Pore Formation by Cholesterol Dependent Cytolysins

 描述（由申请人提供）：来自链球菌属、李斯特菌属、短杆菌属、梭菌属、芽孢杆菌属、神秘杆菌属和加德纳菌属的40多种革兰氏阳性致病菌种表达成孔胆固醇依赖性溶细胞素（CDC）毒素家族的成员。总的来说，这些病原体在全世界造成了重大的健康负担，并引起各种疾病，包括肺炎、链球菌性咽喉炎、耳痛、伤口感染和食源性疾病。CDC促成这些疾病，并由这些病原体分泌为可溶性单体蛋白，其通过膜受体（主要是胆固醇）结合哺乳动物宿主细胞。这些细胞结合的单体寡聚成由35-40个单体组成的寡聚体复合物，然后组装并将大的β-桶孔插入真核细胞的质膜中。我们对CDC孔复合物组装的研究为基于CDC的新疫苗的合理设计提供了见解，并为哺乳动物免疫防御蛋白的机制提供了第一个见解。在此，我们将继续研究CDCs识别细胞和组装大β-桶孔的分子基础。在目的1中，我们将解决这样的假设，即CDC的膜结合界面的结构差异指导它们与存在于细胞上的特定结构域中的胆固醇结合，其中孔形成诱导不同CDC特有的细胞效应。了解这些差异的基础将改变CDC如何识别和改变细胞功能的基本范式。在目标2中，将研究CDC控制β-桶孔的组装及其插入膜的基本特征。这些研究不仅将加深我们对CDC孔形成机制的理解，而且将与各种细菌和哺乳动物免疫防御孔形成蛋白相关。在目标3中，我们将继续研究变构途径，通过该途径，CDC在细胞结合后激活单体以组装成巨大的CDC孔。总的来说，这些研究的结果将揭示 CDC孔形成的新范例，提供了一个更深入的了解CDC如何识别和改变细胞功能，并影响其他孔形成毒素和蛋白质的研究。

 DESCRIPTION (provided by applicant): Over 40 gram-positive pathogenic species from genera such as Streptococcus, Listeria, Brevibacterium, Clostridium, Bacillus, Arcanobacterium and Gardnerella express a member of the pore-forming cholesterol-dependent cytolysin (CDC) toxin family. Collectively, these pathogens represent a significant health burden worldwide and cause a wide variety of diseases, which include pneumonia, strep throat, earaches, wound infections and food-borne illnesses. The CDCs contribute to these diseases and are secreted by these pathogens as soluble monomeric proteins that bind mammalian host cells via membrane receptors, primarily cholesterol. These cell-bound monomers oligomerize into an oligomer complex comprised of 35-40 monomers, which then assembles and inserts a large β-barrel pore into the plasma membrane of eukaryotic cells. Our studies of the assembly of the CDC pore complex have provided insight into the rational design of a new CDC-based vaccine and provided the first insights into the mechanism of mammalian immune defense proteins. Herein we will continue to investigate the molecular basis of cellular recognition and assembly of the large β-barrel pore by the CDCs. In Aim 1, we will address the hypothesis that the structural differences in the membrane binding interface of the CDCs directs their binding to cholesterol that resides in specific domains on the cell wherein pore formation induces cellular effects that are unique to different CDCs. Understanding the basis of these differences will change the fundamental paradigms of how CDCs recognize and alter cellular function. In Aim 2 the fundamental features by which the CDCs control the assembly of the β-barrel pore and its insertion to the membrane will be investigated. These studies will not only deepen our understanding of the CDC mechanism of pore formation, but will have relevance to a wide variety of bacterial and mammalian immune defense pore-forming proteins. In Aim 3, we will continue the investigation of the allosteric pathway by which the CDCs activate monomers upon cell binding to assemble into the giant CDC pore. Overall, the results of these studies will reveal new paradigms of CDC pore formation, provide a deeper understanding of how CDCs recognize and alter cellular function and impact the study of other pore-forming toxins and proteins.