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. In Escherichia 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 E. coli cells expressing a periplasmic ß-Galactosidase sensor (ß-Galdbs), which is only active when disulfide bond formation is inhibited. Here, we report the construction of plasmids that allows fine-tuning of the expression of the ß-Galdbs sensor and can be mobilized into other gram-negative organisms. As an example, when expressed in Pseudomonas aeruginosa UCBPP-PA14, which harbors two DsbB homologs, ß-Galdbs behaves similarly as in E. coli, and the biosensor responds to the inhibition of the two DsbB proteins. Thus, these ß-Galdbs reporter plasmids provide a basis to identify novel inhibitors of DsbA and DsbB/VKOR in multidrug-resistant gram-negative pathogens and to further study oxidative protein folding in diverse gram-negative bacteria. IMPORTANCE: Disulfide 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 because 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.

Engendering reactivity at group 5-heteroatom multiple bonds via π-loading.

In this Perspective, we discuss the strategy of π-loading, i.e., coordination of two or more strongly π-donating ligands to a single metal center, as it applies to promoting reactivity at group 5 transition metal-imido groups. When multiple π-donor ligands compete to interact with the same symmetrically-available metal dπ orbitals, the energy of the imido-based frontier molecular orbitals increases, leading to amplified imido-based reactivity. This strategy is of particular relevance to group 5 metals, as mono(imido) complexes of these metals tend to be inert at the imido group. Electronic structure studies of group 5 bis(imido) complexes are presented, and examples of catalytically and stoichiometrically active group 5 bis(imido) and chalcogenido-imido complexes are reviewed. These examples are intended to encourage future work exploring π-loaded bis(imido) systems of the group 5 triad.