Screening And Mechanistic Study Of Anti-virulence Drugs Targeting Pseudomonas Aeruginosa Quorum-sensing System

Bacterial resistance has become one of the serious public health issues worldwide and is harmful to the health of humans and animals,as well as the ecological environment.Pseudomonas aeruginosa(PA)is a common clinical pathogen and a huge obstacle in clinical infection treatment because of its tremendous capacity to develop resistance to antibiotics.Both the production of biofilm and extracellular virulence factors of PA are associated to the large and complex quorum-sensing(QS).Therefore,the development of anti-virulence drugs targeting QS system is of great significance to combat drug resistant PA.In this project,the reference strain of PA,PAO1,was used as the model strain to screen the anti-virulence drugs with significant QS inhibition activity in vitro and in vivo.The main contents and conclusions of the research are as follows:(1)M9-adenosine and LB media were used to test the QS inhibition activities of 30 new structural compounds.Finally,five oxazolidinone new compounds,including SIIA-MA5,SIIA-MA7,SIIA-MA8,SIIA-MA9 and SIIA-MA12 which had no significant effect on the growth of PAO1 in LB broth and but could inhibit PAO1 in M9-adenosin,were obtained.Subsequently,dose-dependent effects of the five compounds were determined by growing PAO1 in M9-adenosine containing different concentrations(0,25,50,100 and 200 μM)of the compounds,and 50 μM was found to be the optimal inhibitory concentration.Finally,the results of anti-virulence activity analysis suggested that compounds SIIA-MA9 and SIIA-MA12 could inhibit the production of biofilm,pyocyanidin and proteolytic enzyme in PAO1,as well as the motility.(2)Caenorhabditis elegans infection model was utilized to evaluate the protective effects of compounds SIIA-MA9 and SIIA-MA12 on C.elegans against PAO1-infected.The results showed that SIIA-MA9 could significantly protect C.elegans from PAO1 acute infection,while SIIA-MA12 had significant protect activity during chronic PAO1 infection.Finally,compounds SIIA-MA9 and SIIA-MA12 were found to inhibit the las-QS system of PAO1 by using a lasR-nuh indicator bacteria contructed in this study.(3)RNA-sequencing was then conducted to explore the underlying mechanism of anti-virulence activities of the oxazolidinone compounds SIIA-MA9 and SIIA-MA12.The results showed that both compounds could inhibit the expression of genes positively controlled by the QS system of PAO1,as confirmed by using real-time PCR to determine the expression levels of typical QS-activated genes in SIIA-MA9 or SIIA-MA12-treated PAO1.In this study,the new oxazolidinone compounds SIIA-MA5,SIIA-MA7,SIIA-MA8,SIIA-MA9,SIIA-MA12 had significant anti-QS activities on PAO1 were obtained by a variety of screening methods.Among the five compounds,SIIA-MA9 and SIIA-MA12 could protect C.elegans from PAO1 infection.Transcriptome analysis and q PCR further confirmed that SIIA-MA9 and SIIA-MA12 could significantly inhibit the expression of QS-related genes by targeting the las-QS system.This study reveals the application potential of oxazolidinones in the treatment of PA infection by inhibiting the QS system,and provides a methodological support for the development of anti-virulence drugs to combat drug-resistant bacteria.