Control of Virus Induced Lysis

描述（由申请人提供）： 重点是宿主裂解的机制，由细菌病毒（病毒）。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）。最近的进展表明，这种蛋白质编码三种类型的蛋白质，每种蛋白质负责攻击细胞壁的三种成分之一。整个过程是由洞蛋白控制的，洞蛋白是一种小蛋白质，通常在IM中积累15到60分钟，然后突然触发形成洞。抗洞蛋白是洞蛋白的特异性抑制剂，有助于调节其致死功能。这些膜孔的突然形成会杀死细胞，并立即停止所有能量代谢，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网络被破坏，一种新发现的蛋白质，称为Spanins，进行最后的行动。Spanins通过PG网络将IM连接到OM。一旦这种网络被破坏，Spanins就会发生构象变化并形成大的复合物，从而以某种方式破坏OM。已经提出了一种模型，即OM的破坏是膜与IM融合的结果，从而消除了病毒释放的最后障碍。 该建议侧重于两个具体领域。第一个也是最全面的目的是在分子和结构水平上表征spanins，使用遗传学，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。第二个目的有两个部分，涉及到的分子功能的holins。首先，将研究来自经典噬菌体T4的抗洞蛋白-洞蛋白系统，目的是了解抗洞蛋白如何阻断洞蛋白功能。本目标的第二部分重点是开发一种体外系统来研究上述触发过程。触发是发生在所有噬菌体感染中的神秘事件。holin蛋白质突然从无害的膜蛋白质转变为致命的空穴，这些蛋白质对膜的完整性或能量没有影响，以一种依赖于膜能量状态的方式停止所有大分子合成。提出了在通电条件下将纯化的holin蛋白质放入人工膜中的方法。如果这些研究成功，将为探索触发事件打开大门，而触发事件可能是生物学中最简单、最普遍的分子计时过程。这些研究将进一步加深我们对细菌病毒如何杀死它们的猎物并影响其后代的传播的理解。除了阐明许多基本过程之外，这可能具有直接的实际益处，因为越来越多的共识是，作为天然抗菌剂的抗生素将成为对抗细菌病原体的重要工具，这些病原体对可用的抗生素越来越耐药。

DESCRIPTION (provided by applicant): The focus is on the mechanism of host lysis by bacterial viruses (phages). 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 phages 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. Antiholins are specific inhibitors of holins that contribute to regulation of their lethal function. 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 act. Spanins connect the IM to the OM through the PG meshwork. Once that meshwork is destroyed, the spanins undergo a conformational change and form large complexes, which somehow disrupt the OM. A model has been advance that the destruction of the OM is the result of membrane fusion with the IM, thus removing the last barrier to virus release. The proposal focuses on two specific areas. The first and most comprehensive 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 has two parts, concerned with the molecular function of holins. First, the antiholin-holin system from the classic phage T4 will be studied with the goal of understanding how the antiholin blocks holin function. The second part of this Aim is focused on developing an in vitro system to study the triggering process cited above. Triggering is mysterious event that occurs in all phage infections. The holin proteins are suddenly transformed from harmless membrane proteins, that have no effect on membrane integrity or energy, to lethal holes that stop all macromolecular synthesis, in a manner that depends on the energy state of the membrane. Experiments are proposed to find ways to put purified holin proteins into artificial membranes under energized conditions. If these are successful, it will open the door to probing the triggering event that defines possibly the simplest, and most ubiquitous, molecular timing processes in biology. 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.