Suitability of RelBE and MazEF toxin-antitoxin systems as antibacterial targets
Toxin–antitoxin (TA) systems are unique modules that effect plasmid stabilization via post-segregational killing of the bacterial host. The genes encoding TA systems also exist on bacterial chromosomes, and it has been speculated that these are involved in a variety of cellular processes. Interest in TA systems has increased dramatically over the past five years as the ubiquitous nature of TA genes on bacterial genomes has been revealed. The exploitation of TA systems as an antibacterial strategy via artificial activation of the toxin has been proposed and has considerable potential; however, efforts in this area remain in the early stages and several major questions remain. This thesis research investigated the tractability of targeting the TA systems RelBE and MazEF and these systems were found to be ubiquitous in clinical isolates of Pseudomonas aeruginosa and Staphylococcus aureus. A high-throughput in vitro screen to identify small molecule disruptors of RelBE was developed using the photonic crystal biosensor technology. Peptide activators of the toxin RelE were sought by screening phage-displayed peptide against the RelB antitoxin. Additionally, a kinetic assay using a fluorogenic substrate was designed for the ribonuclease toxin MazFSa and applied in a screen for peptide enhancers of MazFSa enzymatic activity. A highly sensitive radiometric gel-based assay allowed for the first detection of endogenous MazFSa activity in S. aureus lysate, which will enable further studies to identify both artificial and natural activators of the MazFSa toxin. Further defining the prevalence of TA systems in clinical isolates and developing a variety of assays for assessment of MazFSa enzymatic activity have advanced our progress towards the goal of identifying TA system activators for use as an antibacterial therapy.
Toxin-antitoxin, antibacterial, RelBE, MazEF, Pseudomonas aeruginosa, Acinetobacter baumannii, ribonuclease, enzymatic assay, plasmid, multi-drug resistance
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign