Bacteria and pathogen characterizations using outer membrane vesicles

使用外膜囊泡表征细菌和病原体

• 批准号: 10602343
• 负责人: GREGORY W FARIS
• 金额: $ 29.88万
• 依托单位: NUMENTUS TECHNOLOGIES INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-03 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10602343
• 关键词: Address; Antibodies; Back; Bacteria; Bacterial Infections; Biological; Biological Markers; Biology; Biotechnology; Blood; Cell Culture Techniques; Cells; Centrifugation; Cerebrospinal Fluid; Classification; Clinical; Coated vesicle; Communication; Complex; Cost Analysis; Coupled; Data; Detection; Diagnosis; Diagnostic; Equilibrium; Escherichia coli; Eukaryotic Cell; Feces; Goals; Gram-Positive Bacteria; Health; Heterogeneity; Homeostasis; Human; Human Microbiome; Human body; Image; Immunoprecipitation; In Situ; Individual; Infection; Life; Lipid Bilayers; Lipids; Liquid substance; Mammalian Cell; Mediating; Medicine; Membrane; Membrane Proteins; Methods; Microbe; Monitor; Normal Cell; Nucleic Acids; Organ; Organism; Outcome; Pathogenesis; Pathology; Patients; Phase; Physiological Processes; Physiology; Plasma; Population; Process; Prognosis; Protein Analysis; Proteins; RNA; Reporting; Research; Reverse Transcriptase Polymerase Chain Reaction; Saliva; Sampling; Serum; Small RNA; Specimen; Speed; Staphylococcus aureus; Surface; Technology; Testing; Time; Urine; Vesicle; amplification detection; antibody detection; cell type; design; detection method; exosome; experimental study; extracellular vesicles; fluorescence imaging; high throughput analysis; host microbiome; imaging platform; imaging system; improved; innovation; intercellular communication; interest; liquid biopsy; meter; microbial; microbiome; minimally invasive; nanoparticle; nanoscale; new technology; novel; novel diagnostics; particle; pathogen; pathogenic bacteria; potential biomarker; prevent; prognostic; prognostic tool; technology platform; vesicular release

PROJECT SUMMARY There is growing recognition that extracellular vesicles (EVs)–micrometer- or nanometer-sized lipid particles containing protein and nucleic acid cargoes–are shed by all domains of life. The ubiquity of prokaryotic EVs suggests that the presence of bacterial EVs in biofluids could be exploited for new diagnostics/prognostics and even therapies for diverse pathogenic bacteria. There is, however, an unmet need for methods able to solve a fundamental problem confounding the exploitation of EV information in biology and medicine--EVs are naturally highly heterogeneous particles and thus those of interest may be present only at very low abundance in a mixture of other EVs. In this Phase I project, our overall experimental goal is to address the need for analyzing EV heterogeneity (subpopulations) by focusing on an important class of bacterial nanoparticles called outer membrane vesicles (OMVs). Specifically, our novel technology platform is designed to address the problem of resolving OMV subpopulations at the single OMV level by directly correlating surface protein and nucleic acid cargoes (here small RNAs or sRNAs) of dispersed OMVs, through highly multiplexed fluorescence imaging analysis coupled with amplified readouts of both cargoes. Our ultimate goal is to develop a unique imaging platform for the high-content, high-throughput analysis of the protein and nucleic acid cargoes of single OMVs (and other EVs) obtained from any biological sample. Our platform could enable novel diagnostic/prognostic “liquid biopsy” tests for bacterial infections by providing a minimally invasive and more informative alternative (or supplement) to conventional methods, which often rely on lengthy culturing of target organisms from clinical samples or specimens. Our innovative platform simultaneously analyzes, in one pass, up to 10 potential OMV biomarkers in as many as 106 dispersed OMVs obtained from a biofluid such as blood, saliva, or stool. Unlike conventional methods, our platform can rapidly: 1) analyze diverse EVs; 2) simultaneously read multiple OMV surface markers and thus detect subpopulations; 3) read single OMV cargoes, greatly raising information yield com- pared to typical methods producing pooled cargo data; and 4) identify OMV subpopulations based on unique combinations of biomarkers from points 1-3. We therefore propose three stepwise objectives. First, to optimize the combined protein/nucleic acid analyses of OMVs shed by representative examples of Gram-negative and Gram-positive bacteria (E. coli and S.aureus, respectively). Second, to test the capability of our platform for the analysis in situ of sRNA cargo using dispersed single OMVs representing these major group classifications. Third, combine our surface molecule and sRNA analysis methods for the correlated direct detection in situ of both protein and nucleic acid cargoes in dispersed single OMVs representing the two major groups. We anticipate that our platform could become a new research/diagnostic/prognostic tool for managing pathologies in which OMV analysis is clinically informative, and for monitoring normal or aberrant microbiome status.

项目概要 人们越来越认识到细胞外囊泡（EV）——微米或纳米大小的脂质 含有蛋白质和核酸货物的颗粒——由生命的各个领域释放。 原核 EV 表明生物体液中细菌 EV 的存在可用于新的研究 然而，对多种病原菌的诊断/预后甚至治疗的需求尚未得到满足。 能够解决困扰生物学中 EV 信息利用的基本问题的方法 医学——EV 本质上是高度异质的颗粒，因此感兴趣的颗粒可能仅存在于 在这个第一阶段的项目中，我们的总体实验目标是 通过关注一类重要的变量来满足分析 EV 异质性（亚群）的需求 具体来说，我们的新技术平台是称为外膜囊泡（OMV）的细菌纳米颗粒。 旨在通过直接解决在单个 OMV 水平上解析 OMV 亚群的问题 将分散的 OMV 的表面蛋白和核酸货物（此处为小 RNA 或 sRNA）相关联，通过 高度多重荧光成像分析以及两种货物的放大读数。 目标是开发一个独特的成像平台，用于蛋白质和蛋白质的高内涵、高通量分析 我们的平台可以从任何生物样本中获得单个 OMV（和其他 EV）的核酸货物。 通过提供微创方法，实现细菌感染的新型诊断/预后“液体活检”测试 以及传统方法的更多信息替代（或补充），传统方法通常依赖于冗长的 从临床样本或标本中培养目标生物体。 我们的创新平台一次性同时定位多达 10 个潜在的 OMV 生物标志物 与传统方法不同，从血液、唾液或粪便等生物流体中获得多达 106 个分散的 OMV。 方法，我们的平台可以快速：1）分析不同的 EV；2）同时读取多个 OMV 表面； 标记，从而检测亚群；3）读取单个OMV货物，大大提高信息产量 与生成汇总货物数据的典型方法进行比较；4) 基于独特的 OMV 亚群 因此，我们提出了三个逐步目标：首先，优化。 对革兰氏阴性和代表性例子排出的 OMV 进行组合蛋白质/核酸分析 其次，测试我们平台的革兰氏阳性菌（分别为大肠杆菌和金黄色葡萄球菌）的能力。 使用代表这些主要组分类的分散的单个 OMV 对 sRNA 货物进行原位分析。 第三，结合我们的表面分子和sRNA分析方法，进行相关的原位直接检测 分散的单个 OMV 中的蛋白质和核酸货物代表两个主要群体。 预计我们的平台可以成为管理病理学的新研究/诊断/预后工具 其中 OMV 分析可提供临床信息，并可用于监测正常或异常的微生物组状态。