Pore Formation by Cholesterol Dependent Cytolysins

描述（由申请方提供）：胆固醇依赖性溶细胞素（CDC）是一大类致孔毒素，可导致许多革兰氏阳性病原菌的发病。我们对CDC孔形成机制的研究使我们更好地理解了它们在发病机制中的作用，并揭示了细胞识别和组装膜孔的新范例。这些研究使我们能够合理地设计一种基于CDC的肺炎链球菌组分疫苗，该疫苗目前正被盖茨基金会用于肺炎链球菌的疫苗研究。肺炎。此外，我们对CD 59结合CDC的发现使其他人能够开发出一种可能的专门针对HIV的治疗方法。在目前的更新中，我们将继续研究CDC机制，以了解触发孔形成的分子事件。了解CDC如何启动孔形成将有助于我们设计治疗或改进疫苗，专门针对其功能所需的高度敏感的结构成分。我们还将继续研究CDC对胆固醇的膜识别。我们的研究揭示了CDC与胆固醇相互作用的一个显著方面：一些CDC可以结合到膜胆固醇的扩增群体，其中只有一部分支持孔形成。似乎在不支持孔形成的位点处将显著部分的单体与胆固醇结合的能力对于特定CDC功能可能是重要的。最近研究表明，化脓性链球菌的CDC--链球菌溶血素O（streptolysin O，SLO）通过非孔依赖性机制将一种特异性蛋白质转运到真核细胞中。这些研究表明，CDC的孔非依赖性作用可能是CDC促成发病机制的重要但未探索的机制。了解CDCs如何与膜胆固醇结合而不引发孔形成，将揭示CDCs在分子水平上如何发挥作用以及它们如何促进发病机制的新范式。最后，我们将利用我们对CDC机制的深刻理解，开始研究膜攻击复合物/穿孔素（MACPF）蛋白质家族的孔形成机制。MACPF蛋白分布广泛，有助于哺乳动物的免疫防御和引起原核和真核病原体的疾病。最近的结构研究的MACPF蛋白和我们的工作的CDC促使其他人推测，MACPF孔形成机制的CDC孔形成机制的功能，可能导致从一个共同的起源的两个蛋白质家族。我们将利用我们在CDC的专业知识，开始研究MACPF蛋白的分子机制，以验证这一假设。我们期望对MACPF孔形成机制的研究将成为研究其他MACPF蛋白的重要努力的基础，这些蛋白在免疫防御（补体膜攻击复合物）、发育（astrotactins）、由真核病原体（如弓形虫和疟疾）引起的疾病以及肿瘤破坏（穿孔素）中发挥重要作用。

DESCRIPTION (provided by applicant): The cholesterol-dependent cytolysins (CDCs) are a large group of pore-forming toxins that contribute to the pathogenesis of many Gram-positive pathogenic bacteria. Our studies of the CDC pore-forming mechanism have led to a better understanding of their role in pathogenesis and have revealed new paradigms for cellular recognition and assembly of a membrane pore. These studies enabled us to rationally design a CDC-based component vaccine for Streptococcus pneumoniae, which is currently being used by the Gates Foundation in their vaccine effort for S. pneumoniae. Also, our discovery of a CD59 binding CDC has allowed others to develop a possible therapeutic that specifically targets HIV. In the current renewal we will continue our studies into the CDC mechanism to understand the molecular events that trigger pore formation. Understanding how the CDCs initiate pore formation will help us design therapies or improved vaccines that specifically target highly sensitive structural components necessary for their function. We will also continue our studies into the membrane recognition of cholesterol by the CDCs. Our studies have revealed a remarkable aspect of the CDCs' interaction with cholesterol: some CDCs can bind to an expanded population of membrane cholesterol of which only a fraction supports pore formation. It appears that the ability to bind a significant fraction of monomers to cholesterol at sites that do not support pore formation may be important to specific CDC functions. It was recently shown that the CDC of Streptococcus pyogenes, streptolysin O (SLO), transports a specific protein into eukaryotic cells by a pore-independent mechanism. These studies suggest that pore- independent effects of the CDCs may be a significant, but unexplored, mechanism by which the CDCs contribute to pathogenesis. Understanding how CDCs can bind to membrane cholesterol without triggering pore formation will reveal new paradigms for how CDCs function at the molecular level and how they contribute to pathogenesis. Finally, we will leverage our deep understanding of the CDC mechanism to initiate studies into the pore forming mechanism of the membrane attack complex/perforin (MACPF) family of proteins. The MACPF proteins are widespread and contribute to mammalian immune defense and disease causing prokaryotic and eukaryotic pathogens. Recent structural studies of MACPF proteins and our work on the CDCs has prompted others to speculate that the MACPF pore forming mechanism exhibits features of the CDC pore forming mechanism, possibly resulting from a common origin of the two protein families. We will leverage our expertise with the CDCs to initiate studies into the molecular mechanism of the MACPF proteins to test this hypothesis. We expect the study of the MACPF pore forming mechanism will form the basis of a significant effort to investigate other MACPF proteins that play important roles in immune defense (complement membrane attack complex), development (astrotactins), diseases caused by eukaryotic pathogens such as Toxoplasma and malaria, and tumor destruction (perforin).