Granulysin is a protein that is bactericidal for a broad range of Gram-positive and Gram-negative bacterial pathogens. This spectrum of activity, the rapid rate of killing and the likelihood that resistance cannot be readily engineered make granulysin an unusually promising lead for the development of anti-infectives for the prophylaxis or treatment of infections caused by bioterrorism agents. The purpose of this project is to investigate the mechanisms of action of granulysin compounds in order to produce information that will guide this drug development effort. A combination of functional genomic, molecular genetic, ultrastructural, and biochemical methods will be used to determine how granulysin derived compounds bind and inhibit critical cellular targets and why this event leads to the disruption of essential physiological processes. In Aim 1, microarray expression profiling of granulysin peptide treated bacteria will be conducted to identify affected metabolic/biosynthetic pathways, characterize physiological responses and explore mechanisms of bacterial statis and death. In Aim 2, genes required for the susceptibility of bacteria to granulysin peptides will be sought by screening transposon libraries for granulysin resistant mutants. In Aim 3, mutations that might confer an increased level of resistance will be investigated by determining if previously described and characterized Salmonella mutants with increased resistance to other antimicrobial peptides are also more resistant to granulysin peptides. Aim 4 will use ultrastructural and cell fractionation methods to localize granulysin peptides to a particular cellular compartment. In addition, radiolabeled photoreactive crosslinking derivatives of granulysin derivatives will be used to identify protein and/or LPS species with which granulysin interacts. The results of these localization and biochemical studies should provide topopgraphical information about the granulysin site of action and identify potential granulysin peptide cellular targets.