The destruction of microorganisms within phagolysosomes is an essential immunological function of macrophages and other phagocytes that protects us from invading pathogens. Over the past several years we have established that Perforin-2 (PRF2), a recently described effector of the innate immune system, is pivotal for the destruction of phagocytosed bacteria. For example, we have published studies demonstrating that PRF2 knockout mice succumb to infectious doses that the majority of their wild-type littermates survive when challenged with bacterial pathogens. This is accompanied by replication and dissemination of bacteria to deeper tissues. With cell based studies we established bacteria that would normally be destroyed are able to replicate and persist within macrophages that lack PRF2. It has also recently been shown that polymorphisms within human PRF2 increase an individual's susceptibility to persistent nontuberculous mycobacterial infections. Most recently the results of our collaboration with structural biologists were published demonstrating that PRF2 polymerizes to form rings of 16 subunits. This study also revealed that the transition from pre-pore to pore is dependent upon low pH; such as would be encountered within acidifying phagosomes. Thus, our investigations spanning from the atomic to experimental mice have established that PRF2 underpins an essential function of macrophages as a pore-forming protein that permeabilizes the envelope of phagocytosed bacteria. Our working HYPOTHESIS is that PRF2-dependent killing of bacteria is a multistep process that begins with the intracellular trafficking of PRF2 as an inactive transmembrane (TM) protein in response to exogenous stimuli such as infection or pathogen- associated molecular patterns. Subsequent cleavage of PRF2 from its TM domain releases it to polymerize as a pre-pore structure on the membrane of phagocytosed bacteria. Acidification of the maturing phagosome triggers a dramatic reorganization of PRF2 that culminates in membrane penetrating pores through which other antimicrobials pass. Within this overall hypothesis are two areas of uncertainty –trafficking and proteolytic processing– that are appropriate for exploratory investigations. To redress these gaps Aim 1 will identify the protein-protein interactions that drive the intracellular trafficking of PRF2. Aim 2 will characterize the proteolytic processing events that regulate the activation of PRF2 within phagocytes. Significant impacts of this study will include a more complete understanding of macrophage mediated killing of phagocytosed bacteria and the molecular events that govern bactericidal pore formation.