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.

项目摘要 人们越来越认识到细胞外蔬菜（EV） - 微米或纳米大小的脂质 含有蛋白质和核酸货物的颗粒是由生命的所有领域脱落的。无处不在 核核电电动汽车表明，可以探索生物流体中细菌电动汽车的存在 诊断/预后，甚至针对潜水员致病细菌的疗法。但是，有未满足的需求 对于方法，可以解决一个基本问题，以混淆生物学中电动汽车信息的开发和 药物 - eVs自然是高度异质的颗粒，因此感兴趣的颗粒可能只存在于 其他电动汽车的混合物中的抽象非常低。在这个阶段的项目中，我们的总体实验目标是 通过专注于一类重要类别的一类 细菌纳米颗粒称为外膜蔬菜（OMV）。具体来说，我们的新技术平台是 旨在直接解决单个OMV级别解决OMV亚群的问题 通过分散OMV的表面蛋白和核酸货物（此处为小的RNA或SRNA）通过 高度多路复用的荧光成像分析，加上两种货物的扩增读数。我们的最终 目标是开发一个独特的成像平台，用于对蛋白质和蛋白质的高通量分析 从任何生物样品获得的单个OMV（和其他EV）的核酸货物。我们的平台可以 通过提供最小的侵入性，启用新型诊断/预后“液体活检”测试，以对细菌感染进行细菌感染 以及对常规方法的更有信息的替代方案（或补充），这些方法通常依赖于冗长 从临床样品或标本中培养靶生物。 我们的创新平台仅在一次通行证中简单地分析了多达10个潜在的OMV生物标志物AS 从血液，唾液或粪便等生物流体获得的OMV分散了多达106个OMV。与传统不同 方法，我们的平台可以迅速：1）分析潜水员电动汽车； 2）只需阅读多个OMV表面 标记，从而检测亚群； 3）阅读单个OMV货物，大量提高信息收益率com- 涉及产生合并货物数据的典型方法； 4）基于唯一的识别OMV亚群 生物标志物的组合来自点1-3。因此，我们提出了三个逐步目标。首先，要优化 OMV的蛋白质/核酸分析，通过革兰氏阴性和代表性的例子 革兰氏阳性细菌（分别是大肠杆菌和金黄色葡萄球菌）。第二，测试我们平台的功能 使用代表这些主要组类别的分散的单个OMV进行分析SRNA货物的原位。 第三，结合我们的表面分子和SRNA分析方法，以原位相关的直接检测 分散的单个OMV中的蛋白质和核酸货物都代表了两个主要基团。我们 预计我们的平台可能会成为用于管理病理的新研究/诊断/预后工具 其中OMV分析在临床上有用，并且用于监测正常或异常的微生物组状态。