Commensal Bacteroides signal through sphingolipid rafts during intestinal stress

描述（由申请人提供）：常驻肠道微生物群与宿主生理和体内平衡密切相关。这些微生物如何在复杂和频繁应激的胃肠道环境中建立和维持它们的种群是一个重要的问题。拟杆菌属是胃肠道的主要细菌组成部分，在其他地方很少发现。基因组分析和遗传学研究表明，这些细菌具有非凡的能力，成功定植在哺乳动物的肠道。拟杆菌类在其膜中也有一种独特的脂质成分-鞘脂，否则被认为只存在于真核细胞的膜中。真核鞘脂类已被确定为响应各种细胞胁迫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。胆固醇通过在真核生物膜上形成信号传导平台，在应激反应中起着关键的协调作用。有趣的是，作为哺乳动物肠道的居民，拟杆菌很容易从宿主的饮食摄入或胆道分泌物中获取丰富的胆固醇。因此，这些细菌具有真核类鞘脂介导的信号传递所需的基本成分。初步研究表明，模式生物脆弱拟杆菌（Bacteroides fragilis）以鞘脂/胆固醇依赖的方式显著提高了在4种不同胁迫下的存活率。此外，原子力显微镜还证明，在纯化的脆弱芽孢杆菌膜脂中加入胆固醇，会显著改变这些脂质的物理组织，出现大量富含鞘脂的结构域。当施加压力时，荧光显微镜研究证实，在生长培养基中含有外源性胆固醇的细菌细胞在细胞膜上形成富含胆固醇的脂质团簇。如果将胆固醇添加到鞘脂阴性突变株的培养中或在无压力条件下，则不会发生这种脂质聚集。我们还发现了一个候选鞘脂信号分子，以及可能进行胆固醇摄取和鞘脂信号产生的基因。我们推测拟杆菌类利用鞘脂/胆固醇依赖的信号通路来抵御各种压力，并在潜在的敌对肠道环境中建立自己。在这项研究中，我们的具体目的是确定1)通过定义电路中的关键成分来确定信号通路；2)信号通路是否对维持拟杆菌在哺乳动物胃肠道中的主要细菌成分的地位很重要。

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.