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. PUBLIC HEALTH RELEVANCE: Study of host lysis is important for a number of reasons: (a) understanding the molecular basis of any process that is lethal to bacteria may ultimately help us combat bacterial disease; (b) many of the fundamental processes involved are mirrored in human viruses and in fundamental aspects of basic cell biology; (c) phage lysis has a direct role in several pathogenic processes, including the release of important human toxins; and (d) phages are being intensively investigated as potential antibacterial agents, so understanding the fundamentals of the phage infection cycle is critical.
