during in vitro cultivation of B. burgdorferi. A major focus of our research is to determine the contributions of individual B. burgdorferi genes and plasmids at each stage of the infectious cycle, taking a molecular genetic approach. The genome of the type strain (B31) of B. burgdorferi has 12 linear and 9 circular plasmids, in addition to the linear chromosome. Previous studies from our lab and others have determined the roles of two linear plasmids, lp25 and lp28-1, in the mammalian host. The linear plasmid lp28-1 is required by B. burgdorferi to establish persistent infections in immunocompetent mice following needle inoculation, whereas lp25 is essential for B. burgdorferi growth in mammals under all conditions. Of the specific genes carried by these plasmids, others have shown that the bbe22 gene on lp25, encoding a nicotinamidase (PncA), is sufficient to restore infectivity in mammals to B. burgdorferi clones lacking lp25. In contrast, the essential gene or genes on lp28-1 for mouse infectivity have not been identified. However, the requirement in ticks for lp25 and lp28-1, or any of the genes they carry, has not been investigated. In a recent study, Grimm and colleagues analyzed the requirement for plasmids lp25 and lp28-1 by B. burgdorferi during colonization of ticks, a site where B. burgdorferi must survive and replicate to be maintained in its natural infectious cycle. This was accomplished by experimentally infecting ticks with wild type B. burgdorferi, with clones lacking lp28-1 or lp25, and with isogenic clones in which the respective plasmid had been restored. To do so, we utilized recently developed tools for the genetic manipulation of B. burgdorferi in combination with an efficient method for experimental tick infection. Many prior studies have examined the phenotypes of various B. burgdorferi clones lacking specific plasmids in mice, but it has been difficult to address their phenotypes in ticks, particularly when a clone is non-infectious in mice. We found that lp28-1 is not essential for survival and replication in the tick midgut, for migration to the salivary glands, or for transmission via the saliva to the mammalian host, although lp28-1 is required for establishing a persistent infection in mammals, regardless of the route of infection. In contrast, we found that lp25 is required for proficient infection of ticks and therefore is essential for persistence in both hosts of the infectious cycle. We also investigated the requirement for the nicotinamidase PncA by B. burgdorferi in the tick vector by infecting ticks with spirochetes that lack lp25, but contain the bbe22 gene (encoding PncA) on a shuttle vector. We found that complementation of B. burgdorferi lacking lp25 with just bbe22 restores the ability of spirochetes to colonize ticks, and infect mice after tick bite. Spirochetes lacking this enzyme presumably cannot utilize nicotinamide for the synthesis of NAD. These results indicate that B. burgdorferi requires bbe22 for infectivity in ticks but do not rule out the possibility that additional genes on lp25 may contribute to survival and replication of B. burgdorferi in the tick midgut. Further studies are needed to determine if any of the remaining genes on lp25, while not essential, provide an advantage for spirochete growth or survival in ticks and mice.This is the first study to investigate the requirement for specific plasmids by B. burgdorferi within the tick vector and it begins to establish the genomic components required for persistence of this pathogen throughout its natural infectious cycle.

伯氏疏螺旋体是莱姆病的病原体，通过野生哺乳动物和蜱虫之间的感染循环在自然界中得以维持。与许多细菌病原体一样，伯氏疏螺旋体必须应对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基因组结构，由线性染色体和大量线性和环状质粒组成。大量证据表明，质粒编码基因对于感染周期的适应至关重要。与它们在体内的重要作用相反，质粒在伯氏疏螺旋体的体外培养过程中经常丢失。我们研究的一个主要重点是采用分子遗传学方法确定单个伯氏疏螺旋体基因和质粒在感染周期每个阶段的贡献。 伯氏疏螺旋体典型菌株(B31)的基因组除线性染色体外还有12个线性质粒和9个环状质粒。我们实验室和其他实验室之前的研究已经确定了两种线性质粒 lp25 和 lp28-1 在哺乳动物宿主中的作用。伯氏疏螺旋体需要线性质粒 lp28-1 在针头接种后在免疫活性小鼠中建立持续感染，而 lp25 对于哺乳动物在所有条件下的伯氏疏螺旋体生长都是必需的。在这些质粒携带的特定基因中，其他人已经表明，lp25 上的 bbe22 基因编码烟酰胺酶 (PncA)，足以恢复哺乳动物对缺乏 lp25 的伯氏疏螺旋体克隆的感染性。相比之下，lp28-1 上与小鼠感染性相关的必需基因尚未确定。然而，蜱对 lp25 和 lp28-1 或它们携带的任何基因的需求尚未得到研究。 在最近的一项研究中，Grimm 及其同事分析了伯氏疏螺旋体在蜱的定植过程中对质粒 lp25 和 lp28-1 的需求，在蜱虫定植期间，伯氏疏螺旋体必须生存和复制以维持其自然感染周期。这是通过用野生型伯氏疏螺旋体、缺乏lp28-1或lp25的克隆以及其中已恢复相应质粒的等基因克隆实验感染蜱来完成的。为此，我们利用最近开发的伯氏疏螺旋体基因操作工具与实验性蜱感染的有效方法相结合。许多先前的研究已经在小鼠中检查了各种缺乏特定质粒的伯氏疏螺旋体克隆的表型，但很难确定它们在蜱中的表型，特别是当克隆在小鼠中不具有感染性时。 我们发现，lp28-1 对于蜱中肠的生存和复制、迁移到唾液腺或通过唾液传播到哺乳动物宿主来说并不是必需的，尽管 lp28-1 对于在哺乳动物中建立持续感染是必需的，无论感染途径如何。相比之下，我们发现 lp25 是蜱虫有效感染所必需的，因此对于感染周期的两个宿主中的持续存在至关重要。我们还通过用缺乏 lp25 但在穿梭载体上含有 bbe22 基因（编码 PncA）的螺旋体感染蜱来研究蜱载体中伯氏疏螺旋体对烟酰胺酶 PncA 的需求。我们发现，缺乏 lp25 的伯氏疏螺旋体与仅 bbe22 的互补可以恢复螺旋体定殖蜱的能力，并在蜱叮咬后感染小鼠。缺乏这种酶的螺旋体可能无法利用烟酰胺来合成 NAD。这些结果表明伯氏疏螺旋体需要 bbe22 才能在蜱中具有感染性，但不排除 lp25 上的其他基因可能有助于伯氏疏螺旋体在蜱中肠中的存活和复制的可能性。需要进一步研究以确定 lp25 上的任何剩余基因（虽然不是必需的）是否为蜱和小鼠中螺旋体的生长或生存提供优势。这是第一项研究蜱载体内伯氏疏螺旋体对特定质粒的需求，并开始建立该病原体在其整个自然感染周期中持续存在所需的基因组成分。