The Study Of The Regulation Of Conjugation By Quorum Sensing And It's Related SRNA Under The Intervention Of Antibiotics

ObjectivePseudomonas aeruginosa(PA)is a gram-negative non-ferment bacteria,which is a common opportunistic pathogen.It can cause infection in many parts like the respiratory tract,urinary tract,skin,abdominal cavity and blood.Moreover,PA can get acquired resistance through mutation and other mechanisms under the influence of antibiotics so that clinical treatment is now in great difficulty.Plasmids mediated horizontal gene transfer is an important mechanism for PA to get drug resistance.The bacteria can release the specific signal molecules to the surrounding during growth,to perceive the change in the density of bacteria.When the signal molecules accumulate to a certain concentration as the density of bacteria increases to a certain threshold,the combination of signaling molecules and receptor proteins changes the conformation of the receptor protein and activates a series of gene expressions,it then regulates the group behavior of bacteria,this phenomenon is known as quorum sensing.In gram negative bacillus,acyl homoserine lactone(AHL)was used as the signal molecule QS system.In gram negative bacilli,acyl homoserine lactone was used as the signal molecule of QS system,AHL can bind to the bacteria's LuxR protein receptor,achieve the regulation of a series of genes downstream.Some bacteria such as Escherichia coli and Vibrio cholerae do not contain the QS system,But they express the SdiA protein which is homologous molecules of LuxR protein,the SdiA protein can binds to AHL in the surrounding.SdiA is also a transcription factor.Conjugation is the process of horizontal gene transfer(HGT)by the bacteria hairs.It is an important mechanism for the transmission of drug-resistant gene by plasmid.It is generally believed that conjugation is mainly regulated by conjugative elements carried by plasmids.In the earlystage of our research group,the Conjugative model of Escherichia coli-Pseudomonas aeruginosa was constructed to study the role of receptor in conjugative regulation.We found that the signal molecule AHL of PA QS system plays an important role in inhibiting conjugation.This part of the experiment use the same conjugative model,to detece the changes of conjugative efficiency and the changes in the expression level of gene lasI?rhlI?traI under the intervention of antibiotics.And take the non coding small RNA of bacteria as the breakthrough point to explore the specific mechanism for the change of conjugation under the intervention of antibiotic.MethodsIn this experiment we use SM10??as the donor,the conjugative plasmid RP4(contain the gene traI)is integrated on the chromosomes of the SM10??,and the recombinant plasmid pUCP24T is contained in it.The pUCP24T was constructed by our laboratory,we used the Plasmid pUCP24 as frame,inserted the oriT fragment in the plasmid pCVD442 genome which is the starting sequence of the conjugation,and oriT contains the identification site of relaxation enzyme,which is the key to determine whether plasmids can conduct conjugation.Plasmid pUCP24T contains gentamicin resistance gene,which can be used as a marker for the screening of transconjugants.In addition,SM10??sdiA knockout strain SM10???sdiA is used as donor,and protein SdiA is the receptor protein of QS system signaling molecule in Escherichia coli.The combination of AHL and SdiA can inhibits the expression of traI.In this experiment,the SM10???sdiA mutant was used to eliminate the effect of signal molecules secreted by the receptor to the conjugation.The wild strain PAO1 of Pseudomonas aeruginosa and mutant strains PAO1?rhlI?PAO1?lasI were selected as the receptors in thse experiment,and rhlI and lasI regulate the synthesis and secretion of QS system signal molecules C4-HSL and3-oxo-C12-HSL.Due to the PAO1 has natural resistance to ampicillin(APM),only the transconjugants obtained the plasmid pUCP24T has both Gm and APM resistance can grow on the Gm+APM screen plate.In order to observe the effect of antibiotics on conjugative efficiency without QS system,the SM10??and EC600 were respectively used as donor and recipor,EC600 was naturally resistant to rifampicin(RIF),and the Gm+RIF select plate can used to screen the transconjugants.Conjugation:The donor and receptor were cultured to logarithmic growth period,then use the automatic urine analyzer to measure the concentration of the bacterial fluid,mixing the donor and receptor according to the 1:1 ratio,mating the donor and receptor(0.5×10~7 CFU/mL each)at 37?for 6h,screening and counting the transconjugants.Use qPCR to detect the expression of conjugation related gene:SM10??and PAO1 co-cultures were treated with 0.3?g/mL of Gm for 6 hours,then extract RNA and reverse transcript to cDNA,using cDNA as the template,followed by real-time PCR etect the expression of gene lasI,rhlI and tarI.The genome of PA does not contain tarI,and Escherichia coli has no QS system,Therefore,the whole RNA group was extracted from the mixture of two bacteria will not effect the results of qPCR.Screening and identification the related sRNA with QS system:Using the bioinformatics software IntaRNA to forecast the target gene from the annotated sRNA in PA,Screening the related sRNA with QS system,use qPCR to detected the relationship between the selected candidate sRNA and the key genes of the QS system.Knockout the prrH gene:The upstream and downstream homologous arms of prrH were amplified by PCR and then were ligated to the vector pGSM which has the gentamicin resistance gene aacC1,sucrose sensitive gene sacB and a conjugative transfer gene mob to constructed suicide plasmids pGSM–prrH.The plasmids were transformed into Escherichia coli strainSM10??and then transfered to PAO1 by conjugation.Under the pressure of antibiotics,prrH genes were knocked out by homologous recombination,and then the resistance selection markers were deleted by using sucrose medium.The recombinant strains were identified by PCR and DNA sequencing.ResultsUnder the intervention of antibiotics that cause survival pressure to the receptor,The expression of lasI and rhlI in PAO1 was significantly down-regulated and the expression of traI in SM10??was up-regulated.The SM10??-PAO1 conjugative efficiency can be increased about 10 times(P<0.01),by knockout lasI/rhlI or sdiA blocking AHLs-sdiA-TraI signaling pathway can weaken antibiotic-induced conjugation.However,it has no such effect on the SM10??-EC600,which lacks the QS system(P<0.01).Through bioinformatics analysis,we found that several key genes of QS system,such as qscR,lasI,rhlI and rhlR,are regulated by sRNA.Five sRNA,such as PrrF,PrrH,CrcZ,RgsA and P34,were screened for the potential regulatory functions of the QS system.LasI may be the target for sRNA PrrF and PrrH,RhlI and C4-HSL receptor RhlR may be the target for sRNA RgsA,CrcZ or P34 respectively.The PAO1 prrH gene knockout strain was successfully constructed.ConclusionThis experiment constructed two kinds of conjugative models,It is proved that under the stress of antibiotics,the expression of QS system is down regulated,and it,s inhibition of conjugation can be reduced,and the bacteria can get exogenous resistance genes to adapt to the environment.