Friday, December 6, 2:25pm – 2:45pm via Zoom Meeting ID: 921 0377 3127 Passcode: 879566
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Sophie Pruett
Dr. Caitlin McMahon
The rise of antibiotic-resistant bacteria calls for new methods for combating infections. Traditional antibiotics place evolutionary pressure on a species to evade the antibiotic, leading to the development of resistance. Alternatively, anti-virulence drugs target non-essential traits that cover a wide range of adaptations in pathogenic bacteria that facilitate host colonization and resistance to treatment, including biofilm formation. These antivirulence drugs put less selective pressure on the bacteria and are less likely to drive resistance. Quorum sensing (QS) is an intercellular communication strategy using signaling molecules called autoinducers that lead to bacterial biofilm formation, making these pathways prime candidates for therapeutic inhibition. Autoinducer-2 (AI-2) is an interspecies QS signal formed by the metalloenzyme LuxS. This study uses irreversible covalent inhibition to target the nucleophilic Cys84 residue in the LuxS active site with electrophilic warheads. Developing a convenient assay to screen potential covalent inhibitors is essential to determine probe viability. Electrophilic probes were designed with terminal alkynes to enable enzyme detection with fluorescent labeling via a click chemistry reaction between the alkyne and an azide fluorescent dye. In-gel fluorescence was used to determine which electrophiles reacted with LuxS. Of tested electrophilic groups, a maleimide probe resulted in successful fluorescence labeling in the optimized assay. Other electrophilic groups, including chloroacetamide, vinyl sulfonamide, and acrylamide, did not show LuxS labeling. Site-directed mutagenesis was used to confirm the specificity of the probes. The C84A mutant LuxS protein showed diminished fluorescent labeling with the maleimide compared to the wild-type enzyme, implicating reactivity and possibly selectivity for Cys84. Further exploration of the protein-maleimide probe conjugation via dialysis and mass spectroscopy will reveal the nature of the probe’s addition as well as the number of additions and location within LuxS. A successful probe will ultimately be used to evaluate potential quorum sensing inhibitors in a simple gel-based assay.