Biogenesis and functions of outer membrane vesicles in Bacteroidetes

摘要 人类肠道中定植有约1014种微生物，构成肠道微生物群。约 居住在人类肠道中的40%的细菌属于拟杆菌门，它可以促进这两种细菌的生长。 健康和疾病状态。拟杆菌在人类肠道中的长期定殖主要是由于它们的 利用宿主和内源性宿主聚糖不能消化的膳食多糖的能力。 拟杆菌中的多糖降解受多种多样的多基因多糖控制 利用基因座（PUL），其编码感测和转运所需的受体、糖苷酶（GH）和转运蛋白。 消化各种聚糖。通过蛋白质组学分析，我们确定了许多生长激素优先 包装在外膜囊泡（OMV）中。OMV是球形的膜状结构， 革兰氏阴性菌外膜（OM）起泡。拟杆菌OMV已被提议 在免疫调节、维持肠道内稳态和促进 细菌间的互利相互作用尽管其重要性日益增加，但OMV的确切机制 生物起源已经建立，OMV生物学仍然是研究最少的基本过程之一， 微生物学.我们以前已经证明脆弱类杆菌和B。纳米粒子生产 显著量的OMV在大小和形状上是均匀的。OMV特异性蛋白质组显示出较高的 GH和蛋白酶的患病率。专门打包到OMV中的GH通常在PUL中编码， 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，并利用它们 作为活体荧光显微镜的标记。这是我们第一次看到OMV的形成。使用 不同的荧光标记物使我们能够区分真正的OMV和裂解副产物。我们 初步数据表明，在拟杆菌中，OMV是高度协调的生理过程的结果。 过程在本提案的目标1中，我们将采用生物化学和质谱法来研究OMV 调节货物以使膳食和内源性宿主聚糖的消化最大化。在目标2中，我们将使用 OMV蛋白和荧光素酶之间的融合，以研究OMV所需的分子机制 生物起源了解这些过程可能会导致，在未来，操纵细菌囊泡形成， 创新的干预措施，以治疗涉及肠道生态失调的病理，如克罗恩病和IBD。

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.