Molecular Genetic Basis Of The Infectious Cycle Of 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。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。 伯氏杆菌含有一个分段的基因组，包括一个900kb的小的线形质粒和多达23个环状和线形的质粒，大小从5kb到56kb不等。伯氏杆菌基因组在体外传代过程中是不稳定的，在这一过程中许多质粒可能会丢失。某些质粒的丢失与小鼠和扁虱的感染性和持久性的丧失密切相关。例如，我们和其他人之前已经证明，线性质粒(LP)25对于小鼠和扁虱的感染是关键的，而线性质粒lp28-1对于小鼠的持久性是唯一需要的。与lp25和lp28-1不同的是，伯氏杆菌在体外传代过程中不会频繁丢失线形质粒lp36，其在感染循环中的潜在作用以前还没有被研究过。我们最近发现了一种罕见的伯氏杆菌变异体，它丢失了lp36，但保留了所有已知的对毒力重要的其他质粒，从而使人们能够研究这种质粒在感染循环中的作用(Juett等人。2007)。 测序的伯氏杆菌B31菌株的lp36质粒是一个约36kb的线性质粒，编码54个开放阅读框，其中7个似乎是假基因。Lp36上的大多数基因在数据库中缺乏同源性，也没有预测的功能。我们发现，缺乏lp36的螺旋体在哺乳动物中不容易存活，但在扁虱中没有表现出缺乏(朱伊特等人。2007)。通过在突变克隆中重组lp36质粒，恢复了小鼠的感染，证明了感染性缺陷是由于lp36的缺失造成的。此外，我们证实了lp36的bbk17基因编码腺嘌呤脱氨酶(ADEC)，并且是lp36上的一个足以恢复小鼠对缺乏lp36的螺旋体的感染性的遗传成分。我们的工作确立了lp36质粒在感染循环中的关键作用(朱厄特等人。2007)。 伯氏杆菌外膜的组成发生了深刻的变化，因为螺旋体从扁虱媒介传播到哺乳动物宿主。质粒编码的OspA蛋白丰富地存在于伯氏杆菌的表面，存在于受感染的硬蜱的中肠中，但随着硬蜱的进食，OspA被下调，并被OspC取代，OspC在不同的质粒上编码。螺旋体表面蛋白的这种转换被认为是伯氏杆菌从中肠迁移到扁虱的唾液腺或在传播后适应哺乳动物宿主所必需的。 我们之前通过研究SPC在扁虱载体和哺乳动物宿主中的体内作用来验证这一假说。我们发现，从OspA到OspC的转换不是螺旋体从中肠迁移到扁虱唾液腺所必需的，而是为传播到哺乳动物宿主做准备。由于OspC似乎在确定哺乳动物感染中起着关键作用，我们最近进一步描述了该蛋白必须存在的关键期(Tilly等人。2007)。为了解决这个问题，我们将野生型和SpC突变螺旋体接种到小鼠皮肤中，并评估了接种后不同时间(从3小时到21天)重新分离螺旋体的能力。我们还通过确定细菌最初从远端部位分离出来的时间来分析螺旋体何时传播。所有接种点均分离到野生型和补体突变型螺旋体，接种后第8天从较远的接种点分离到。相比之下，SpC突变螺旋体在接种后24小时(当时5/6只小鼠检测为阴性)从接种地点分离到，但此后从未分离到。我们得出结论，SpC在将螺旋体引入哺乳动物后的第一个48小时内，在宿主传播或血清转换之前的第一个48小时内，在Burgdorferi芽孢杆菌感染中发挥关键作用(Tilly等人。2007)。 早期对SPC的要求排除了该蛋白在逃避获得性免疫反应中的作用，并表明它参与了逃避宿主的先天免疫反应。我们的工作模型是，OspC抑制伯氏杆菌的吞噬作用，可能是通过限制补体的调理作用，允许细菌在传播到哺乳动物宿主后立即逃避清除。 这些发现，加上以前关于扁虱和哺乳动物螺旋体基因表达差异调控的观察，表明宿主适应不仅发生在不同的节肢动物和哺乳动物环境中，而且在每个宿主内的不同阶段也有差异，大致对应于定殖期、持久性和传播期。

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. 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. B. burgdorferi harbors a segmented genome that includes a small 900 kb linear plasmid and as many as 23 circular and linear plasmids, ranging in size from 5kb to 56 kb. The B. burgdorferi genome is unstable during in vitro passage and many of the plasmids can be lost during this process. Loss of certain plasmids is tightly correlated with loss of infectivity and persistence in mice and ticks. For example, we and others have previously demonstrated that the linear plasmid (lp) 25 is critical for infection of the mouse and tick, whereas linear plasmid lp28-1 is required exclusively for persistence in the mouse. Unlike lp25 and lp28-1, the linear plasmid lp36 is not frequently lost by B. burgdorferi during in vitro passage and its potential role in the infectious cycle has not previously been examined. We recently identified a rare B. burgdorferi variant that has lost lp36 but retains all other plasmids known to be important for virulence, thereby allowing investigation of the role of this plasmid in the infectious cycle (Jewett et al. 2007). The lp36 plasmid of the sequenced B. burgdorferi strain B31 is a linear plasmid of approximately 36 kb encoding 54 open reading frames, seven of which appear to be pseudogenes. Most of the genes on lp36 lack homologs in the database and have no predicted function. We found that spirochetes lacking lp36 did not readily survive in the mammal but displayed no deficiency in the tick (Jewett et al. 2007). Mouse infection was restored by reconstitution of the lp36 plasmid in the mutant clone, demonstrating that the infectivity defect resulted from the loss of lp36. Furthermore, we established that the bbk17 gene of lp36 encodes an adenine deaminase (AdeC) and is a genetic component on lp36 that is sufficient to restore mouse infectivity to spirochetes lacking lp36. Our work establishes a critical role for the lp36 plasmid in the infectious cycle (Jewett et al. 2007). The composition of the outer membrane of B. burgdorferi undergoes a profound change as the spirochete is transmitted from the tick vector to a mammalian host. The plasmid-encoded OspA protein is abundant on the surface of B. burgdorferi residing in the midguts of infected ticks but as ticks feed, OspA is down-regulated and replaced with OspC, which is encoded on a different plasmid. This switch in spirochetal surface proteins was hypothesized to be required for migration of B. burgdorferi from the midgut to the salivary glands of the tick or adaptation to the mammalian host after transmission. We previously tested this hypothesis with an investigation of the in vivo role of OspC in both the tick vector and mammalian host. We found that the switch from OspA to OspC is not required for spirochete migration from the midgut to the salivary glands of ticks, but is in preparation for transmission to the mammalian host. Since the essential role of OspC appears to be in establishing mammalian infection, we recently further delineated the critical period in which the protein must be present (Tilly et al. 2007). To address this question, we inoculated wild type and ospC mutant spirochetes into mouse skin and assessed the ability to reisolate spirochetes at various times after inoculation, ranging from 3 hours to 21 days. We also assayed when spirochetes disseminate, by determining when bacteria were first isolated from distal sites. Wild type and complemented mutant spirochetes were isolated from inoculation sites at all time points and first isolated from distant sites at 8 days post-inoculation. In contrast, ospC mutant spirochetes were isolated from inoculation sites through 24 hours post-inoculation (at which time 5/6 mice tested were negative), but never thereafter. We conclude that OspC plays a crucial role in B. burgdorferi infection within the first 48 hours after introduction of spirochetes into the mammal, prior to dissemination or seroconversion of the host (Tilly et al. 2007). The early requirement for OspC excludes a role for the protein in evading the acquired immune reponse and suggests that it is involved in evasion of the host innate immune response. Our working model is that OspC inhibits phagocytosis of B. burgdorferi, perhaps by limiting opsonization by complement, allowing the bacteria to evade clearance immediately after transmission to the mammalian host. These findings, together with previous observations regarding differential regulation of spirochetal gene expression in ticks and mammals, indicate that host adaptation not only occurs in response to disparate arthropod and mammalian environments, but also varies within each host at stages roughly corresponding to colonization, persistence and transmission.