Antimicrobial peptides (AMPs) represent an essential component of the innate immune system that provides protection from bacterial infections. However, the development of AMPs as i.v. antibiotics has encountered difficulties from in vivo toxicity and limited efficacy. Therefore, in the previous funding periods we designed small molecule mimics of AMPs with robust activity against Staphyococcal aureus. One of these compounds (PMX30063) has now proven safe and efficacious in phase II trials in humans (conducted by PolyMedix). Membrane-binding appears to be an essential part of their antimicrobial mechanism. However, the precise mechanism by which these compounds and many AMPs kill bacteria has not been determined. Much can be inferred concerning the modes of action from studies of the transcriptional and translational response of bacteria to sub-killing concentrations of these agents. Bacteria have developed an elaborate response to AMPs over millions of years of co-evolution with their hosts. Many of these responses rely on bacterial histidine kinases (HKs), which are membrane-spanning protein kinases. These proteins, together with their partner response regulators, make up two-component systems (TCSs) that mediate antibacterial resistance, quorum sensing, nutrient utilization, and virulence. We will profile the transcriptional and translational responses of S. aureus and E. coli to AMPs, cyclic lipopeptide antibiotics, and AMP mimetics. These studies will not only provide a better understanding of the antimicrobial mechanisms, but will also define new targets to enhance the susceptibility of bacteria to synthetic drugs as well as our own endogenous AMPs. Our aims are: Aim 1) AMPs and mimetics cause large changes in expression of genes associated with membrane stress and periplasmic protein misfolding stress in E. coli. We will extend this analysis and compare the corresponding response of S. aureus. The degree of protection or sensitivity to AMPs and AMP mimetics imparted by each gene will be evaluated in vitro by determining bacterial growth and viability of strains that over-express or have the identified genes knocked out. Aim 2) We will examine the degree to which the identified gene products examined in Aim 1 affect virulence, colonization, and antimicrobial-susceptibility in mouse models. Aim 3) We will test a mechanism of antimicrobial recognition and signal transduction in the AMP sensing HK, PhoQ. The mechanism will be tested using quantitative disulfide mapping, hydrogen-deuterium exchange, and X-ray crystallography. Aim 4) We will discover and characterize peptide and small molecule modulators of HKs. PhoQ interacts with small endogenous regulatory proteins, and the mechanism of this regulation will be elucidated. We will also discover small molecule inhibitors of this and other HKs, and use them to probe the roles of HKs in colonization and virulence in animal models.