Targeting extracellular tRNA-derived RNA fragments (tRFs) to protect against fatal rickettsiosis

靶向细胞外 tRNA 衍生的 RNA 片段 (tRF) 以预防致命的立克次体病

• 批准号: 10042688
• 负责人: Bin Gong
• 金额: $ 23.7万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-23 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10042688
• 关键词: Adherens Junction; Antibiotic Therapy; Arbovirus Infections; Atomic Force Microscopy; Bacteria; Binding; Biochemical; Biomechanics; Bovine Serum Albumin; Brain; Cells; Development; Early Diagnosis; Endothelial Cells; Endothelium; Environment; Exhibits; Exposure to; Formulation; Functional disorder; Goals; Human; Infection; Lateral; Lead; Liquid substance; Measurement; Measures; Mediating; Molecular; Molecular Sieve Chromatography; Morbidity - disease rate; Mus; Oligonucleotides; Outcome; Pathogenesis; Plasma; Proteins; RNA; Reporting; Ribonucleases; Rickettsia; Rickettsia Infections; Signs and Symptoms; Structure; Technology; Testing; Therapeutic; Tight Junctions; Transfection; Transfer RNA; Umbilical vein; Vaccines; Vascular Endothelial Cell; brain endothelial cell; clinical Diagnosis; exosome; experimental study; extracellular; in vivo; insight; mortality; nanoparticle; novel; overexpression; prophylactic; spotted fever; uptake

Rickettsioses represent devastating human infections. These arthropod-borne diseases are caused by obligatory intracellular bacteria of the genus Rickettsia (R.). A vaccine is not available for rickettsioses. Disseminated vascular endothelial cell (EC) infection and EC barrier dysfunction are the central pathophysiologic features of human lethal spotted fever group rickettsial (SFGR) infections. Typically, infection with SFGR is controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early. Nevertheless, SFGR infections present with nonspecific signs and symptoms rendering early clinical diagnosis difficult. Untreated or misdiagnosed SFGR infections are frequently associated with severe morbidity and mortality. Comprehensive understanding of rickettsial pathogenesis is urgently needed for the development of novel prophylactics and post-infection (p.i.) therapeutics. We reported that, upon SFGR, RNase-mediated tRNA cleavage occurs and a specific subset of tRNA-derived RNA fragments (tRFs) are induced. Among them, 5'-end fragment from tRNAGlyGCC is the most prominently induced tRF, termed as tRFGlyGCC. We found that tRFGlyGCC exhibits trans-silencing activity in a sequence-specific manner and induces EC barrier dysfunction. Several lines of new evidence from our preliminary studies suggest that Exos derived from R. parkeri-infected human umbilical vein EC (HUVEC) (RCExos) at 72hr p.i. or Exos derived from plasma of 2LD50 R. parkeri-nfected mice (RMExos) on day 4 p.i. can induce dysfunction of normal recipient human brain microvascular ECs (BMECs) in a tRFGlyGCC-dependent manner. Compared with naked format, bovine serum albumin-nanoparticlized anti- tRFGlyGCC oligonucleotides (BSAanti-tRFGlyGCCs) in normal media with sera can maintain BMEC barrier functions after exposure to RCExos. These findings suggest that RCExos/RMExos-packed tRFGlyGCC may induce normal recipient EN dysfunction during SFGRs. Our goal in this proposal is to seek more experimental evidence to support our central hypothesis that targeting identified SFGR-induced tRFGlyGCC in exosomes can provide protection against SFGR by maintaining recipient EC barrier function. To test this hypothesis, we will pursue three Specific Aims: (1) biochemically corroborate that RCExos/RMExos-packed tRFGlyGCC alters the recipient EC barrier structure(s), (2) biomechanically corroborate that RCExos/RMExos-packed tRFGlyGCC causes the recipient EC barrier dysfunction, and (3) evaluate whether targeting Exos-packed tRFGlyGCC with anti-tRFGlyGCC nanoparticles can protect against lethal rickettsial infection by maintain the endothelial barrier function. We will test our hypothesis by employing cutting-edge approaches (FluidFM technology, size-exclusion chromatography, and formulation of nanoparticles for optimizing delivery of anti-tRFGlyGCC into ECs). Outcomes will provide deeper insights into the biomechanical and molecular mechanisms of SFGR infection, ultimately leading to the identification of a druggable host-dependent factor during lethal SFGR infections.

立克索氏症代表了毁灭性的人类感染。这些节肢动物传播的疾病是由 里克西亚属的强制性细胞内细菌（R.）。可用于立克的疫苗不可用。 传播血管内皮细胞（EC）感染和EC屏障功能障碍是中枢性病理生理学 人致死性斑点发烧组立克（SFGR）感染的特征。通常，SFGR感染是 如果早点诊断出，则由适当的广谱抗生素治疗控制。然而，SFGR感染 出现难以早期临床诊断的非特异性体征和症状。未经处理或 误诊的SFGR感染经常与严重的发病率和死亡率有关。综合的 迫切需要对立克性发病机理的理解，以开发新的预防学和 感染后（P.I.）治疗学。我们报告说，在SFGR时，会发生RNase介导的tRNA裂解，A 诱导了TRNA衍生的RNA片段（TRF）的特定子集。其中，来自5'末端的片段 TrnaglyGCC是最突出的TRF，称为TRFGLYGCC。我们发现trfglygcc展品 以序列特异性的方式进行反沉降活性，并诱导EC屏障功能障碍。几行新行 我们初步研究的证据表明，来自R. parkeri感染的人脐静脉的外来 EC（HUVEC）（RCEXOS）在72小时P.I.或源自2LD50的等离子体R. Parkeri-Nfected小鼠（RMEXOS） 在第4天P.I.可以诱导正常受体人脑微血管EC（BMEC）的功能障碍 trfglygcc依赖性方式。与赤裸的格式相比，牛血清白蛋白 - 纳米叶酸 带有血清的正常介质中的TrfGlyGCC寡核苷酸（Bsaanti-Trfglygccs）可以保持BMEC屏障 暴露于RCEXOS后的功能。这些发现表明rcexos/rmexos包装的trfglygcc可能 在SFGRS期间诱导正常的受体功能障碍。 我们在该提案中的目标是寻求更多的实验证据，以支持我们的中心假设 靶向识别的SFGR诱导的外泌体中的trfglygcc可以通过 维护受体EC障碍功能。为了检验这一假设，我们将追求三个具体目标：（1） 在生化上证实了RCEXOS/RMEXOS包装的TrfGlyGCC改变受体EC屏障结构， （2）生物力学证实了rcexos/rmexos包装的trfglygcc导致受体EC障碍 功能障碍，（3）评估是否针对抗trfglygcc纳米颗粒靶向外部包装的TRFGLYGCC 可以通过维持内皮屏障功能来防止致命的立克感染。我们将测试我们的 假设通过采用尖端方法（流体FM技术，尺寸 - 排斥色谱法和 纳米颗粒的配方，以优化抗trfglygcc到ECS中的递送）。结果将更深入 对SFGR感染的生物力学和分子机制的见解，最终导致 在致命的SFGR感染期间鉴定可药物依赖宿主依赖性因子。