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Thesis Defense – October 13, 2017

Daniel Shin
Title: “AHL based Modulators for RhlI, a Quorum Sensing Signal Synthase in P. aeruginosa
Time: 1:30 pm
Location: Liberal Arts Building, Room 106


Gram-negative bacteria use N-acyl homoserine lactone (AHL) autoinducer based signal system, known as quorum sensing (QS), to modulate the gene expression for such traits as biofilm formation, toxin production, and antibiotic resistance. Therefore, there is great potential in pursuing quorum sensing inhibition (QSI) as a means of achieving antivirulence. Pseudomonas aeruginosa, an opportunistic pathogen commonly found in healthcare-related infections, use two LuxI/R type systems to regulate AHL-based quorum sensing, LasI/R and RhlI/R. LasI (initiator protein/signal synthase) and LasR (receptor) use 3-oxododecanoyl homoserine lactone signal molecule while RhlI and RhlR use butanoyl homoserine lactone autoinducer. Thus far, most of the studies have focused on inhibiting the Las system, in particular by using AHL signal analogs to interfere with signal-receptor binding. Recently, RhlI/R system has gain attention as potentially having greater effect in P. aeruginosa virulence. In this study, we have tested the effect of AHL analogs on RhlI, as product inhibitors with the goal of targeting both RhlI and RhlR for increased potency. Screening of compounds have revealed three variations to have the great effect on RhlI inhibition: longer/bulkier acyl- chain, D-stereocenter in the headgroup, and a less polar thiolactone head-group. Surprisingly, the addition of a carbonyl at the C3 position was found to activate the enzyme. Moreover, we measured kinetic constants of RhlI with various acyl-substrates and performed inhibition assays with inert acyl-substrate analogs to determine how RhlI activity changes to variations in the acyl-chain length. We found that the catalytic efficiency of acyl-substrate and inhibition potency of the corresponding inert acyl-substrate analogs surges with increase in the length of the acyl-chain. These patterns suggest that long acyl-chains most likely bind to an alternate binding site with marked increase in both kon and koff rate constants. Our findings with AHL derivatives provide a basis for rational design of quorum sensing inhibitors to better combat P. aeruginosa bacterial infections.

Program: Master of Science in Chemistry
Advisor: Dr. Rajesh Nagarajan, Chemistry and Biochemistry
Committee: Dr. Henry A. Charlier, Chemistry and Biochemistry and Dr. Michael P. Callahan, Chemistry and Biochemistry