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: 7592280
• 负责人: PATRICIA A ROSA
• 金额: $ 103.73万
• 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/7592280
• 关键词: Address; Adenine; Arthropods; Bacteria; Biological Assay; Borrelia; Borrelia burgdorferi; Chromosomes; Complement; Condition; Cues; Databases; Deaminase; Defect; Distal; Distant; Elements; Environment; Future; Gene Expression; Genes; Genetic; Genome; Genomics; Homologous Gene; Hour; Immune; Immune response; In Vitro; Individual; Infection; Investigation; Lyme Disease; Mammals; Membrane; Membrane Proteins; Methods; Midgut; Modeling; Molecular Genetics; Mus; Nature; Numbers; Open Reading Frames; Order Spirochaetales; OspA protein; OspC protein; Phagocytosis; Plasmids; Play; Preparation; Process; Proliferating; Proteins; Pseudogenes; Range; Regulation; Replicon; Research; Research Design; Role; Salivary Glands; Series; Signal Transduction; Site; Skin; Staging; Structure; Surface; Testing; Ticks; Time; Variant; Virulence; Work; base; coping; critical developmental period; day; feeding; in vivo; interest; migration; mutant; pathogen; pathogenic bacteria; reconstitution; research study; response; size; tool; transmission process; vector

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.

莱姆病的病原体伯氏疏螺旋体通过野生哺乳动物和蜱之间的传染循环在自然界中维持。像许多细菌病原体一样，B。伯氏菌必须科普一系列不断变化的环境条件，以便成功地持续存在、增殖和在宿主之间传播。B。Burgdorferi具有由线性染色体和大量线性和环状质粒组成的不寻常的基因组结构。大量证据表明，质粒编码的基因在感染循环中对适应至关重要。我们研究的一个主要焦点是确定个体B的贡献。通过分子遗传学方法，研究了感染周期每个阶段的伯氏螺旋体基因和质粒。 B。Burgdorferi具有分段的基因组，其包括一个小的900 kb线性质粒和多达23个大小在5 kb至56 kb范围内的环状和线性质粒。B。伯氏螺旋体基因组在体外传代过程中不稳定，许多质粒在此过程中可能丢失。某些质粒的丢失与小鼠和蜱的感染性和持久性的丧失密切相关。例如，我们和其他人先前已经证明线性质粒（lp）25对于小鼠和蜱的感染是关键的，而线性质粒lp 28 -1对于小鼠中的持久性是唯一需要的。与lp 25和lp 28 -1不同，线性质粒lp 36不经常被B丢失。在体外传代过程中，其在感染周期中的潜在作用先前尚未被研究。我们最近发现了一种罕见的B。已经丢失lp 36但保留已知对毒力重要的所有其他质粒的伯氏螺旋体变体，从而允许研究该质粒在感染循环中的作用（朱厄特et al. 2007）。 测序的B的lp 36质粒。burgdorferi菌株B31是约36 kb的线性质粒，编码54个开放阅读框，其中7个似乎是假基因。lp 36上的大多数基因在数据库中缺乏同源物，并且没有预测的功能。我们发现缺乏lp 36的螺旋体在哺乳动物中不易存活，但在蜱中没有表现出缺陷（朱厄特et al. 2007）。通过在突变体克隆中重建lp 36质粒来恢复小鼠感染，证明感染性缺陷是由lp 36的缺失引起的。此外，我们确定了lp 36的bbk 17基因编码腺嘌呤脱氨酶（AdeC），并且是lp 36上的遗传组分，其足以恢复小鼠对缺乏lp 36的螺旋体的感染性。我们的工作确立了lp 36质粒在感染循环中的关键作用（朱厄特et al.2007）。 B的外膜成分。当螺旋体从蜱媒传播到哺乳动物宿主时，伯氏螺旋体经历了深刻的变化。质粒编码的OspA蛋白在B的表面上是丰富的。OspA在感染蜱的中肠中存在，但当蜱进食时，OspA下调并被OspC取代，OspC在不同的质粒上编码。假设螺旋体表面蛋白的这种转换是B迁移所必需的。从蜱的中肠到唾液腺或在传播后适应哺乳动物宿主。 我们以前测试了这一假设与调查的OspC在蜱载体和哺乳动物宿主的体内作用。我们发现，开关从OspA到OspC是不需要的螺旋体从中肠迁移到蜱的唾液腺，但在准备传输到哺乳动物宿主。由于OspC的基本作用似乎是建立哺乳动物感染，我们最近进一步描述了蛋白质必须存在的关键时期（蒂利等人，2007）。 为了解决这个问题，我们将野生型和ospC突变体螺旋体接种到小鼠皮肤中，并评估在接种后3小时至21天的不同时间重新分离螺旋体的能力。 我们还通过确定何时首次从远端部位分离出细菌来检测螺旋体何时传播。在所有时间点从接种部位分离野生型和互补突变体螺旋体，并在接种后8天首先从远处部位分离。 相比之下，ospC突变体螺旋体从接种部位分离至接种后24小时（此时5/6只测试小鼠为阴性），但此后从未分离。我们的结论是OspC在B中起着至关重要的作用。在宿主传播或血清转化之前，在将螺旋体引入哺乳动物后的前48小时内发生伯氏螺旋体感染（蒂利等人，2007年）。 OspC的早期需求排除了该蛋白在逃避获得性免疫应答中的作用，并表明其参与逃避宿主先天免疫应答。我们的工作模型是OspC抑制B的吞噬作用。这可能是通过限制补体的调理作用，使细菌在传播到哺乳动物宿主后立即逃避清除。 这些研究结果，连同以前的观察，蜱和哺乳动物中的螺旋体基因表达的差异调节，表明宿主适应不仅发生在不同的节肢动物和哺乳动物的环境，但也在每个主机内大致对应的殖民化，持久性和传输的阶段。