Molecular Genetics Of Infectious Borrelia Burgdorferi

伯氏疏螺旋体是莱姆病的致病因子，在自然界中是通过野生哺乳动物和扁虱之间的感染循环保持的。像许多细菌病原体一样，伯氏杆菌必须应对一系列不断变化的环境条件，才能成功地持续、增殖并在宿主之间传播。伯氏假单胞菌具有一种不寻常的基因组结构，由一条线性染色体和大量的线性和环状质粒组成。大量证据表明，质粒编码的基因在感染周期中对适应至关重要。与它们在体内的重要作用相反，在伯氏杆菌的体外培养过程中，质粒往往会丢失。我们研究的一个主要重点是采用分子遗传学的方法，确定单个伯氏杆菌基因和质粒在感染周期的每个阶段的贡献。 伯氏杆菌模式株(B31)的基因组除含有线形染色体外，还含有12个线形和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个线性质粒lp25和lp28-1在哺乳动物宿主中的作用。伯氏假单胞菌需要线形质粒lp28-1，才能在具有免疫能力的小鼠中建立针状接种后的持续感染，而在所有条件下，lp25对伯氏杆菌在哺乳动物中的生长是必不可少的。在这些质粒携带的特定基因中，其他的已经表明，编码烟酰胺酶(PncA)的lp25上的bbe22基因足以恢复哺乳动物对缺乏lp25的伯氏杆菌克隆的感染力。相比之下，lp28-1上与小鼠感染性有关的一个或多个必需基因尚未被鉴定。然而，扁虱对lp25和lp28-1或它们携带的任何基因的需求还没有被调查。 在最近的一项研究中，Grimm和他的同事分析了Burgdorferi在蜱类定植期间对质粒lp25和lp28-1的需求，Burgdorferi必须在这个位置生存和复制，才能维持其自然感染周期。这是通过用野生型Burgdorferi(缺乏lp28-1或lp25的克隆)和恢复了各自的质粒的等基因克隆感染扁虱来实现的。为了做到这一点，我们利用最近开发的工具对伯氏杆菌进行遗传操作，并结合一种有效的方法来进行实验性的蜱虫感染。许多先前的研究已经在小鼠身上检测了各种缺乏特异质粒的伯氏杆菌克隆的表型，但很难确定它们在硬蜱中的表型，特别是当一个克隆在小鼠中是非传染性的时候。 我们发现，尽管无论感染途径如何，lp28-1都是哺乳动物持续感染所必需的，但lp28-1对于扁虱中肠的生存和复制、向唾液腺的迁移或通过唾液传播到哺乳动物宿主并不是必不可少的。相反，我们发现lp25是熟练感染硬蜱所必需的，因此对于感染周期中的两个宿主的持久性是必不可少的。我们还通过在穿梭载体上感染缺乏lp25但含有bbe22基因(编码pncA)的螺旋体感染扁虱，研究了B.burgdorferi对烟草酰胺酶pncA的需求。我们发现，缺乏lp25的伯氏杆菌仅与bbe22互补，可以恢复螺旋体定植扁虱的能力，并在扁虱叮咬后感染小鼠。缺少这种酶的螺旋体可能不能利用烟酰胺合成NAD。这些结果表明，伯氏伯氏杆菌需要bbe22才能在硬蜱中感染，但不排除lp25上的额外基因可能有助于伯氏杆菌在硬蜱中肠的存活和复制。还需要进一步的研究来确定lp25上的任何剩余基因，虽然不是必需的，但是否对螺旋体的生长或在扁虱和小鼠中的生存提供了优势。这是第一项研究伯氏杆菌在扁虱载体中对特定质粒的需求，并开始建立这种病原体在其自然感染周期中持续存在所需的基因组成分。

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.