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 人类肠被用构成肠道微生物群的〜1014种微生物定植。大约 居住在人肠道中的40％的细菌属于类菌群，这可以促进两者 健康且犹豫不决的状态。人类肠道中菌植物的长期定植在很大程度上是由于它们 能够利用宿主和内源性宿主聚糖无法消除的饮食多糖。 细菌植物中的多糖降解由多糖的潜水员阵列控制 使用局部（PUL），它编码接收器，糖苷酶（GH）和转运蛋白需要感知和 消化各种聚糖。通过蛋白质组学分析，我们确定了许多GH优先 包装到外膜蔬菜（OMV）中。 OMV是球形的，膜结构 革兰氏阴性细菌的外膜（OM）的爆炸。已经提出了omv的细菌群 在免疫调节，维持肠内稳态和促进中发挥重要作用 无菌互动。尽管重要性越来越大，但没有确定的OMV机制 已经建立了生物发生，OMV生物学仍然是研究中最少的基本过程之一 微生物学。我们以前已经证明了菌脂菌和芽孢杆菌的生产 大量的OMV大小和形状是同质的。 OMV特异性蛋白质组显示高 GH和蛋白酶的患病率。专门包装到OMV中的GH通常在PUL中编码，并且 OMV未检测到它们的同源转运蛋白，这表明OM和 OMV。我们与荧光蛋白构建了OMV和OM特异性蛋白的融合，并采用了它们 作为活荧光显微镜的标记。我们第一次可以看到OMV的形成。使用 不同的荧光标记使我们能够区分真正的OMV和产品裂解。我们的 初步数据表明，在杆菌中，OMV是精心策划的生理的结果 过程。在该提案的目标1中，我们将采用生物化学和质谱法来研究OMV 货物受到调节，以最大化饮食和内源性宿主聚糖的消化。在AIM 2中，我们将雇用 OMV蛋白和荧光素酶之间的融合以研究OMV所需的分子机械 生物发生。了解这些过程可能会导致对细菌的操纵 治疗涉及肠道营养不良的病理的创新干预措施，例如克罗恩病和IBD。

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