Control of Virus Induced Lysis

项目摘要 重点是细菌病毒（BTV）破坏（裂解）宿主的机制 细胞和病毒后代的释放。革兰氏阴性细菌细胞有三层：细胞质或 内膜（IM）、细胞壁或肽聚糖（PG）和外膜（OM）。最新进展 研究表明，主导生物圈的dsDNA噬菌体编码三种类型的蛋白质，每种 负责攻击细胞壁的三个组成部分之一。整个过程由 holins是一种小蛋白质，通常在IM中积累15到60分钟，突然，它们触发 形成洞。突然形成的这些膜孔杀死细胞，并立即停止所有的能量 新陈代谢，标志着感染周期的结束。这使得另一类称为内溶素的蛋白质 能够攻击细胞壁，降解糖-糖或肽键。一旦PG网络被摧毁， 一种新发现的蛋白质，称为spanins，进行最后一步。Spanins将IM连接到 通过PG网络的OM。一旦这个网络被破坏，Spanins就会经历一个构象变化， 改变和破坏了OM。我们将测试一个模型，即OM的破坏是膜的结果， 与IM融合，从而消除病毒释放的最后障碍。第一个目的是表征Spanins 在分子和结构水平上，利用遗传学、分子生物学、细胞生物学、超微结构 显微镜、生物化学和结构研究。第二个目标是与共同主要研究者的一项重大合作努力， 博士约翰霍普金斯大学的J. Xiao专注于使用超分辨率显微镜实现完整的 在四维（细胞中的空间和位置，以及感染中的时间）中描述裂解过程 cycle.） 这些研究将进一步加深我们对细菌病毒如何杀死猎物的理解， 影响其后代的传播。除了阐明许多基本过程外，这可能具有直接的 实际的好处，因为有一个越来越多的共识，即作为天然抗菌剂， 成为对抗细菌病原体的重要工具，这些病原体对现有的 抗生素

Project Summary The focus is on the mechanism by which bacterial viruses (phages) achieve destruction (lysis) of the host cell and release of the viral progeny. The Gram-negative bacterial cell has three layers: the cytoplasmic or inner membrane (IM), the cell wall or peptidoglycan (PG), and the outer membrane (OM). Recent progress has revealed that dsDNA phages that dominate the biosphere encode three types of proteins, each responsible for attacking one of the three components of the cell wall. The overall process is controlled by holins, small proteins that accumulate in the IM for typically 15 to 60 minutes, when, suddenly, they trigger to form holes. The sudden formation of these membrane holes kills the cell and instantly stops all energy metabolism, marking the end of the infection cycle. This allows another class of proteins called endolysins are able to attack the cell wall, degrading the sugar-sugar or peptide linkages. Once the PG network is destroyed, a newly discovered class of proteins, called the spanins, conducts the final step. Spanins connect the IM to the OM through the PG meshwork. Once that meshwork is destroyed, the spanins undergo a conformational change and disrupt the OM. We will test a model that the destruction of the OM is the result of membrane fusion with the IM, thus removing the last barrier to virus release. The first Aim is to characterize the spanins at the molecular and structural levels, using genetics, molecular biology, cell biology, ultrastructural microscopy, biochemistry and structural studies. The second Aim, a major collaborative effort with the co-PI Dr. J. Xiao of Johns Hopkins, is focused on using super-resolution microscopy in achieving a complete description of the lysis process in four dimensions (space and place in the cell, as well as time in the infection cycle.) These studies will further our understanding of how bacterial viruses, or phages, kill their prey and effect dispersal of their progeny. Besides illuminating many fundamental processes, this may have direct practical benefits because there is a growing consensus that phages, as natural antibacterial agents, will become an important tool in combating bacterial pathogens, which are increasingly resistant to available antibiotics.