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.

项目总结 越来越多的人认识到细胞外小泡(EVS)-微米或纳米级的脂质 含有蛋白质和核酸的颗粒--被生命的所有领域所释放。无处不在的 原核EVS提示，体液中细菌EVS的存在可能被开发为新的 诊断/预后，甚至各种致病细菌的治疗。然而，还有一种未得到满足的需求 对于能够解决在生物学和生物领域中利用电动汽车信息的根本问题的方法 药物--电动汽车自然是高度不均匀的颗粒，因此那些感兴趣的可能只存在于 在其他电动汽车的混合物中非常低的丰度。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EV异质性(亚群)来解决分析需求 细菌纳米颗粒被称为外膜泡(OMV)。具体来说，我们的新技术平台是 旨在通过直接解决在单个OMV水平上解决OMV亚群的问题 分散的OMV的表面蛋白和核酸货物(这里是小RNA或sRNA)之间的关联，通过 高度多元化的荧光成像分析与两种货物的放大读数相结合。我们的终极 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含量、高通量分析 从任何生物样本中获得的单一OMV(和其他EVS)的核酸货物。我们的平台可以 通过提供一种微创的细菌感染的新的诊断/预后“液体活组织检查”测试 以及对传统方法的更多信息的替代(或补充)，传统方法通常依赖于冗长的 从临床标本或标本中培养目标生物。 我们的创新平台一次性同时分析AS中多达10个潜在的OMV生物标志物 从血液、唾液或粪便等生物流体中获得的OMV多达106种。与传统不同 方法：我们的平台可以快速地：1)分析各种电动汽车；2)同时读取多个OMV表面 3)读取单个OMV货物，大大提高了信息量。 与产生汇集货物数据的典型方法相比较；以及4)基于唯一的 1-3点的生物标志物组合。因此，我们提出了三个分步骤的目标。第一，优化 革兰氏阴性杆菌和革兰氏阴性杆菌OMV的蛋白质/核酸联合分析 革兰氏阳性菌(分别为大肠埃希菌和金黄色葡萄球菌)。第二，测试我们平台的能力 使用代表这些主要类别的分散的单一OMV对SRNA货物进行现场分析。 第三，将我们的表面分子和sRNA分析方法相结合，用于相关的直接原位检测 蛋白质和核酸都装在代表这两个主要群体的分散的单一OMV中。我们 期待我们的平台能够成为管理病理的新研究/诊断/预测工具 其中OMV分析在临床上是有用的，并用于监测正常或异常的微生物组状态。