Identification And Functional Study Of Quorum-sensing Small Non-coding RNAs In P.aeruginosa

ObjectiveQuorum sensing(QS)system and small regulatory RNA(sRNA)play key roles in metabolism,lipopolysaccharide modification,outer membrane protein synthesis,biofilm formation,virulence generation and drug resistance of P.aeruginosa.However,there is no systematic study on whether sRNA is involved in the regulation of QS and its specific mechanism and function in P.aeruginosa.In the early stage,the research group used IntaRNA,bioinformatics analysis software,to predict the target gene of the annotated sRNA in P.aeruginosa.It was found that key genes of QS system,such as lasI,rh1I and rh1R,were potentially regulated by sRNA.Based on this,this study starts from the QS-related sRNA PrrH predicted by IntaRNA,studies its function and defines the regulatory relationship between it and QS system;Then further extensive screening of QS-related sRNA,by RNA sequencing(RNA-seq)to lay a foundation for further systematic discussion of QS-related sRNA.The purpose of this study is to clarify the regulatory role of sRNA in P.aeruginosa QS system and its related functions,so as to enrich the regulatory network of QS system and the biological function of sRNA,and provide laboratory basis for the development of antibacterial strategies based on sRNA and QS system.Methods1.To clarify the effect of PrrH on the downstream virulence of QS and its regulation mechanism.(1)Construction and verification of prrH gene overexpression and knockout strains:using pROp200 plasmid as skeleton,prrH overexpression plasmid was constructed by Seamless Cloning Kit,and prrH knockout strain of P.aeruginosa was constructed by homologous recombination.qRT-PCR was used to verify whether the over-expressed and knockout strains were successfully constructed.(2)The effect of PrrH on the virulence of P.aeruginosa:the main virulence factors(pyocyanin,elastase and rhamnolipid)regulated by QS system were detected in wild-type strain,prrH knockout strain and overexpression strain,and the biofilm and motility which were closely related to its pathogenicity were also detected.In vitro bloodstream infection model was used to detect the viability of the above strains in blood which is conducted by bacterial count test and the relative expression of prrH,1asI and rh1I genes by qRT-PCR.(3)PrrH target gene verification experiments:according to the PrrH and target mRNA binding sites predicted by bioinformatics software,pUCP30T-mRNA-gfp and pUCP30T-mRNA-mut-gfp green fluorescent protein report vectors were constructed to verify the interaction between PrrH and target mRNA,and the effect of PrrH on target mRNA level was verified by qRT-PCR.2.To explore the transcriptional regulation mechanism of PrrH.Using qRT-PCR to verify whether 1asI and rh1I,the key genes of QS system,regulate PrrH,and then using bioinformatics tool PRODORIC to analyze the PrrH transcription factor recognition site.Further then the prrH promoter was PCR-amplified and inserted into BamHⅠ/HindⅢsites upstream of the lacZ reporter in pQF50 to construct prrH promoter report plasmid.Analyzing the effect of transcription factors on prrH promoter activity by β-galactosidase activity analysis experiment and qRT-PCR experiment.3.RNA-seq was used to screen sRNA related to QS system.(1)Transcriptome sequencing was carried out by RNA-seq technology to further screen the expression changes sRNA of QS activation process and after knockout of lasI and rh1,and verified by qRT-PCR.(2)Construction candidate sRNA gene overexpression and knockout strains.(3)Detection the effect of candidate sRNA on the virulence of QS downstream.Results1.(1)The prrH overexpression strain and knockout strain were successfully constructed.(2)Gain-and loss-of-function studies showed that PrrH affects pyocyanin,elastase and rhamnolipid production;biofilm formation;and swimming and swarming motility,and impaired the viability of P.aeruginosa in vitro bloodstream infection model.(3)IntaRNA predicted that PrrH targeted 1asI and phzC/D,green fluorescent protein reporter system and qRT-PCR further confirmed that LasI and PhzC/D were inhibited by PrrH.2.PRODORIC found two binding sites of Rh1R,the transcription factor of the rh1 system,on the promoter region of prrH.Further β-galactosidase reporter and qRT-PCR assays confirmed that PrrH was transcriptionally repressed by Rh1R.3.(1)Through RNA-seq and qRT-PCR,five sRNA:P26,P5316.1,P34,P30 and AmiL related to QS system were screened.(2)The sRNA overexpression strains of P26,P5316.1,P34,P30 and AmiL and sRNA knockout strains of P34,P30 and AmiL were successfully constructed.(3)The effect of sRNA on the synthesis of pyocyanin synthesis:AmiL inhibition,P30 promotion,while P34 had little effect on the production of pyocyanin.Conclusion1.PrrH is an important quorum regulatory RNA(Qrr)in P.aeruginosa,which participates in the regulation of Rh1I/R-PrrH-LasI/PhzC/PhzD signal pathway and inhibits the virulence and bloodstream infection of P.aeruginosa.Down-regulation of PrrH expression may be an important mechanism of P.aeruginosa infection.2.Transcriptome sequencing can make up for the deficiency of bioinformatics prediction,and more comprehensively and systematically screen out a number of QS-related sRNA.Preliminary virulence tests of several sRNA,related to QS system show that AmiL and P30 are involved in the virulence regulation of downstream QS,which is a potential QS related sRNA,and its specific mechanism needs to be further studied.