Quinoline-based Derivatives Regulate Quorum Sensing In Pseudomonas Aeruginosa By Inhibiting PqsR And Their Interaction With Bacterial C-di-GMP

Antibiotic resistance has threaten greatly the global public health.It renders an urgent action to discover innovative antimicrobial interventions to help solve the bacterial resistance crisis.Anti-virulence signaling strategies reduce bacterial pathogenicity by specifically interfering with bacterial signal transduction systems.This strategy does not directly kill bacteria and exerts less selective pressure on bacterial drug resistance.Quorum sensing（QS）is an intercellular signal transduction mechanism that relies on bacterial density to govern group behavior among bacteria by using signal molecules.pqs quorum sensing system governs multiple virulence traits and biofilm formation through the interaction with PqsR,a Lys-R type transcriptional regulator,with its cognate signal molecules 2-heptyl-4-hydroxyquinoline（HHQ）and 2-heptyl-3-hydroxy-4-quinolone（PQS）.c-di-GMP is a ubiquitous second messenger in bacteria involved in the regulation of virulence,bacterial motility,and biofilm formation.Both QS and c-di-GMP have important roles in bacterial signaling and virulence secretion.In this thesis,PqsR was used as a drug target to study the inhibitory effect of quinoline-based derivatives on quorum sensing of Pseudomonas aeruginosa.The main contents include:（1）design and synthesis of quinoline-based derivatives;（2）the minimum inhibitory concentration（MIC）and growth curve of the compounds to P.aeruginosa PAO1were determined to analyse the antibacterial effect of compounds;（3）the cytotoxicity of the compounds was determined by normal human cells;（4）the inhibitory effect of the compounds on the QS system was evaluated by GFP reporter strain and q RT-PCR test;（5）the effect of the compounds on the secretion of virulence factors,motility and biofilm formation of PAO1 was evaluated;（6）the synergistic effect of compounds and tetracycline was evaluated;（7）the binding mode between the representative compound and PqsR was studied by molecular docking study.The results show that the compound may affect the QS system by inhibiting the pqs pathway.Compound 1 with morpholine side chain inhibited the expression of QS-related genes and effectively inhibited the pqs system of P.aeruginosa PAO1 with an IC₅₀of 20.22μM.Compounds can effectively inhibit the secretion of QS-related virulence factors,including pyocyanin,total protease,elastase and rhamnolipid.Among them,the inhibitory effect on pyocyanin controlled by the pqs system is particularly obvious.In addition,compound 1 in combination with tetracycline inhibited synergistically the bacterial growth and suppressed the biofilm formation of P.aeruginosa PAO1.The molecular docking studies also suggested that compound 1 could potentially interact with the ligand-binding domain of PqsR as a competitive antagonist.The relatively short side chain of the compound 1 unable to occupy the adjacent hydrophobic pocket may be responsible for its lower PqsR antagonistic activity.In addition,the interaction of these compounds with c-di-GMP was preliminarily studied in this thesis.The main contents include:（1）the interaction between the compounds and c-di-GMP was studied by UV spectroscopy and fluorescence spectroscopy;（2）the dissociation constants of the compounds and c-di-GMP was determined by Isothermal Titration Calorimetry（ITC）;（3）The inducing ability and stabilizing effect of the compounds on c-di-GMP G-quadruplex were determined by circular dichroism spectroscopy;（4）The configuration of the c-di-GMP G-quadruplex induced by compound 4 was determined by 1D NMR spectroscopy.The results show that there is interaction between these compounds and c-di-GMP.Compounds containing straight-chain amino side chains,especially compound 4,have a strong affinity for c-di-GMP.The compound 4 can induce c-di-GMP to form all-syn G-quadruplex and improve the stability of c-di-GMP G-quadruplex structure.