Date of Graduation
Master of Science (MS)
Megan Eileen Bolitho
Quorum sensing in bacteria is a process of cell-to-cell communication facilitated by small molecules called autoinducers. Interspecies quorum sensing is facilitated by the autoinducer AI-2. The enzyme LuxS catalyzes the formation of AI-2 from S-ribosyl homocysteine (SRH) in a wide variety of bacterial species. Inhibition of LuxS would therefore inhibit interspecies quorum sensing. The goal of this project is to establish biochemical assays for the evaluation of small molecules as potential LuxS inhibitors. The first assay is a conventional colorimetric assay that utilizes Ellman’s reagent to quantify the homocysteine byproduct of DPD production by LuxS. For this assay purified enzyme (LuxS), a negative control (LuxS C84A), and the substrate (SRH) are required. His-tagged LuxS and LuxS C84A have been purified from overexpression cultures of Escherichia coli cells freshly transformed with a vector harboring the appropriate gene. Chemically-synthesized and quantified SRH was also acquired after extensive efforts of our fellow organic chemists. Ellman’s assay was then performed to determine the activity of house-purified LuxS. This assay would be optimized in future to be performed in 96 well plate to avoid excess consumption of enzyme and substrate. In addition, a fluorescence proximity assay is envisioned for the evaluation of a subset of LuxS inhibitors that function by preventing protein dimerization. This assay requires that purified LuxS be conjugated with an appropriate fluorophore, likely via a cysteine residue. An appropriate LuxS variant i.e. LuxS Y71C to which a single fluorophore would attach was acquired after extensive troubleshooting. An appropriate fluorophore would be attached to this LuxS variant for determination of potential dimerization inhibition.
gupta, richa, "Biochemical Evaluation of SRH Analogs as Potential LuxS Inhibitors" (2012). Master's Theses. 27.