PROJECT SUMMARY / ABSTRACT Gram-negative bacteria are uniquely equipped to defeat antibiotics. Their outermost layer, the cell envelope, is a natural permeability barrier that contains an array of resistance proteins capable of neutralizing most existing antimicrobials. As a result, its presence creates a major obstacle both for the treatment of resistant infections and the development of new antibiotics. The cell envelope is also home to numerous conserved pathways that safeguard the integrity of its proteome. Despite the central role of these systems in maintaining protein homeostasis, their interaction with resistance proteins localizing in the cell envelope has not been examined. We hypothesized that the activity of cell envelope folding catalysts may be important for the function of resistance determinants, and we tested this hypothesis on a key proteostasis player, the disulfide bond formation system. We discovered that the oxidative-protein-folding activity of this pathway is essential for the function of some of the most epidemiologically relevant and clinically challenging resistance proteins, namely β-lactamases, colistin resistance enzymes, and efflux pumps. Guided by strong preliminary data obtained from model laboratory strains and from clinical isolates, we propose an in- depth investigation of the role of cell envelope proteostasis systems in antibiotic resistance. We will use a combination of bacterial genetics, microbiology, biochemistry, proteomics, experimental evolution, and human disease modeling to pursue three specific aims: 1) Identify the components of the resistome that rely on oxidative protein folding, by assessing the requirement for disulfide bond formation on hundreds of clinically important resistance proteins. 2) Evaluate the impact of oxidative protein folding on resistant infections, by testing our biochemical findings in clinical isolates and in a relevant murine chronic infection model. 3) Explore the role of other cell envelope folding catalysts in antibiotic resistance, by probing their function in multidrug-resistant clinical strains of pathogenic bacteria and validating our results in model laboratory strains. We expect that our holistic approach, spanning multiple resistance determinants and folding catalysts, will break new ground in our understanding of the role of cell envelope proteostasis in resistance. This knowledge will be applicable to many high-priority Gram-negative pathogens and, in the long term, may inspire novel broad-acting strategies for overcoming antibiotic resistance.