Ordered Membrane Domain Formation and Function in Pathogenic Bacteria

描述(申请人提供)：富含胆固醇的膜微区的形成、性质和生物学功能是膜结构和功能中最令人兴奋和最具争议的话题之一。1994年，我们提出(与黛博拉·布朗，石溪大学)目前对真核细胞中这类结构域性质的工作模型是：它们是富含胆固醇和饱和脂肪的液体有序状态脂类结构域，与富含不饱和脂类的无序脂类区域分开。许多致病细菌，包括疏螺旋体属的细菌，从它们的宿主那里获得胆固醇。作者Jorge Benach博士(石溪大学)作为莱姆病病原伯氏疏螺旋体的共同发现者，我们发现伯氏疏螺旋体形成了含有胆固醇糖脂的外膜微区，其大小足以在电子显微镜下直接观察到。该项目的目标是与Benach和Brown实验室合作，了解疏螺旋体膜中胆固醇糖脂和游离胆固醇结构域组织的物理基础，并确定这些结构域的功能重要性。这些研究将有助于我们理解膜结构域组织和功能的一般基本原理。电子显微镜、光谱和生化方法将被用来检验伯氏杆菌结构域是自组织有序类脂结构域的假设。为此，将利用脂类替代实验对伯氏杆菌膜进行修饰，以确定甾醇和其他脂类在体外形成有序结构域的能力是否是它们在伯氏杆菌细胞中形成膜域的必要条件和充分条件。下一步，是否只有那些先前被证明与模型膜中的有序膜结构域和定位于伯氏杆菌膜结构域中的真核细胞中的有序膜结构域紧密相关的分子将被确定。然后，将确定伯氏杆菌外膜蛋白是否影响结构域的形成。为了验证特定涉及胆固醇糖脂的相互作用对结构域形成至关重要的假设，我们将在体外确定哪些伯氏杆菌脂类有能力在模型膜囊泡中形成膜结构域。最终目的将是研究伯氏杆菌膜结构域的功能。根据初步研究，膜结构域是否是维持伯氏杆菌所必需的。膜完整性和/或抗体敏感性，将进行测试。这些研究可能为生物医学应用指明方向，包括通过改变膜结构域来预防/治疗莱姆病。沿着这些思路，将测试一种通过使用对真核细胞无毒但我们最近发现对伯氏杆菌细胞有毒性的类固醇来对抗伯氏杆菌感染的策略。最终，该项目中提出的研究可能会导致对其他含胆固醇病原菌的膜的新理解，以及对抗它们引起的疾病的新策略。 公共卫生相关性：将研究莱姆病的病原体伯氏疏螺旋体中膜胆固醇的组织。胆固醇和胆固醇衍生的脂类自组织成具有重要功能的有序膜结构域的假设将得到检验。此外，还将探索伯氏杆菌膜结构域的功能以及如何改变结构域以对抗伯氏杆菌感染。

DESCRIPTION (provided by applicant): The formation, properties and biological functions of membrane microdomains rich in cholesterol is one of the most exciting and controversial topics in membrane structure and function. In 1994 we proposed (with Deborah Brown, Stony Brook U.) the current working model for the nature of such domains in eukaryotic cells: that they are cholesterol and saturated lipid-rich liquid ordered state lipid domains that segregate from disordered lipid regions rich in unsaturated lipids. A number of pathogenic bacteria, including those in the genus Borrelia, obtain cholesterol from their hosts. In collaboration with Dr. Jorge Benach (Stony Brook U.) the co-discoverer of Borrelia burgdorferi as the causative agent of Lyme disease, we found that B. burgdorferi forms cholesterol glycolipid-containing outer membrane microdomains large enough to directly visualize by electron microscopy. The goal of this project is, in collaboration with the Benach and Brown labs, to understand the physical basis of domain organization of cholesteryl glycolipids and free cholesterol in Borrelia membranes, and to define the functional importance of these domains. Such studies will contribute greatly to our understanding of the general underlying principles of membrane domain organization and function. Electron microscopy, spectroscopic and biochemical approaches will be used to test the hypothesis that B. burgdorferi domains are self-organized ordered lipid domains. To do this, lipid substitution experiments will be used to modify B. burgdorferi membranes in order to determine whether the ability of sterols and other lipids to form ordered domains in vitro is necessary and sufficient for them to form membrane domains in B.burgdorferi cells. Next, whether only those molecules previously shown to strongly associate with ordered membrane domains in model membranes and eukaryotic cells localize within B.burgdorferi membrane domains will be determined. Then, whether B. burgdorferi outer membrane proteins influence domain formation will be determined. To test the hypothesis that interactions specifically involving cholesteryl glycolipids are crucial for domain formation, we will determine which B. burgdorferi lipids have the ability to form membrane domains in model membrane vesicles in vitro. The final aim will be to investigate the function of B. burgdorferi membrane domains. Based on preliminary studies, whether membrane domains are necessary to maintain B.burgdorferi. membrane integrity and/or antibody susceptibility, will be tested. These studies may point the way towards biomedical applications, including prevention/treatment of Lyme disease, by modifying membrane domains. Along these lines, a strategy for combating B. burgdorferi infections by using sterols that are not toxic to eukaryotic cells, but which we have very recently discovered to be toxic to B.burgdorferi cells, will be tested. Ultimately, the studies proposed in this project may lead to a new understanding of the membranes of other cholesterol-containing pathogenic bacteria, and new strategies to combat the diseases that they cause. PUBLIC HEALTH RELEVANCE: The organization of membrane cholesterol in the bacterium Borrelia burgdorferi, the agent of Lyme disease, will be studied. The hypothesis that cholesterol and cholesterol derived lipids self-organize into functionally-important ordered membrane domains will be tested. In addition, the function of B. burgdorferi membrane domains and how domains may be altered in order to combat B. burgdorferi infections will be explored.