Commensal Bacteroides signal through sphingolipid rafts during intestinal stress

描述(申请人提供)：寄生肠道微生物群与宿主生理和动态平衡密切相关。这些微生物如何在复杂且经常受到压力的胃肠道(GI)环境中建立和维持其种群是一个重要的问题。类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，这些细菌具有在哺乳动物肠道成功定植的非凡能力。拟杆菌属在它们的膜中也有一种独特的脂类成分-鞘磷脂，否则被认为只存在于真核细胞的膜中。真核生物中的鞘脂已经被公认为是响应各种细胞压力的关键信号转导分子。该信号的生物学结果是深刻和多样的，从细胞凋亡到血管生成。胆固醇也是应激反应的关键协调者，帮助在真核细胞膜上形成信号平台。有趣的是，作为哺乳动物肠道的居民，类杆菌属(Bacteroids spp.)从宿主的饮食摄取或胆汁分泌物中很容易获得丰富的胆固醇。因此，这些细菌配备了真核细胞样鞘磷脂介导的信号传递所需的基本成分。初步研究表明，模式生物脆弱类杆菌以鞘脂/胆固醇依赖的方式显着提高了对4种不同压力的存活率。此外，原子力显微镜表明，在纯化的脆弱杆菌膜脂中加入胆固醇后，这些脂类的物理结构发生了很大的变化，出现了大量的鞘磷脂富集区。当施加压力时，荧光显微镜研究证实，在生长介质中含有外源胆固醇的细菌细胞在细胞膜中形成富含胆固醇的脂簇。如果将胆固醇添加到鞘磷脂阴性突变菌株的培养中或在无应激条件下，这种脂质聚集不会发生。我们还确定了一个候选的鞘脂信号分子，以及可能的能够进行胆固醇摄取和鞘脂信号产生的基因。我们推测拟杆菌属(Bacteroids spp.)利用鞘脂/胆固醇依赖的信号通路来抵抗各种压力，并在潜在的敌意肠道环境中建立自己。在这项研究中，我们的具体目标是通过定义回路中的关键组件来确定1)信号通路，以及2)信号对于维持类杆菌作为哺乳动物胃肠道主要细菌成分是否重要。 公共卫生相关性：理解微生物共生的一个关键问题是微生物如何长期在胃肠道中作为共生生物生存。拟杆菌属是极其重要的共生体，其膜上含有鞘磷脂，这在细菌物种中是罕见的。在这项资助中，我们将确定这些微生物是否通过鞘脂/胆固醇介导的信号通路存活并抵御肠道环境的压力，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.