Development of a sensor for disulfide bond formation in diverse bacteria

In bacteria, disulfide bonds contribute to the folding and stability of proteins important for processes in the cellular envelope. InE. 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, usingEscherichia colicells expressing a periplasmic β-Galactosidase sensor (β-Gal^dbs) 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 β-Gal^dbssensor and can be mobilized into other gram-negative organisms. As an example, when harbored inP. aeruginosaUCBPP-PA14, β-Gal^dbsbehaves similarly as inE. coliand the biosensor responds to the inhibition of the two DsbB proteins. Thus, these β-Gal^dbsreporter 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.