Commensal Bacteroides signal through sphingolipid rafts during intestinal stress

描述（由申请人提供）：常驻肠道微生物组密切参与宿主生理学和体内平衡。这些微生物如何在复杂且频繁应激的胃肠道（GI）环境中建立和维持其种群是一个重要的问题。拟杆菌属是胃肠道的主要细菌成分，在其他地方很少发现。基因组分析和遗传研究表明，这些细菌具有非凡的能力，成功地在哺乳动物肠道定植。拟杆菌属在它们的膜中还具有独特的脂质组分-鞘脂，其被认为仅存在于真核细胞的膜中。真核鞘脂类物质已被公认为响应各种细胞应激的关键信号转导分子。信号传导的生物学结果是深刻而多样的，从细胞凋亡到血管生成。胆固醇也是压力反应中的关键协调者，有助于在真核细胞膜中形成信号平台。有趣的是，作为哺乳动物肠道的居民，拟杆菌属。容易从宿主的饮食摄入或胆汁分泌物中获得丰富的胆固醇。因此，这些细菌具有真核生物样鞘脂介导的信号传导所需的基本组分。初步研究表明，模式生物脆弱拟杆菌以鞘脂/胆固醇依赖性方式显著提高了对4种不同应激的存活率。此外，原子力显微镜证实，胆固醇的加入到纯化的B。fragilis膜脂质显著改变了这些脂质的物理组织，出现了大的鞘脂富集结构域。当施加应力时，荧光显微镜研究证实，生长培养基中含有外源性胆固醇的细菌细胞在细胞膜中形成富含胆固醇的脂质簇。如果将胆固醇添加到鞘脂阴性突变株的培养物中或在无应力条件下，则不会发生这种脂质聚集。我们还确定了一个候选的鞘脂信号分子，以及可能的基因，可以进行胆固醇摄取和鞘脂信号的生产。我们假设拟杆菌属。使用鞘脂/胆固醇依赖性信号通路来承受各种压力，并在潜在的不利肠道环境中建立自己。在这项研究中，我们的具体目标是确定1）通过定义电路中的关键组件的信号传导途径和2）信号传导是否是维持拟杆菌作为哺乳动物胃肠道中的主要细菌组分的重要。 公共卫生相关性：在理解微生物肠道菌群的过程中，一个关键的未回答的问题是微生物如何在胃肠道中长时间存活。拟杆菌属是极其重要的细菌，其膜中含有鞘脂，这在细菌物种中是罕见的。在这项研究中，我们将确定这些微生物是否能通过鞘脂/胆固醇介导的信号通路存活并承受肠道环境的压力，否则只能在真核细胞中发现。

DESCRIPTION (provided by applicant): The resident gut microbiome is intimately involved in host physiology and homeostasis. How these microbes establish and maintain their population in the complex and frequently stressed environment of the gastrointestinal (GI) tract is an important question. The genus Bacteroides is a major bacterial component of the GI tract and is rarely found elsewhere. Genomic analyses and genetic studies revealed that these bacteria have extraordinary ability for successful colonization in the mammalian intestine. Bacteroides spp. also have in their membranes a distinct lipid component-sphingolipids, which are otherwise thought to only be present in the membranes of eukaryotic cells. Eukaryotic sphingolipids species have been well-established as key signal transduction molecules in response to various cell stresses. The biological outcome of the signaling is profound and diverse, ranging from apoptosis to angiogenesis. Cholesterol is also a key coordinator in stress responses by helping to form the signaling platform in the eukaryotic membranes. It is interesting that as inhabitants of mammalian intestine, Bacteroides spp. have easy access to abundant cholesterol from dietary intake of the host or from biliary secretions. Therefore these bacteria are equipped with the basic components required for eukaryotic-like sphingolipid-mediated signaling. Preliminary studies show that the model organism Bacteroides fragilis has significantly improved survival against 4 different stresses in a sphingolipids/cholesterol-dependent manner. In addition, it is demonstrated by atomic force microscopy that the addition of cholesterol to purified B. fragilis membrane lipids considerably changes the physical organization of these lipids with the appearance of large sphingolipid- enriched domains. When stress is applied, fluorescence microscopy studies confirm that bacterial cells with exogenous cholesterol in the growth medium develop cholesterol-enriched lipid clusters in the cell membrane. This lipid clustering does not occur if cholesterol is added to the culture of a sphingolipid-negative mutant strain or in stress-free conditions. We have also identified a candidate sphingolipid signaling molecule, as well as possible genes that can carry out cholesterol uptake and sphingolipid-signal production. We hypothesize that Bacteroides spp. use sphingolipids/cholesterol-dependent signaling pathway to withstand various stresses and establish itself in the potentially hostile intestinal environment. In this study, our specific aims are to determine 1) the signaling pathway by defining the key components in the circuitry and 2) whether the signaling is important for maintaining Bacteroides as a major bacterial component in the mammalian GI tract. PUBLIC HEALTH RELEVANCE: A critical unanswered question in understanding microbial commensalism is how microbes survive as commensals in the gastrointestinal tract over long periods of time. Bacteroides spp. are extremely important commensals that have sphingolipids in their membranes, a rare occurrence in bacterial species. In this grant we will determine whether these microbes survive and withstand the stress of the intestinal environment through a sphingolipid/cholesterol mediated signaling pathway otherwise only found in eukaryotic cells.