Screening And Mechanistic Study Of Inhibitory Compounds Targeting Pseudomonas Aeruginosa Quorum-sensing System

Pseudomonas aeruginosa is a clinically important Gram-negative opportunistic pathogen and can be frequently isolated from immunocompromised patients.This microorganism has relatively larger genome size and more genes than other common pathgenic bacteria,and these enable the rapid adaptation of P.aeruginosa to a varity of environmental habitats and host tissues.It is recognized that quorum-sensing(QS)system plays a vital role in regulating the expression and production of virulence factors in P.aeruginosa.Development of novel antibacterial drugs by targeting the QS system has become a promissing strategy in recent years.By using P.aeruginosa as model,the current study screened the compounds with QS inhibitory activity from different compounds,and explored the action mechanism of candidate drugs.The results are as follows:By using the 51 compounds for commercial use,D-cyclosine,Ribavirin,Dimetronidazole,Bilobalide and AICAR phosphate which could significantly inhibit the growht of P.aeruginosa on QS-required medium(QSM)but had no effect on nutrient medium(LB),were finally screened for further study.By measuring the production of pyocyanin,hydrolase,extracellular proteases and biofilm,the in vitro QS inhibitory activities of the five compounds on P.aeruginosa were comprehensively evaluated.The results showed that D-cycloserine had certain activities of pyocyanin,extracellular proteases,hydrolase and biofilm inhibition;Ribavirin could significantly inhibit the production of pyocyanin,extracellular proteases,hydrolase and biofilm,In addition,ribavirin had a clear dose-effect relationship on the inhibition of pyocyanin and extracellular proteases,with the lowest effective inhibitory concentration of 50 μM.Dimetronidazole could significantly inhibit the production of pyocyanin,extracellular proteases and hydrolase,however,the inhibitory activity against biofilm was not significant.Moreover,the inhibition of extracellular proteases by dimetronidazole had a clear dose-effect relationship,with the lowest effective inhibitory concentration of 50 μM.Besides,Bilobalide and AICAR phosphate had significant inhibitory activities of pyocyanin and biofilm inhibition.Among them,bilobalide had a clear dose-effect relationship on the inhibition of pyocyanin and,with the lowest effective inhibitory concentration of 50 μM.Caenorhabditis elegans-based fast-killing and slow-killing infection models were established to evaluate the protection effect of the five compounds against P.aeruginosa challenge.The results showed that D-cycloserine,Ribavirin,and Bilobalide had more significant protection activity on C.elegans than Dimetronidazole and AICAR phosphate against P.aeruginosa infection in the fast-killing assay,and the survival rates of C.elegans were 89%,78% and 78%.Moreover,Ribavirin,Dimetronidazole,Bilobalide and AICAR phosphate showed more significant protection activity against P.aeruginosa infection than Dcycloserine in the slow-killing assay,and the survival rate of C.elegans in the dimetronidazoletreated group was the highest(78%).Finally,Ribavirin and Dimetronidazole were selected to further study their action mechanisms.The results of transcriptomic analysis revealled 403 up-regulated and 412 down-regulated genes in Ribavirin-treated P.aeruginosa.The cluster analysis of differentially expressed genes showed that the significantly down-regulated DEGs mostly enriched in cellular component,and the metabolic pathway prediction results showed that ribosome-related genes were completely suppressed.For the 131 differentially expressed genes positively controlled by the QS of P.aeruginosa,61 genes of them were up-regulated and 70 genes were down-regulated.Notably,the QS transcription regulators and auto-inducible synthetic protein regulatory genes,as well as downstream elastase,rhamnolipid,movement and attachment and other related functional genes were all significantly down-reuglated against Ribavirin treatment.Additionally,the treatment of Dimetronidazole up-regualted the expression of 581 genes and down-regulated the expression of 1,027 genes in P.aeruginosa.The prediction of KEGG metabolic pathways showed that biofilm formation and bacterial secretion and other related genes were significantly inhibited.For the 145 differentially expressed genes positively controlled by the QS of P.aeruginosa,only 11 genes of them were up-regulated and 134 genes were down-regulated.Notably,the QS transcription regulators and auto-inducible synthetic protein regulatory genes,as well as downstream elastase,rhamnolipid,methyl chemotactic protein,major facilitator superfamily,and movement and attachment-related gene clusters were all significantly downreuglated against Dimetronidazole treatment.The result of Real-time fluorescent quantitative PCR further confirmed that the expression of typical QS-controlled genes(las R,las B,rhl R,rhl A,pqs R,pqs A,and pqs D)could be simultaneously inhibited by either Ribavirin or Dimetronidazole.In conclusion,by using a series of commercial compounds,the current study screened the compounds that could inhibit the QS system of P.aeruginosa and explored their action mechanism.Finally,a total of five candicate compounds,especially Ribavirin and Dimetronidazole,which could significantly inhibit the QS-related phenotypes of P.aeruginosa and protect C.elegans from P.aeruginosa infection were obtained.Comparative-transcriptomic analysis and quantitative PCR based transcriptional profiling further confirmed that Ribavirin and Dimetronidazole could inhibit the regulation of QS system in P.aeruginosa.These findings provide important methodological and structural basis for the development of anti-virulence drugs in the future.