Control of Virus Induced Lysis

描述(由申请人提供)： 重点放在细菌病毒(噬菌体)裂解宿主的机制上。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)。最近的研究表明，噬菌体编码三种蛋白质，每种蛋白质都负责攻击细胞壁的三个组成部分中的一个。整个过程由Holins控制，Holins是一种小蛋白，通常在IM中积累15到60分钟，然后突然触发形成洞。抗霍林素是霍林素的特异性抑制物，有助于调节其致命功能。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毒素的蛋白质能够攻击细胞壁，降解糖与糖或多肽之间的联系。一旦PG网络被摧毁，一类新发现的蛋白质，称为Spanins，进行最后的行动。西班牙人通过PG网络将IM连接到OM。一旦这个网络被破坏，跨度就会发生构象变化，形成大的复合体，以某种方式破坏OM。提出了一种模型，认为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一道屏障。该提案侧重于两个具体领域。第一个也是最全面的目标是利用遗传学、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，在分子和结构水平上表征Spanins。第二个目标有两个部分，与Holins的分子功能有关。首先，将研究经典噬菌体T4的抗霍林-霍林系统，目的是了解抗霍林是如何阻断霍林的功能的。这一目标的第二部分集中在开发一种体外系统来研究上述触发过程。触发是所有噬菌体感染中都会发生的神秘事件。Holin蛋白突然从对膜完整性或能量没有影响的无害膜蛋白转变为致命的孔，阻止所有大分子的合成，其方式取决于膜的能量状态。提出了在通电条件下将纯化的Holin蛋白放入人工膜中的实验方法。如果这些成功，它将为探测触发事件打开大门，该触发事件可能定义了生物学中最简单、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或噬菌体如何杀死它们的猎物并影响其后代传播的理解。除了阐明许多基本过程外，这可能还有直接的实际好处，因为人们越来越一致地认为，噬菌体作为天然抗菌剂，将成为对抗细菌病原体的重要工具，而细菌病原体对现有抗生素的抗药性越来越强。

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.