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

伯氏疏螺旋体是莱姆病的病原体，在节肢动物媒介和各种哺乳动物宿主中存活和繁殖。在其传播/感染周期中，伯氏杆菌会遇到特定于这些宿主的环境挑战。一个挑战来自活性氧物种(ROS)，如超氧阴离子自由基(O2-)、过氧化氢(H_2O_2)和羟基自由基(OH-)，以及活性氮物种(RNS)，如一氧化氮(NO)、二氧化氮(NO2)、三氧化二氮(N_2O_3)和过氧亚硝酸盐(NO_3)。当伯氏杆菌暴露于ROS/RNS时，感染周期分为两个阶段。第一个阶段是在哺乳动物宿主感染的初始阶段，免疫系统的细胞试图使用包括产生ROS和RNS在内的几种机制来限制和消灭伯氏杆菌。令人惊讶的是，第二个ROS/RNS挑战发生在扁虱喂食期间，以及细菌在传播过程中通过扁虱唾液腺迁移时。伯氏杆菌生存面临的另一个挑战来自营养供应和渗透通量的变化。随着细菌从哺乳动物宿主迁移到肩袖假单胞菌，伯氏杆菌遇到的渗透压分别从大约300mOsm增加到650mOsm。与其他细菌相比，伯氏杆菌具有较窄的渗透耐受性，已被证明可以调节与毒力有关的重要调控途径，以响应渗透压的变化。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获得性会发生戏剧性的变化。这些环境压力共同影响伯氏杆菌的生理，并在调节感染周期所需的重要调节级联反应中发挥作用。 在2020财年，我们研究了渗透压、营养限制和反应氮物种(RNS)在感染周期中对生存和基因调控的作用。伯氏杆菌必须适应不同的媒介环境和不同的哺乳动物宿主。扁虱的有效定植(获取阶段)需要细菌适应喂食后，扁虱中肠生理(营养限制)，而成功传播(传播阶段)哺乳动物需要细菌感知中肠环境线索并做出反应，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控网络，它们促进了伯氏杆菌的获取和长期生存(HK1-Rrp1)和传播(RRP2-RpoN-rpos)。我们实验室的最新数据显示，由Rrp1产生的c-di-GMP可以刺激Hk2的磷酸酶活性，Hk2是被认为激活RRP2的同源组氨酸激酶。这是一个新的观察结果，我们相信，这种串扰对于协调这两个重要的监管体系至关重要。我们目前正在进行实验，以确定Rrp1、Hk2、RRP2在调节哺乳动物宿主中传播和疾病所需的重要毒力因子以及在扁虱载体中获取和维持所需的重要毒力因子方面的关系。 在相关研究中，我们已经表明，只存在于摄食硬蜱中肠的RNS对伯氏杆菌的长期生存构成了重大挑战。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，从而确保最大限度的生长和长期生存。我们的合作者，克雷顿大学的T.Bourret博士的数据表明，在RNS过程中观察到的生理变化是由转录因子Dks A和信号分子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，这表明了一个涉及代谢物水平变化的新的调控回路。这些数据表明：(1)饥饿和/或渗透压触发的c-di-GMP可能是协调HK1/Rrp1和Hk2/RRP2依赖的调控的重要调节因子；(2)RNS刺激依赖于DksA的基因调控，这对伯氏杆菌在硬蜱中的长期生存是必不可少的。我们将继续进行实验，研究饥饿和RNS对伯氏杆菌基因调控的作用。 最后，我们正在研究精氨酸脱亚胺系统(ADS)在伯氏杆菌感染循环中的作用。正在进行的实验表明，ADS有助于维持伯氏杆菌的胞内pH。对细菌细胞内pH的扰动会导致一般的应激反应，导致rpos-RpoN调节级联反应的结构性激活。与广告相关的酶产生瓜氨酸、鸟氨酸和氨，每一种都有独特的细胞命运。目前正在进行研究，以确定B.burgdorferi ADS在存活酸胁迫中的作用。此外，我们正在研究伯氏杆菌精氨酸/鸟氨酸在感染周期中的作用，以确定这些代谢物如何从宿主中隔离出来，从而促进宿主和媒介的定植。

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. In FY 2020, 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.