Biogenesis and functions of outer membrane vesicles in Bacteroidetes

拟杆菌外膜囊泡的生物发生和功能

• 批准号: 10553698
• 负责人: Mario Feldman
• 金额: $ 19.56万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-21 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10553698
• 关键词: Ablation; Amylopectin; Bacteria; Bacterial Physiology; Bacteroides; Bacteroides fragilis; Bacteroides thetaiotaomicron; Bacteroidetes; Biochemistry; Biogenesis; Biological Assay; Biology; Bulla; Carbohydrates; Cell Culture Techniques; Cells; Chromosomes; Complex; Crohn' s disease; Cues; Cytolysis; Data; Defect; Dietary Carbohydrates; Dietary Polysaccharide; Digestion; Disease; Enzymes; Exhibits; Fluorescence Microscopy; Foundations; Future; Genes; Glycosaminoglycans; Glycoside Hydrolases; Gram-Negative Bacteria; High Prevalence; Human; In Situ; In Vitro; Inflammatory Bowel Diseases; Intervention; Intestines; Investigation; Label; Lead; Libraries; Lipoproteins; Luciferases; Maintenance; Mannans; Mass Spectrum Analysis; Mediating; Membrane; Methods; Microbiology; Molecular; Mucins; Mutation; N-terminal; Pathology; Peptide Hydrolases; Physiological; Physiological Processes; Play; Polysaccharides; Process; Production; Protein Sortings; Proteins; Proteome; Proteomics; Reporter; Research; Role; Shapes; Signal Transduction; Structure; System; Time; Vesicle; Visualization; Western Blotting; dietary; differential expression; experimental study; gut bacteria; gut dysbiosis; gut health; gut microbiota; high throughput screening; immunoregulation; improved; in vivo; infancy; innovation; insight; intestinal homeostasis; levan; luminescence; microbiome; microbiota; microorganism; model organism; mutant; mutualism; nano; nanoluciferase; novel; novel therapeutic intervention; receptor; response; sugar

Abstract The human intestine is colonized with ~1014 microorganisms that make up the gut microbiota. Approximately 40% of the bacteria inhabiting the human gut belong to the phylum Bacteroidetes, which can promote both healthy and diseased states. Long-term colonization of Bacteroidetes in the human gut is largely due to their ability to utilize dietary polysaccharides that are indigestible by the host and endogenous host glycans. Polysaccharide degradation in Bacteroidetes is controlled by a diverse array of multi-gene polysaccharide utilization loci (PUL), which encode the receptors, glycosidases (GH), and transporters needed to sense and digest various glycans. Through proteomic analyses, we determined that numerous GH are preferentially packaged into outer membrane vesicles (OMV). OMV are spherical, membranous structures generated by blebbing of the outer membrane (OM) of Gram-negative bacteria. Bacteroidetes OMV have been proposed to play important roles in immune modulation, maintenance of intestinal homeostasis, and promotion of interbacterial mutualistic interactions. Despite their increasing importance, no definitive mechanism for OMV biogenesis has been established, and OMV biology remains one of the least studied fundamental processes in microbiology. We have previously demonstrated that Bacteroides fragilis and B. thetaiotaomicron produce significant amounts OMV that are homogenous in size and shape. The OMV-specific proteome showed a high prevalence of GH and proteases. The GHs specifically packed into OMV are usually encoded within PUL, and their cognate transporters are not detected in OMV, which suggests that PUL systems partition between OM and OMV. We constructed fusions of OMV- and OM-specific proteins with fluorescent proteins and employed them as markers for live fluorescence microscopy. For the first time, we visualized the formation of OMV. The use of different fluorescent markers allowed us to differentiate between bona fide OMV and lysis by products. Our preliminary data demonstrate that, in Bacteroidetes, OMV are the result of a highly orchestrated physiological process. In aim 1 of this proposal we will employ biochemistry and mass spectrometry to investigate how OMV cargo is regulated to maximize the digestion of dietary and endogenous host glycans. In aim 2, we will employ fusions between OMV proteins and luciferase to investigate the molecular machinery required for OMV biogenesis. Understanding these processes may lead, in the future, to manipulation of bacterial vesiculation for innovative interventions to treat pathologies involving gut dysbiosis, such as Crohn’s disease and IBD.

抽象的 人类肠道内栖息着大约 1014 种微生物，这些微生物构成了肠道微生物群。 人类肠道内40%的细菌属于拟杆菌门，它可以促进 健康和患病状态下拟杆菌在人体肠道中的长期定植很大程度上是由于它们的影响。 利用宿主不可消化的膳食多糖和内源性宿主聚糖的能力。 拟杆菌中的多糖降解是由多种多基因多糖控制的 位点利用 (PUL)，编码感知和转运所需的受体、糖苷酶 (GH) 和转运蛋白 通过蛋白质组学分析，我们确定许多 GH 优先消化。 包装成外膜囊泡（OMV）是由 产生的球形膜结构。 已提出革兰氏阴性细菌 OMV 的外膜（OM）起泡。 在免疫调节、维持肠道稳态、促进 尽管细菌间的互利相互作用越来越重要，但 OMV 尚无明确的机制。 生物起源已经确立，OMV 生物学仍然是研究最少的基本过程之一 微生物学。我们之前已经证明脆弱拟杆菌和多形拟杆菌产生。 大量 OMV 的大小和形状均一，OMV 特异性蛋白质组表现出较高的水平。 专门包装到 OMV 中的 GH 和蛋白酶通常在 PUL 中编码，并且 在 OMV 中未检测到它们的同源转运蛋白，这表明 PUL 系统在 OM 和 OMV。我们构建了 OMV 和 OM 特异性蛋白与荧光蛋白的融合体并使用它们。 作为活体荧光显微镜标记，我们首次可视化了 OMV 的形成。 不同的荧光标记使我们能够区分真正的 OMV 和裂解副产物。 初步数据表明，在拟杆菌中，OMV 是高度精心策划的生理作用的结果。 在本提案的目标 1 中，我们将采用生物化学和质谱法来研究 OMV 的作用。 调节货物以最大限度地消化膳食和内源性宿主聚糖在目标 2 中，我们将采用。 OMV 蛋白和荧光素酶之间的融合，以研究 OMV 所需的分子机制 了解这些过程可能会在未来指导细菌囊泡的形成。 治疗涉及肠道菌群失调的疾病的创新干预措施，例如克罗恩病和炎症性肠病。

项目成果

Human gut bacteria tailor extracellular vesicle cargo for the breakdown of diet- and host-derived glycans.

• DOI: 10.1073/pnas.2306314120
• 发表时间: 2023-07-04
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Sartorio, Mariana G.;Pardue, Evan J.;Scott, Nichollas E.;Feldman, Mario F.
• 通讯作者: Feldman, Mario F.

Dual membrane-spanning anti-sigma factors regulate vesiculation in Bacteroides thetaiotaomicron.

双跨膜抗σ因子调节多形拟杆菌中的囊泡形成。

• DOI: 10.1073/pnas.2321910121
• 发表时间: 2024
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Pardue,EvanJ;Sartorio,MarianaG;Jana,Biswanath;Scott,NichollasE;Beatty,WandyL;Ortiz-Marquez,JuanC;VanOpijnen,Tim;Hsu,Fong-Fu;Potter,RobertF;Feldman,MarioF
• 通讯作者: Feldman,MarioF