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

关键转录因子在莱姆病病原体伯氏疏螺旋体发病机制中的作用

• 批准号: 10692075
• 负责人: Frank Gherardini
• 金额: $ 98.41万
• 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/10692075
• 关键词: Affect; Ammonia; Arginine; Arginine deiminase; Arthropod Vectors; Bacteria; Bacteria sigma factor KatF protein; Base Excision Repairs; Biochemical; Black-legged Tick; Borrelia burgdorferi; Calendar; Cells; Chemistry; Citrulline; Cues; DNA; DNA Damage; DNA Repair; Data; Deamination; Disease; Ensure; Environment; Enzymes; Excision Repair; Exposure to; Gene Expression Regulation; Growth; Hydrogen Peroxide; Hydroxyl Radical; Immune system; Infection; Investigation; Laboratories; Lactic acid; Lead; Link; Lyme Disease; Maintenance; Mammals; Mediating; Metabolism; Metalloproteins; Midgut; NBL1 gene; Nitric Oxide; Nitrogen Dioxide; Nucleotide Excision Repair; Nutrient; Order Spirochaetales; Ornithine; Osmolar Concentration; OspC protein; Oxidative Stress; Pathogenesis; Pathway interactions; Peroxonitrite; Phase; Phosphoric Monoester Hydrolases; Physiological; Physiology; Play; Production; Proliferating; Proteins; Reaction; Reactive Nitrogen Species; Reactive Oxygen Species; Regulation; Regulatory Pathway; Role; Salivary Glands; Signal Pathway; Signal Transduction; Signaling Molecule; Starvation; Superoxides; System; Ticks; Universities; Virulence; Virulence Factors; Zinc; acid stress; base; biological adaptation to stress; environmental stressor; experience; experimental study; feeding; mutant; nitrogen trioxide; nitrosative stress; novel; organic acid; protein-histidine kinase; response; subcellular targeting; tick feeding; transcription factor; transmission process; uptake; vector; vector tick

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 2022, 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.

莱姆病的药物Borrelia burgdorferi在节肢动物载体和各种哺乳动物宿主中均能生存和增殖。在其传播/感染周期中，B。Burgdorferi遇到了特定于这些宿主的环境挑战。一个挑战来自活性氧（ROS），例如超氧化物自由基（O2-），过氧化氢（H2O2）和羟基自由基（OH-）和反应性氮种（RNS），例如一氧化氮（NO），二氧化氮（NO2），三氧化氮（N2O3）和过氧亚硝酸盐（NO3）。在感染周期中，B. burgdorferi暴露于ROS/RN时，有两个阶段。第一个是在免疫系统的细胞试图使用多种机制（包括ROS和RNS的产生）限制和消除B. burgdorferi时，在哺乳动物宿主感染的初始阶段。出乎意料的是，第二次ROS/RNS挑战发生在滴答作用期间，并且随着细菌在传播过程中通过滴答唾液腺迁移。 B. burgdorferi生存的另一个挑战是营养物的可用性和渗透通量的变化。当细菌分别从哺乳动物宿主迁移到肩cap骨时，B. burgdorferi遇到的渗透压从大约300 MOSM增加到650 MOSM。 B. burgdorferi与其他细菌相比具有狭窄的渗透压，并且已被证明可以调节涉及毒力的重要调节途径，以响应变化的渗透压。此外，细菌在这些不同的宿主之间移动时，养分浓度和可用性会急剧转移。这些环境应力共同影响了B. burgdorferi生理，并在调节传染性周期所需的重要调节级联反应中发挥作用。 在2022财年，我们研究了渗透压，营养限制和反应性氮（RN）在感染周期期间的生存和基因调节中的作用。 B. Burgdorferi必须适应其tick矢量和各种哺乳动物宿主中明显不同的环境。 tick的有效定植（采集阶段）需要细菌适应喂食后，tick中肠生理（营养限制），而成功的传播（传播阶段）向哺乳动物进行了传播（传播阶段），需要细菌感知并对中肠环境线索进行感知并响应中肠环境线索，并在向新宿主传播之前（反应对RNS）上调关键因素。值得注意的是，这些相对较小的变化影响了两个独立的监管网络，这些网络促进了B. burgdorferi的促进获得和长期生存（HK1-RRP1）以及传播（RRP2-RPON-RPOS）。我们实验室的最新数据表明，由RRP1产生的C-DI-GMP刺激了HK2的磷酸酶活性，HK2的磷酸酶活性，即被认为会激活RRP2的同源组氨酸激酶。这是一个新颖的观察结果，我们认为这种串扰对于协调这两个基本调节系统至关重要。目前，我们正在进行实验，以定义RRP1，HK2，RRP2之间的关系，以调节哺乳动物宿主传播和疾病所需的重要毒力因子以及tick载体中的获取和维持。 在相关研究中，我们表明，仅在喂食壁虱的中肠中存在的RN对B. burgdorferi的长期生存提出了重大挑战。 RNS介导的损伤刺激了核苷酸切除修复（NER），碱基切除修复（BER）和不匹配切除修复（MER）系统，可确保最大的生长和长期存活。我们合作者Creighton University的T. Bourret博士的数据表明，在RN期间观察到的生理变化是由转录因子DKSA以及信号分子PPGPPP介导的（由Rela合成）。有趣的是，C-DI-GMP和PPGPPP的产生都受营养水平的影响，表明一种新型的调节环，涉及变化的代谢物水平。这些数据表明； （1）由饥饿和/或渗透压触发的C-DI-GMP可能是协调HK1/RRP1和HK2/RRP2依赖性调节的重要调节调节剂，（2）RNS刺激DKSA依赖性基因调节，这对于B. Burggdorferferferferferferferferferferferferferferferferferferferferferferferferferferferferferferferferfefi in ticks in tick in tick in tick in tick in tick in tick in tick in tick。我们将继续进行实验，以研究饥饿和RN在伯格多菲利芽孢杆菌中基因调节的作用。 最后，我们正在研究精氨酸脱节酶系统（ADS）在B. burgdorferi感染周期中的作用。正在进行的实验表明，ADS有助于维持B. burgdorferi的细胞内pH值。细菌内pH的扰动导致一般应力反应，从而导致RPOS-RPON调节级联反应。与AD相关的酶产生瓜氨酸，鸟氨酸和氨，每种都有独特的细胞命运。目前正在进行调查，以表征B. burgdorferi广告在存活的酸应激中的作用。此外，我们正在检查在传染性周期中伯格多菲利（Burgdorferi）精氨酸/鸟氨酸利用率的作用，以确定如何从宿主中隔离这些代谢产物，从而促进宿主和载体定殖。