The Roles of Key Transcription Factors on the Pathogenesis of B. burgdorferi, the Causative Agent of Lyme Disease

莱姆病的病原体伯氏疏螺旋体在节肢动物载体和各种哺乳动物宿主中存活和增殖。在其传播/感染周期中，B. burgdorferi遇到了这些宿主特有的环境挑战。一个挑战来自活性氧物质（ROS），例如超氧化物自由基（O2-）、过氧化氢（H2 O2）和羟基自由基（OH-），以及活性氮物质（RNS），例如一氧化氮（NO）、二氧化氮（NO2）、三氧化二氮（N2 O3）和过氧亚硝酸根（NO3）。B. burgdorferi暴露于ROS/RNS。第一种是在哺乳动物宿主感染的初始阶段，此时免疫系统的细胞试图限制和消除B。利用包括ROS和RNS的产生在内的几种机制来抑制burgdorferi。令人惊讶的是，第二次ROS/RNS攻击发生在蜱喂食期间，并且在传播期间细菌通过蜱唾液腺迁移。对B的另一个挑战。burgdorferi存活来自于营养物质可利用性和渗透通量的变化。B.当细菌从哺乳动物宿主迁移到I.肩胛肌。B。与其它细菌相比，伯氏菌具有窄的耐药性，并且已经显示出响应于改变的渗透压来调节涉及毒力的重要调节途径。此外，当细菌在这些不同的宿主之间移动时，它们的营养浓度和可用性会发生巨大的变化。这些环境压力共同影响B。Burgdorferi生理学，并在感染周期所需的重要调节级联的调节中发挥作用。 我们研究了渗透压，营养限制和活性氮（RNS）在感染周期中的生存和基因调控的作用。B。burgdorferi必须适应其蜱媒介和各种哺乳动物宿主中明显不同的环境。蜱虫的有效定殖（获取阶段）需要细菌适应进食后蜱虫中肠生理学（营养限制），而成功传播（传播阶段）到哺乳动物需要细菌感知和响应中肠环境线索，并在传播到新宿主之前上调关键毒力因子（对RNS的反应）。值得注意的是，这些相对较小的变化影响了两个独立的调节网络，促进B的获得和长期生存（Hk 1-Rrp 1）以及传播（Rrp 2-RpoN-RpoS）。burgdorferi。我们实验室的最新数据显示，由Rrp 1产生的c-di-GMP刺激Hk 2的磷酸酶活性，Hk 2是被认为激活Rrp 2的同源组氨酸激酶。这是一个新颖的观察结果，我们相信这种串扰对于协调这两个重要的监管系统至关重要。我们目前正在进行实验，以确定Rrp 1，Hk 2，Rrp 2之间的关系，在调节重要的毒力因子所需的传播和疾病的哺乳动物宿主，以及收购和维护的蜱载体。 在相关研究中，我们已经表明，RNS仅存在于喂食蜱的中肠中，对B的长期存活提出了重大挑战。burgdorferi。由RNS介导的损伤刺激核苷酸切除修复（NER）、碱基切除修复（BER）和错配切除修复（MER）系统，从而确保最大的生长和长期存活。数据来自我们的合作者，T博士。Creighton大学的Bourret提出，在RNS期间观察到的生理变化是由转录因子DksA以及信号分子ppGppp（由RelA合成）介导的。有趣的是，c-di-GMP和ppGppp的产生都受到营养水平的影响，这表明了一种涉及改变代谢物水平的新的调节回路。这些数据表明，（1）c-di-GMP，由饥饿和/或渗透压触发，可能是协调Hk 1/Rrp 1和Hk 2/Rrp 2依赖性调节的重要调节因子，（2）RNS刺激DksA依赖性基因调节，这对B的长期存活至关重要。蜱虫中的伯氏菌。我们将继续进行实验，研究饥饿和RNS对B基因调控的作用。burgdorferi。 最后，我们正在研究精氨酸脱亚胺酶系统（ADS）在B中的作用。伯氏菌感染循环正在进行的实验表明，ADS有助于维持B的细胞内pH。burgdorferi。细菌细胞内pH的扰动导致一般应激反应，引起RpoS-RpoN调节级联的组成性激活。与ADS相关的酶产生瓜氨酸、鸟氨酸和氨，每种酶都具有独特的细胞命运。目前正在进行调查，以确定B的作用。burgdorferi ADS在酸胁迫下的存活率。此外，我们正在研究B的作用。本发明的目的在于确定在感染周期中伯氏菌精氨酸/鸟氨酸的利用，以确定这些代谢物如何从宿主隔离，从而促进宿主和载体定殖。 注：主要研究者已于2022年12月退休，该项目将被终止/停用。

Borrelia burgdorferi, the agent of Lyme disease, survives and proliferates in both an arthropod vector and various mammalian hosts. During its transmission/infective cycle, B. burgdorferi encounters environmental challenges specific to those hosts. One challenge comes from reactive oxygen species (ROS) e.g. superoxide radicals (O2-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH-) and reactive nitrogen species (RNS) e.g. nitric oxide (NO), nitrogen dioxide (NO2), nitrogen trioxide (N2O3) and peroxynitrite (NO3). There are two stages in the infective cycle when B. burgdorferi is exposed to ROS/RNS. The first is during the initial stages of infection of the mammalian host when cells of the immune system attempt to limit and eliminate B. burgdorferi using several mechanisms including the production of ROS and RNS. Surprisingly, the second ROS/RNS challenge occurs during tick feeding and as the bacteria migrate through the tick salivary glands during transmission. Another challenge to B. burgdorferi survival comes from changes in nutrient availability and osmotic fluxes. The osmolarity that B. burgdorferi encounters increases from approximately 300 mOsm to 650 mOsm as the bacteria migrate from the mammalian host to I. scapularis, respectively. B. burgdorferi has a narrow osmotolerance compared to other bacteria and has been shown to modulate important regulatory pathways involved in virulence, in response to changing osmolarity. In addition, the bacteria experience dramatic shifts in nutrient concentration and availability as they move between these disparate hosts. Together these environmental stresses affect B. burgdorferi physiology and play a role in the modulation of important regulatory cascades required for the infectious cycle. We investigated the roles of osmolarity, nutrient limitation and reactive nitrogen species (RNS) in survival and gene regulation during the infective cycle. B. burgdorferi must adapt to distinctly different environments in its tick vector and various mammalian hosts. Effective colonization (acquisition phase) of a tick requires the bacteria to adapt to post feeding, tick midgut physiology (nutrient limitation) while successful transmission (transmission phase) to a mammal requires the bacteria to sense and respond to the midgut environmental cues and up-regulate key virulence factors before transmission to a new host (reaction to RNS). Remarkably, these relatively small changes affect two independent regulatory networks that promote acquisition and long-term survival (Hk1-Rrp1) as well as transmission (Rrp2-RpoN-RpoS) of B. burgdorferi. Recent data from our laboratory shows that c-di-GMP, produced by Rrp1, stimulates the phosphatase activity of Hk2, the cognate histidine kinase thought to activate Rrp2. This is a novel observation and we believe this cross-talk is essential for coordinating these two essential regulatory systems. We are currently conducting experiments to define the relationship between Rrp1, Hk2, Rrp2 in modulating important virulence factors required for transmission and disease in the mammalian host as well as for acquisition and maintenance in the tick vector. In related studies, we have shown that RNS that are only present in the midgut of feeding ticks, presents a significant challenge to long-term survival of B. burgdorferi. The damage mediated by RNS stimulates the nucleotide excision repair (NER), base excision repair (BER) and mismatch excision repair (MER) systems which ensures maximum growth and long-term survival. Data from our collaborator, Dr. T. Bourret at Creighton University, suggests that the physiological changes observed during RNS are mediated by the transcription factor, DksA, as well as the signalling molecule, ppGppp (synthesized by RelA). Interestingly, the production of c-di-GMP and ppGppp are both affected by nutrient levels suggesting a novel regulatory loop involving changing metabolite levels. These data suggest that; (1) c-di-GMP, triggered by starvation and/or osmolarity, might be an important regulatory modulator that coordinates Hk1/Rrp1 and Hk2/Rrp2-dependent regulation, and (2) RNS stimulates DksA-dependent gene regulation that is essential for the long-term survival of B. burgdorferi in ticks. We will continue to conduct experiments that investigate the role of starvation and RNS on gene regulation in B. burgdorferi. Finally, we are investigating the role of the arginine deiminase system (ADS) in the B. burgdorferi infectious cycle. Ongoing experiments have shown that the ADS contributes to the maintenance of the intracellular pH of B. burgdorferi. Perturbations to the bacterial intracellular pH lead to a general stress response, causing constitutive activation of the RpoS-RpoN regulatory cascade. The enzymes associated with the ADS generate citrulline, ornithine, and ammonia, each with a unique cellular fate. Investigations are currently underway to characterize the role of the B. burgdorferi ADS in surviving acid stress. In addition, we are examining the role of B. burgdorferi arginine/ornithine utilization during the infectious cycle to determine how these metabolites might be sequestered from the host thereby promoting host and vector colonization. NOTE: The Principal Investigator retired in December 2022 and this project will be terminated/inactivated.