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: 10014092
• 负责人: Frank Gherardini
• 金额: $ 113.17万
• 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/10014092
• 关键词: Acetates; Acetyl Coenzyme A; Acetylation; Affect; Anabolism; Arginine; Arthropod Vectors; Bacteria; Bacteria sigma factor KatF protein; Base Excision Repairs; Biochemical; Biological Assay; Biological Process; Blood; Borrelia burgdorferi; Calendar; Cell physiology; Cells; Chemistry; Coenzyme A; Cues; DNA; DNA Damage; DNA Repair; Data; Deacetylase; Deacetylation; Deamination; Ensure; Environment; Enzymes; Excision Repair; Exposure to; Future; Gene Expression; Gene Expression Regulation; Genetic Transcription; Genome; Growth; Hydrogen Peroxide; Hydroxyl Radical; Immune system; Infection; Laboratories; Lactic acid; Lyme Disease; Lysine; Mammals; Mass Spectrum Analysis; Mediating; Metabolic; Metabolism; Metalloproteins; Midgut; NBL1 gene; Nitric Oxide; Nitrogen Dioxide; Nucleotide Excision Repair; Nutrient; Order Spirochaetales; Osmolar Concentration; OspC protein; Oxidative Stress; Pathogenesis; Pathway interactions; Peroxonitrite; Phase; Phosphoric Monoester Hydrolases; Physiology; Play; Postdoctoral Fellow; Production; Proliferating; Protein Acetylation; Proteins; Proteomics; Reaction; Reactive Nitrogen Species; Reactive Oxygen Species; Regulation; Regulatory Pathway; Reporting; Role; Salivary Glands; Signal Transduction; Site; Starvation; Superoxides; System; Ticks; Translations; Universities; Virulence Factors; Work; Zinc; acid stress; base; biological adaptation to stress; experimental study; feeding; information processing; mutant; nitrogen trioxide; nitrosative stress; organic acid; protein B; response; subcellular targeting; transcription factor; transmission process; vector tick

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).

莱姆病的药物A. 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。 在2019财年，我们确定了营养限制和反应性氮（RN）在感染周期期间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）。赖氨酸乙酰化在营养限制期间是tick中肠饥饿的信号和翻译后调节反应。最重要的是，我们实验室的最新数据表明，由RRP1产生的Dicyclic-GMP刺激了HK2的磷酸酶活性。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。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。 Creighton University的T. Bourrets实验室的数据表明，对RNS的反应是由DKSA和PPGPPP介导的（由RERA合成）。这些数据表明； （1）由饥饿触发的Dicyclic GMP可能是一个重要的调节调节器，它可以协调HK1/RRP1和HK2/RRP2依赖性调节，并且（2）RNS刺激DKSA依赖性基因调节，对B. Burgdorferi的长期生存至关重要，这对于B. Burgdorferi的长期生存至关重要。 在GRC博士后研究员S. Bontemps-Gallo博士领导的2019财年平行研究中，我们确定了影响基因表达和长期生存的环境状况：营养限制（1）。我们的研究确定了B. burgdorferi中的营养限制/固定相是触发赖氨酸乙酰化的关键因素。使用高度敏感的质谱基蛋白质组学方法，我们表征了B. burgdorferi的乙酰基团。如先前报道的其他细菌，乙酰基乙酰化的相对较少的潜在基因组编码蛋白的数量相对较少（5％）。其中，绝大多数参与中央代谢和细胞信息处理（转录，翻译等）。有趣的是，这些关键细胞功能在中期和固定阶段的生长阶段都是针对的。然而，在固定相期间，在多个位点，在多个位点对这些相同的蛋白质进行了乙酰蛋白的乙酰化限于单赖氨酸残基，而这些相同的蛋白质被乙酰化。为了确定B. burgdorferi中的乙酰供体，我们使用了缺乏乙酸代谢的突变体。 B. burgdorferi菌株B31-A3，B31-A3 ACKA（乙酰-P-和乙酰-COA-）和B31-A3 PTA（乙酰-P+和乙酰-COA-）被种植至固定相，并分析乙酰化谱。虽然在ACKA突变体中仅使用2种蛋白质，但在PTA突变体中乙酰化了140个蛋白质，这表明乙酰-P是B. burgdorferi中的主要乙酰基供体。使用特定的酶试验，我们能够证明固定期蛋白质的高乙酰化似乎在降低大多数糖酵解蛋白的酶活性中起作用。目前，我们假设乙酰化用于在血液粉中tick虫中肠中长期生存期间，将乙酰化用于灭活酶。 该策略将使细菌能够通过脱乙酰化激活关键的糖酵解酶，而不是消耗过多的能量合成新蛋白质。对于具有有限代谢能力的细菌，这将是一种吸引人的低能策略。未来的工作着重于确定潜在的蛋白质脱乙酰基酶，以完成我们对这一重要生物学过程的理解（1）。