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。 2017财年，与T.Bourret博士(系微电子。和免疫学)，我们证明在摄食过程中肩部硬蜱的唾液腺和中肠含有显著水平的ROS和RNS[1]。我们比较了含有DNA修复基因突变的伯氏杆菌菌株在瑞士韦氏小鼠和我肩虱中完成感染循环的能力，这些基因突变对ROS或RNS的杀死高度敏感。我们发现，甲基导向错配修复(MMR)基因muS1和核苷酸切除修复(NER)基因uvrB对于小鼠的感染是必不可少的，而uvrB促进了我肩蜱螺旋体的存活。UvrB缺陷伯氏杆菌存活率的下降与摄食过程中肩部、中肠和唾液腺中RNS的产生有关。总而言之，这些数据表明伯氏假单胞菌相当于在感染我肩部硬蜱过程中产生的RNS的细胞毒性水平(1)。 B.burgdorferi的适应能力和在非常不同的环境中生存的能力(扁虱和哺乳动物)归因于它能够感知温度、pH、细胞密度、氧气、锰和/或接触宿主因素的变化，并相应地改变基因表达。以往的报道表明，调节这些反应的中心是西格玛因子，即RpoN和rpos。重要的是，RpoN依赖的rpos调节负责在感染周期中感染和传播所需的关键毒力因子(如OspC、OspA和DbpA)的表达。RpoN的活性受到严格控制，需要依赖于ATP的激活。RpoN-RNA聚合酶(RNAP)的许多激活剂是双组分调控系统的反应调节器(RR)，这些RR的磷酸化导致它们的激活。这些受体被小分子磷酸供体或更常见的同源蛋白组氨酸激酶(HK)磷酸化，以响应环境信号。B.burgdorferi中RpoN的激活子RRP2由bb0763(RRP2)编码，也是一个双组分系统的RR，RRP2是一个操纵子，带有一个编码其同源蛋白组氨酸激酶的基因，Hk2由bb0764(HK2)编码。这些调控成分形成了RRP2-RpoN-rpos信号级联反应，协调了伯氏假单胞菌从节肢动物载体向哺乳动物宿主成功转移所需的毒力因子的表达。RRP2的激活是启动这一调控途径所必需的。触发RRP2激活的细胞内信号和磷酸化过程知之甚少。已发表的报告表明，乙酰磷酸(ACP)是许多细菌中的一种全球调节分子，是RRP2激活的信号和高能磷酸供体。 在GRC工作人员科学家D.Dulebohn博士领导的2017财年研究中，我们确定了一种影响基因表达和长期生存的环境条件：酸应激(2)。我们的研究在伯氏杆菌中发现了一种酸胁迫反应，它激活了RRP2-RpoN-rpos信号转导级联反应，一些BosR指导的氧化应激反应基因，并增加了参与恢复pH动态平衡的编码蛋白的基因表达。这些基因的转录增加是由膜透性的单羧酸刺激的，其中一些(醋酸盐和乳酸)在摄食后的硬蜱中肠中被鉴定出来。此外，我们现在已经证明，pH动态平衡的扰动激活了以前被认为是乙酰-P的RRP2-RpoN-rpos/BosR调节级联(S)。我们没有确定最终上调这些途径的细胞内信号，然而，phi降低对液泡ATPase的影响可能表明ATP或GTP可能作为有效的细胞内能量感受器。虽然酸胁迫反应和phi在体外似乎明显影响伯氏杆菌毒力因子的表达，但这些参数似乎不太可能影响体内传播。由于毒力因子的表达，如OspC，与rpos紧密相连，在体外触发RRP2-RpoN-rpos和BosR依赖的基因调控的条件并不一定表明这些条件是成功传播所必需的，也不一定在成功传播中发挥作用。考虑到蜱中肠的复杂性，似乎需要多种因素才能最大限度地使毒力因子的表达与蜱的饲养周期同步，以促进成功的传播(2)。

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 2017, in collaboration with Dr. T. Bourret (Dept. of Micro. and Immunol., Cheighton Medical School), we demonstrated that the salivary glands and midgut of Ixodes scapularis contained significant levels of ROS and RNS during feeding (1). We compared the ability of B. burgdorferi strains harboring mutations in DNA repair genes that are hypersensitive to killing by ROS or RNS to complete their infectious cycle in Swiss Webster mice and I. scapularis ticks. We showed that the methyl-directed mismatch repair (MMR) gene mutS1 and the nucleotide excision repair (NER) gene uvrB are dispensable for infection of mice, while uvrB promotes the survival of spirochetes in I. scapularis ticks. The decreased survival of uvrB-deficient B. burgdorferi was associated with the generation of RNS in I. scapularis midguts and salivary glands during feeding. Collectively, these data suggested that B. burgdorferi ecounters cytotoxic levels of RNS produced during infection of I. scapularis ticks (1). B. B. burgdorferi's ability to adapt and survive in very different environments (tick versus mammal) is attributed to its ability to sense changes in temperature, pH, cell density, oxygen, manganese and/or exposure to host factors and alter gene expression accordingly. Previous reports have demonstrated that central to the regulation of these responses are the sigma factors, RpoN and RpoS. Importantly, RpoN-dependent regulation of RpoS is responsible for the expression of key virulence factors (e.g., OspC, OspA and DbpA) required for infectivity and transmission during the infective cycle. The activities of RpoN are tightly controlled and require ATP-dependent activation. Many of the activators of RpoN-RNA polymerase (RNAP) are response regulators (RR) of two-component regulatory systems and phosphorylation of these RR results in their activation. These RRs are phosphorylated by small molecular weight phosphate donors or, more commonly, by their cognate protein histidine kinase (HK) in response to an environmental signal. The activator of RpoN in B. burgdorferi, Rrp2 encoded by bb0763 (rrp2), is also a RR of a two-component system and rrp2 is in an operon with a gene encoding its cognate protein histidine kinase, Hk2 encoded by bb0764 (hk2). These regulatory components form the Rrp2-RpoN-RpoS signaling cascade that coordinates the expression of virulence factors required for successful transition of B. burgdorferi from its arthropod vector to mammalian hosts. Activation of Rrp2 is essential to initiate this regulatory pathway. The intracellular signal and phosphorylation processes triggering Rrp2 activation are poorly understood. Published reports have suggested that acetyl-phosphate (AcP), a global regulatory molecule in many bacteria, serves as a signal and high energy phosphate donor for Rrp2 activation. In a FY 2017 study led by GRC staff scientist, Dr. D. Dulebohn, we identified an environmental condition that affects gene expression and long-term survival: acid stress (2). Our study identified an acid stress response in B. burgdorferi that activated the Rrp2-RpoN-RpoS signal transduction cascade, some BosR-directed oxidative stress response genes, and increased the expression of genes encoding proteins involved in restoring pH homeostasis. An increase in the transcription of these genes was stimulated by membrane-permeable monocarboxylic acids, some of which (acetate and lactate) were identified in the tick midgut following feeding. Moreover, we have now demonstrated that perturbations in pH homeostasis activated the Rrp2-RpoN-RpoS/BosR regulatory cascade(s) that was previously attributed to Acetyl-P. We did not identify the intracellular signal that ultimately up-regulates these pathways, however, the effects of the decrease in pHi on the vacuolar ATPase might suggest that ATP or GTP may serve as effective intracellular energy sensors. While it seemed clear that the acid stress response and pHi effected virulence factor expression in B. burgdorferi in vitro, it seemed unlikely that these parameters affect transmission in vivo. Since the expression of virulence factors, like OspC, are so tightly linked to RpoS, in vitro conditions that trigger Rrp2-RpoN-RpoS and BosR-dependent gene regulation don't necessarily indicate that these conditions are required for, or play a role in, successful transmission. Considering the complexity of the tick midgut, it seems likely that multiple factors are required to maximize and synchronize the expression of virulence factors to the tick feeding cycle to promote successful transmission (2).