Development of a sensor for disulfide bond formation in diverse bacteria

Abstract

In bacteria, disulfide bonds contribute to the folding and stability of proteins important for processes in the cellular envelope. In E. coli, disulfide bond formation is catalyzed by DsbA and DsbB enzymes. DsbA is a periplasmic protein that catalyzes disulfide bond formation in substrate proteins while DsbB is an inner membrane protein that transfers electrons from DsbA to quinones, thereby regenerating the DsbA active state. Actinobacteria including mycobacteria use an alternative enzyme named VKOR which performs the same function as DsbB. Disulfide bond formation enzymes, DsbA and DsbB/ VKOR represent novel drug targets because their inhibition could simultaneously affect the folding of several cell envelope proteins including virulence factors, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. We have previously developed a cell-based and target-based assay to identify molecules that inhibit the DsbB and VKOR in pathogenic bacteria, using Escherichia coli cells expressing a periplasmic {beta}-Galactosidase sensor ({beta}-Galdbs) which is only active when disulfide bond formation is inhibited. Here we report the construction of plasmids that allow fine-tuning of the expression of the {beta}-Galdbs sensor and can be mobilized into other gram-negative organisms. As an example, when harbored in P. aeruginosa UCBPP-PA14, {beta}-Galdbs behaves similarly as in E. coli and the biosensor responds to the inhibition of the two DsbB proteins. Thus, these {beta}-Galdbs reporter plasmids provide a basis for identifying novel inhibitors of DsbA and DsbB/VKOR against multi-drug resistant, gram-negative pathogens and to further study oxidative protein folding in diverse gram-negative bacteria. ImportanceDisulfide bonds contribute to the folding and stability of proteins in the bacterial cell envelope. Disulfide bond-forming enzymes represent new drug targets against multidrug-resistant bacteria since inactivation of this process would simultaneously affect several proteins in the cell envelope, including virulence factors, toxins, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. Identifying the enzymes involved in disulfide bond formation in gram-negative pathogens as well as their inhibitors can contribute to the much-needed antibacterial innovation. In this work, we developed sensors of disulfide bond formation for gram-negative bacteria. These tools will enable the study of disulfide bond formation and the identification of inhibitors for this crucial process in diverse gram-negative pathogens.