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。 在2019财年，我们确定了营养限制和活性氮物种(RNS)在感染周期中的生存和基因调控中的作用。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)。在营养限制期间，赖氨酸乙酰化作为一种信号和翻译后的调节反应，在硬体中肠对饥饿做出反应。最重要的是，我们实验室的最新数据表明，由Rrp1产生的二环GMP可以刺激Hk2的磷酸酶活性。我们认为，这种串扰对于协调这两个必不可少的监管体系至关重要。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。克雷顿大学T.Bourrets博士实验室的数据表明，对RNS的反应是由DksA和ppGppp(由relA合成)介导的。这些数据表明：(1)饥饿触发的二环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(2)。 在由GRC博士后S.Bontemps-Gallo博士领导的2019财年平行研究中，我们确定了一种影响基因表达和长期生存的环境条件：营养限制(1)。我们的研究发现，伯氏杆菌的营养限制/静止期是触发赖氨酸乙酰化的关键因素。利用基于高灵敏质谱学的蛋白质组学方法，我们对伯氏杆菌的乙酰组进行了研究。与之前报道的其他细菌一样，伯氏杆菌的潜在基因组编码蛋白中有相对较少的数量(5%)被乙酰化。其中，绝大多数涉及中枢新陈代谢和细胞信息处理(转录、翻译等)。有趣的是，这些关键的细胞功能都是在对数生长中期和生长稳定期的目标。然而，目标蛋白在对数中期的乙酰化仅限于单一的赖氨酸残基，而这些相同的蛋白在固定相中被多个位置乙酰化。为了确定伯氏杆菌中的乙酰供体，我们使用了缺乏醋酸盐合成代谢的突变株。将伯氏杆菌菌株B31-A3、B31-A3ackA(乙酰基-P-和乙酰辅酶A-)和B31-A3PTA(乙酰基-P+和乙酰辅酶A-)培养到固定相，并对乙酰化谱进行了分析。在ackA突变体中只有2个蛋白质被乙酰化，而在pta突变体中有140个蛋白质被乙酰化，这表明乙酰基P是伯氏杆菌的主要乙酰基供体。使用特定的酶分析，我们能够证明蛋白质在固定相的过度乙酰化似乎在降低大多数糖酵解蛋白质的酶活性中起作用。目前，我们假设乙酰化被用来灭活细菌在血餐之间在扁虱中肠的长期生存过程中的酶。这一策略将允许细菌通过去乙酰化激活关键的糖酵解酶，而不是花费过多的能量合成新的蛋白质。对于新陈代谢能力有限的细菌来说，这将是一个有吸引力的低能量策略。未来的工作重点是寻找潜在的蛋白质脱乙酰酶(S)，以完成我们对这一重要生物学过程的理解(1)。

A. 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. In FY 2019, we determined the roles of 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. Lysine acetylation serves as a signal and post-translational regulatory response to starvation in the tick midgut during nutrient limitation. Most importantly, recent data from our laboratory shows that dicyclic-GMP, produced by Rrp1, stimulates the phosphatase activity of Hk2. We believe this crosstalk is essential for coordinating these two essential regulatory systems. 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 Dr. T. Bourrets laboratory at Creighton University suggests that the response to RNS is mediated by DksA and ppGppp (synthesized by RelA). These data suggest that; (1) dicyclic GMP, triggered by starvation, 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 (2). In a FY 2019 parallel study led by GRC post-doctoral fellow, Dr. S. Bontemps-Gallo, we identified an environmental condition that affects gene expression and long-term survival: nutrient limitation (1). Our study identified nutrient limitation/stationary phase in B. burgdorferi as a critical factor that triggers lysine acetylation. Using a highly sensitive mass spectrometry-based proteomics approach, we characterized the acetylome of B. burgdorferi. As previously reported for other bacteria, a relatively low number (5%) of the potential genome-encoded proteins of B. burgdorferi were acetylated. Of these, the vast majority were involved in central metabolism and cellular information processing (transcription, translation, etc.). Interestingly, these critical cell functions were targeted during both mid-log and stationary phases of growth. However, acetylation of target proteins in mid-log phase was limited to single lysine residues while these same proteins were acetylated at multiple sites during stationary phase. To determine the acetyl donor in B. burgdorferi, we used mutants deficient in acetate anabolism. B. burgdorferi strains B31-A3, B31-A3 ackA (acetyl-P- and acetyl-CoA-) and B31-A3 pta (acetyl-P+ and acetyl-CoA-) were grown to stationary phase and the acetylation profiles were analyzed. While only 2 proteins were acetylated in the ackA mutant, 140 proteins were acetylated in the pta mutant suggesting that acetyl-P was the primary acetyl donor in B. burgdorferi. Using specific enzymatic assays, we were able to demonstrate that hyperacetylation of proteins in stationary phase appeared to play a role in decreasing the enzymatic activity of most glycolytic proteins. Currently, we hypothesize that acetylation is used to inactivate enzymes during long-term survival of the bacteria in the tick midgut between blood meals. This strategy would allow the bacteria to activate key glycolytic enzymes by deacetylation rather than expending excessive energy synthesizing new proteins. This would be an appealing, low-energy strategy for a bacterium with limited metabolic capabilities. Future work focuses on identifying potential protein deacetylase(s) to complete our understanding of this important biological process (1).