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: 10200652
• 负责人: Bin Gong
• 金额: $ 19.75万
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10200652
• 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.

立克次体病是毁灭性的人类传染病。这些节肢动物传播的疾病是由 立克次体属的专性胞内细菌。立克次体病没有疫苗。 播散性血管内皮细胞（EC）感染和EC屏障功能障碍是其重要的病理生理机制 人类致死性斑点热群立克次体（SFGR）感染的特征。通常，SFGR感染是 如果早期诊断，通过适当的广谱抗生素治疗控制。然而，SFGR感染 存在非特异性体征和症状，使得早期临床诊断困难。未处理或 误诊的SFGR感染经常与严重的发病率和死亡率相关。全面 迫切需要了解立克次氏体的发病机制，以开发新的抗立克次体药物， 感染后（p.i.）治疗学我们报道，在SFGR时，RNA酶介导的tRNA切割发生， 诱导tRNA衍生RNA片段（tRF）的特定子集。其中，5 '端片段来自 tRNAGlyGCC是最显著诱导的tRF，称为tRFGlyGCC。我们发现tRFGlyGCC表现出 在某些实施方案中，该抑制剂以序列特异性方式抑制反式沉默活性并诱导EC屏障功能障碍。几行新的 初步研究表明，Exos来源于R.人脐静脉帕克氏菌感染 注射后72小时的EC（HUVEC）（RCExos）或来源于2LD 50 R的血浆的Exos。parkeri感染小鼠（RMExos） 在第4天p.i.可以诱导正常受体人脑微血管内皮细胞（BMEC）功能障碍， tRFGlyGCC依赖性方式。与裸形式相比，牛血清白蛋白纳米化抗- tRFGlyGCC寡核苷酸（BSA anti-tRFGlyGCC）在含血清的正常培养基中可维持BMEC屏障 在暴露于RCExos之后的功能。这些发现表明RCExos/RMExos包装的tRFGlyGCC可能 在SFGR期间诱导正常受体EN功能障碍。 我们在这个提案中的目标是寻求更多的实验证据来支持我们的中心假设， 靶向外来体中鉴定的SFGR诱导的tRFGlyGCC可以通过以下方式提供针对SFGR的保护： 维持受体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） 生物化学证实RCExos/RMExos包装的tRFGlyGCC改变受体EC屏障结构， (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）评估抗tRFGlyGCC纳米颗粒是否靶向Exos包装的tRFGlyGCC 可以通过维持内皮屏障功能来防止致死性立克次体感染。我们将测试 假设通过采用尖端的方法（FluidFM技术，尺寸排阻色谱法， 用于优化抗tRFGlyGCC向EC中的递送的纳米颗粒制剂）。结果将提供更深入的 深入了解SFGR感染的生物力学和分子机制，最终导致 在致死性SFGR感染期间鉴定可药物化的宿主依赖性因子。

项目成果

Endothelial Exosome Plays a Functional Role during Rickettsial Infection.

• DOI: 10.1128/mbio.00769-21
• 发表时间: 2021-05-11
• 期刊: mBio
• 影响因子: 6.4
• 作者: Liu Y;Zhou C;Su Z;Chang Q;Qiu Y;Bei J;Gaitas A;Xiao J;Drelich A;Khanipov K;Jin Y;Golovko G;Saito TB;Gong B
• 通讯作者: Gong B

Cell-Type Apoptosis in Lung during SARS-CoV-2 Infection.

• DOI: 10.3390/pathogens10050509
• 发表时间: 2021-04-23
• 期刊: Pathogens (Basel, Switzerland)
• 作者: Liu Y;Garron TM;Chang Q;Su Z;Zhou C;Qiu Y;Gong EC;Zheng J;Yin YW;Ksiazek T;Brasel T;Jin Y;Boor P;Comer JE;Gong B
• 通讯作者: Gong B

Extending applications of AFM to fluidic AFM in single living cell studies.

• DOI: 10.1002/jcp.30809
• 发表时间: 2022-08
• 期刊: JOURNAL OF CELLULAR PHYSIOLOGY
• 影响因子: 5.6
• 作者: Qiu, Yuan;Chien, Chen-Chi;Maroulis, Basile;Bei, Jiani;Gaitas, Angelo;Gong, Bin
• 通讯作者: Gong, Bin

Exosomally Targeting microRNA23a Ameliorates Microvascular Endothelial Barrier Dysfunction Following Rickettsial Infection.

• DOI: 10.3389/fimmu.2022.904679
• 发表时间: 2022
• 期刊: Frontiers in immunology
• 影响因子: 7.3

Intracellular receptor EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K-/eNOS-dependent manner during inflammation.

• DOI: 10.1016/j.jbc.2021.101315
• 发表时间: 2021-11
• 期刊: The Journal of biological chemistry
• 作者: Xiao J;Zhang B;Su Z;Liu Y;Shelite TR;Chang Q;Qiu Y;Bei J;Wang P;Bukreyev A;Soong L;Jin Y;Ksiazek T;Gaitas A;Rossi SL;Zhou J;Laposata M;Saito TB;Gong B
• 通讯作者: Gong B