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.
