Ordered Membrane Domain Formation and Function in Pathogenic Bacteria

描述（申请人提供）：富含胆固醇的膜微域的形成、性质和生物学功能是膜结构和功能领域最令人兴奋和有争议的话题之一。1994年，我们（与Deborah Brown， Stony Brook U.）提出了真核细胞中这些结构域性质的当前工作模型：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，从富含不饱和脂质的无序脂质区域分离出来。许多致病菌，包括伯氏疏螺旋体属的细菌，从它们的宿主那里获得胆固醇。在与Dr. Jorge Benach （Stony Brook U.）的合作中，我们发现伯氏疏螺旋体作为莱姆病病原体的共同发现者，我们发现伯氏疏螺旋体形成含有胆固醇糖脂的外膜微域，大到足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，了解伯氏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。这些研究将极大地有助于我们了解膜结构域组织和功能的一般基本原理。电子显微镜，光谱学和生化方法将被用来检验伯氏疏螺旋体结构域是自组织有序的脂质结构域的假设。为此，脂质替代实验将用于修饰伯氏疏螺旋体膜，以确定甾醇和其他脂质在体外形成有序结构域的能力是否必要和充分，以使它们在伯氏疏螺旋体细胞中形成膜结构域。接下来，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密切相关的分子定位于伯氏疏螺旋体膜结构域将被确定。然后确定伯氏疏螺旋体外膜蛋白是否影响结构域的形成。为了验证胆固醇糖脂的相互作用对结构域形成至关重要的假设，我们将在体外模型膜囊中确定哪些伯氏疏螺旋体脂质具有形成膜结构域的能力。最后的目的是研究伯氏疏螺旋体膜结构域的功能。基于初步研究，膜结构域是否为维持伯氏疏螺旋体所必需。将检测膜完整性和/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来预防/治疗莱姆病。沿着这些思路，我们将测试一种对抗伯氏疏螺旋体感染的策略，即使用对真核细胞无毒，但我们最近发现对伯氏疏螺旋体细胞有毒的甾醇。最终，在这个项目中提出的研究可能会导致对其他含胆固醇致病菌膜的新认识，以及对抗它们引起的疾病的新策略。

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.