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.

立克次体代表着毁灭性的人类感染。这些节肢动物传播的疾病是由 立克次体属(Rickettsia(R.)立克次体没有疫苗可用。 弥漫性血管内皮细胞(EC)感染和EC屏障功能障碍是其主要的病理生理机制 致死性斑点热群立克次体(SFGR)感染特征。通常情况下，感染SFGR是 如早期确诊，可采用适当的广谱抗生素治疗。然而，SFGR感染 出现非特异性体征和症状，导致早期临床诊断困难。未经治疗或 误诊的SFGR感染往往与严重的发病率和死亡率有关。全面 迫切需要了解立克次体的致病机理，以开发新的预防和治疗方法 感染后(P.I.)治疗学。我们报道，在SFGR上，RNase介导的tRNA发生切割，并且 诱导tRNA衍生的RNA片段(TRF)的特定子集。其中，5‘端片段来自 TRNAGlyGCC是最显著的TRF诱导物，称为tRFGlyGCC。我们发现tRFGlyGCC展示了 以序列特异性的方式反式沉默，并诱导EC屏障功能障碍。几行新消息 我们初步研究的证据表明，Exos来自感染帕氏杆菌的人脐静脉 EC(HUVEC)(RCExos)72小时P.I.或来源于2LD50帕氏杆菌感染小鼠血浆的Exos(RMExos) 在第四天P.I.可诱导正常受体人脑微血管内皮细胞功能障碍 TRFGlyGCC依赖的方式。与裸载体相比，牛血清白蛋白纳米颗粒抗-HBs 正常血清中tRFGlyGCC寡核苷酸(BSAanti-tRFGlyGCCs)对BMEC屏障的维持作用 在暴露于RCExos后的功能。这些发现表明，RCExos/RMExos包装的tRFGlyGCC可能 在SFGRS期间导致正常的受体EN功能障碍。 我们在这项提议中的目标是寻找更多的实验证据来支持我们的核心假设 在外体中靶向SFGR诱导的tRFGlyGCC可以通过以下方式保护SFGR 维护接收方EC屏障功能。为了验证这一假设，我们将追求三个具体目标：(1) 生化证实RCExos/RMExos包装的tRFGlyGCC改变了受体EC屏障结构(S)， (2)生物力学证实RCExos/RMExos包装的tRFGlyGCC导致受体EC屏障 功能障碍，以及(3)评估是否以抗tRFGlyGCC纳米粒靶向外包装的tRFGlyGCC 可以通过维持内皮屏障功能来预防致命的立克次体感染。我们将测试我们的 通过使用尖端方法(FluidFM技术、大小排除层析和 优化抗tRFGlyGCC进入内皮细胞的纳米颗粒的配方)。结果将提供更深层次的 对SFGR感染的生物力学和分子机制的洞察，最终导致 致死性SFGR感染中一种可用药宿主依赖因子的鉴定。