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

伯氏疏螺旋体，莱姆病的病原体，在节肢动物媒介和各种哺乳动物宿主中生存和增殖。在其传播/感染周期中，伯氏疏螺旋体会遇到这些宿主特有的环境挑战。其中一个挑战来自活性氧（ROS），如超氧自由基（O2-）、过氧化氢（H2O2）和羟基自由基（OH-），以及活性氮（RNS），如一氧化氮（NO）、二氧化氮（NO2）、三氧化二氮（N2O3）和过氧亚硝酸盐（NO3）。当伯氏疏螺旋体暴露于ROS/RNS时，感染周期分为两个阶段。第一种是在哺乳动物宿主感染的初始阶段，此时免疫系统细胞试图通过几种机制，包括ROS和RNS的产生，来限制和消除伯氏疏螺旋体。令人惊讶的是，第二次ROS/RNS挑战发生在蜱虫进食过程中，并且细菌在传播过程中通过蜱虫唾液腺迁移。对伯氏疏螺旋体生存的另一个挑战来自营养物质有效性和渗透通量的变化。当伯氏疏螺旋体从哺乳动物宿主迁移到肩胛螺旋体时，它们所遇到的渗透压分别从大约300 mOsm增加到650 mOsm。与其他细菌相比，伯氏疏螺旋体具有狭窄的渗透耐受性，并且已被证明可以调节与毒力有关的重要调节途径，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用性也会发生巨大的变化。这些环境压力共同影响伯氏疏螺旋体的生理，并在感染周期所需的重要调节级联反应中发挥作用。

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.