New Models and Methods for Characterizing Ligand-induced Luxr-type Quorum Sensing Receptor Activation and Inhibition PDF Download

Author: Matthew Joseph Styles
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Languages : en
Pages : 0

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Book Description
Many common Gram-negative bacteria, including important human pathogens, utilize small molecule signals (N-acyl-L-homoserine lactones, or AHLs) and cognate receptors (LuxR-type receptors) to control group phenotypes, such as virulence factor production, using a cell-to-cell signaling process called quorum sensing (QS). AHLs bind to LuxR-type receptors at high cell densities, which activates a series of macromolecular interactions that lead to altered expression of QS-regulated genes. QS has attracted significant recent attention as a target to block infections in humans, and numerous chemical biology approaches have been aimed towards disrupting this communication process in order to attenuate bacterial virulence. Small molecules that selectively target LuxR-type receptors represent one such approach. To develop these chemical tools, we must significantly expand our understanding of LuxR-type receptor:ligand interactions. These receptors have proven challenging targets for biophysical investigation, especially in the presence of compounds that antagonize receptor activity. This thesis focuses on the development of methods for characterizing LuxR-type receptor:ligand interactions using a combination of chemical biology, biochemical, and biophysical approaches.We began by identifying and developing structure-activity relationships for chemical probes capable of agonizing and antagonizing the orphan LuxR-type QS receptor SdiA in Salmonella and E. coli, characterizing the mechanism of action for a covalent inhibitor, and demonstrating the ability of this inhibitor to interfere with an infection-related phenotype (acid resistance). We next developed a FoÌ8rster Resonance Energy Transfer (FRET) assay to monitor LuxR-type receptor ligand binding in vitro and in intact cells and demonstrated the compatibility of these assays with six LuxR-type receptors. Thereafter, we report a series of biophysical experiments to characterize the interactions of two LuxR-type receptors, QscR from P. aeruginosa and SdiA form E. coli, with a range of small molecules agonists and antagonists. Based on these results, we propose models for the underlying signal transduction mechanism for each receptor. Critically, these results suggest several limitations of standard cell-based reporter assays as proxies for different biophysical parameters, and we provide an alternate path for mechanistic investigations that avoid these limitations. The results presented here provide chemical tools, methods, and conceptual models that will advance chemical approaches to modulate QS.