Ordered Membrane Domain Formation and Function in Pathogenic Bacteria

描述（由申请人提供）：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有争议的主题之一。1994年，我们提议（与斯托尼布鲁克大学的黛博拉·布朗）目前对真核细胞中这些结构域的性质的工作模型是：它们是胆固醇和富含饱和脂质的液体有序状态脂质结构域，其与富含不饱和脂质的无序脂质区域分离。许多致病菌，包括疏螺旋体属的那些，从它们的宿主获得胆固醇。与豪尔赫·贝纳赫博士（斯托尼布鲁克大学）合作莱姆病病原体伯氏疏螺旋体的共同发现者，我们发现B. Burgdorferi形成足够大的含胆固醇糖脂的外膜微区，以通过电子显微镜直接观察。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的结构域组织的物理基础，并定义这些结构域的功能重要性。这些研究将大大有助于我们了解膜结构域组织和功能的一般基本原则。电子显微镜，光谱和生物化学方法将被用来测试的假设，B。Burgdorferi结构域是自组织有序的脂质结构域。为此，将使用脂质替代实验来修饰B。为了确定甾醇和其它脂质在体外形成有序结构域的能力对于它们在B.burgdorferi细胞中形成膜结构域是否是必需的和足够的，接下来，将确定是否只有那些先前显示与模型膜和真核细胞中的有序膜结构域强烈缔合的分子定位在B.burgdorferi膜结构域内。那么，是否B.将确定伯氏外膜蛋白影响结构域形成。为了检验这一假设，特别是涉及胆固醇糖脂的相互作用是至关重要的域的形成，我们将确定哪B。Burgdorferi脂质具有在体外在模型膜囊泡中形成膜结构域的能力。最终的目的是研究B的功能。burgdorferi膜结构域。根据初步研究，是否膜域是必要的，以维持伯氏疏螺旋体。膜完整性和/或抗体敏感性。这些研究可能为生物医学应用指明方向，包括通过修饰膜结构域预防/治疗莱姆病。沿着这些路线，一个战略打击B。将测试通过使用对真核细胞无毒、但我们最近发现对伯氏B氏疏螺旋体细胞有毒的甾醇的伯氏疏螺旋体感染。最终，该项目中提出的研究可能会导致对其他含胆固醇致病菌的膜的新理解，以及对抗它们引起的疾病的新策略。 公共卫生相关性：将研究莱姆病病原体伯氏疏螺旋体中膜胆固醇的组织。将检验胆固醇和胆固醇衍生脂质自组织成功能重要的有序膜结构域的假设。此外，还介绍了B. burgdorferi膜结构域以及如何改变结构域以对抗B。将探讨伯氏菌感染。

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.