Study On The Effects Of AHL And AI-2 Inhibitors On The Quorum Sensing Of Pseudomonas Aeruginosa PAO1

In recent years,the emergence of antibiotic-resistant bacteria due to the overuse and misuse of antibiotics,especially in the clinical treatment of infectious diseases caused by drug-resistant bacteria presents a significant challenge.Therefore,it is of great significance to discover new antibacterial drugs.Studies demonstrated that bacteria can increase their pathogenicity and drug resistance through regulating the formation of biofilms and the production of virulence factors by Quorum Sensing system（QS）.Therefore,Quorum Sensing Inhibitors（QSIs）targeting QS is a new strategy for treating drug-resistant bacterial infections.Pseudomonas aeruginosa is the main opportunistic pathogen in the hospital.In this paper,we designed a series of novel compounds with AHL（Acyl-homoserine Lactones）and AI-2（autoinducer-2）QS as targets,and studied the effects of these compounds on the QS of Pseudomonas aeruginosa PAO1 in two research themes.The first theme,18 novel oxazolidinone compounds were designed and synthesized and their effects on Gram-negative bacteria AHL QS were investigated.Firstly,4-p-chlorophenoxy-n-（2-oxoxazolidone-3-group）-butylamide（ZS-12）that synthesized in our previous work was used as a precursor compound to design and synthesize a series of novel oxazolidinone compounds.Chromobacterium violaceum CV026 as the reporter strain to initially screened compound YXL-13 which showed strong AHL QS inhibitory activity(IC₅₀=3.686±0.579μM).Then,Pseudomonas aeruginosa PAO1was used as the test strain to evaluate the QS inhibitory activities of YXL-13.In vitro,YXL-13 significantly inhibited the formation of biofilm,the production of virulence factors and the swarming motility of PAO1.Moreover,YXL-13 increased the susceptibility of PAO1 biofilm cells to antibiotics.Finally,we used wild-type Caenorhabditis elegans N2 as a model to show that YXL-13 had a significant inhibitory effect on PAO1 QS,thereby prolonging the lifespan of infected nematodes.The second theme,studies indicated that autoinducer-2（AI-2）acts as a signal molecule in QS plays an important role in the communication between multiple species and bacterial resistance.Herein we evaluated the QS inhibitory activity of AI-2 QSI.Firstly,we used the receptor LuxP of the AI-2 signal molecule as a target to screen 8600small molecule compounds from the In-house compound library through virtual screening techniques to obtain AI-2 QSI compounds.Vibrio harveyi BB170 used as the reporter strain to screen the compound Str7410 with strong QS inhibition(IC₅₀=0.3724±0.1091μM).Then,we used Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC25923 as the test strains to test the inhibitory effect of compound Str7410on AI-2 QS between the two strains in vitro and in vivo.In vitro,Str7410 significantly inhibited the formation of mixed strain biofilms during co-cultivation of strains.And the combination of compound Str7410 with known antibiotics can significantly improve the susceptibility of biofilm cells to antibiotics.In vivo,Str7410 significantly inhibited interspecific QS and significantly prolonged the lifespan of nematodes infeacted by P.aeruginosa PAO1 and S.aureus ATCC25923.Next,we studied the effect of compound Str7410 on PAO1 virulence factors（pyocyanin and elastase）and swarming motility in co-culturing of P.aeruginosa PAO1 and S.aureus ATCC25923.The results showed that Str7410 significantly reduced the production of virulence factors and swarming motility of PAO1.Finally,we preliminary evaluated the inhibitory mechanism of interspecific QS by the compound Str7410.The real-time quantitative PCR（Q-PCR）analysis found that compound Str7410 down regulated the expressions of PAO1 QS related genes in the co-culture of P.aeruginosa PAO1 and S.aureus ATCC25923.Preliminary mechanism studies showed that Str7410 inhibited interspecific QS by inhibiting the AI-2 sensation of PAO1 and down regulating the expressions of QS related genes.In conclusion,this paper evaluated the QS inhibitory activities of two QSIs via in vitro and in vivo experiments.It was confirmed that the novel oxazolidinone compound YXL-13 and compound Str7410 are better AHL and AI-2 QSI,respectively.This study not only provides new ideas for the discovery of antibiotics in theory,but also finds new methods for the treatment of clinical drug-resistant bacterial infections in practice.