Screening And Identification Of Proteins Interaction With Quorum Sensing Gene SdiA Of Salmonella Paratyphi A

Salmonella paratyphi A(S.paratyphi A)is one of the main pathogens caused paratyphoid,which is spread through fecal-oral route.Bacteria sense signaling molecules secreted by bacteria themselves or other species andperceive their consentration change,thereby regulating the behavior of microbial population phenomenon,which is called quorum sensing.SdiA,the Gram-negative bacteria quorum sensing classical pathway receptor is closely relate to bacterial behavior regulated by quorum sensing,such as the formation of biofilm and produce for antibiotics and so on.In order to study the function of SdiA and related downstream genes involved in quorum sensing in S.paratyphiA,sdiA deletion mutant was constructed with PCR fusion combination with homologous recombination.Biological characteristics of mutant,including growth status,antiphage effect on biofilm formation and sensitivity to various antibiotics were studyed.Then sdiA was constructed to pKT25 by pMD18-T subclone and the feasibility of bait plasmid pKT25-sdiA was verified by cytotoxicity and autoactivation.A genomic library of S.paratyphi A was constructed,and the protiens interacting with SdiA were screened in this library by using bacterial two-hybrid.Positive clones were picked out to sequence and alignment,and then open reading frames were analyzed to determine the target protein.The validation of interaction between the bait protein and the target protein was taken by c-Myc tagged Co-IP.Finally,the target protein gene was knocked out to studythe prey protein whether plays an important role in erythromycin efflux.The results of sdiA deletion mutant showed that:(1)The deletion of sdiA gene does not affect the growth state of S.paratyphi A;(2)The deletion of sdiA gene has no effect on the formation of bacterial biofilms,while adding an appropriate concentration of AHL could promote the formation of biofilm.So there might be other AHL receptor protein.AHL concentration will not enhance the ability of biofilms forming accordingly;(3)The sdiA deletion mutant exhibited significant anti-phage phenomenon.The bacteriophage LSPA1 adsorption rates of wild-type and mutant S.paratyphi A have no differences,so anti-phage of mutant strain was not caused by the process of phage LSPA1 adsorption to host;(4)Analysis of antibiotic sensitivity to wild strains and mutant strains,the results show that both wild strains and mutant strains showed resistance to erythromycin,and the sensitive to ciprofloxacin,tetracycline,chloramphenicol.Bacterial two-hybrid screening and identification results showed that:(1)The bait plasmid constructed for screening is not toxic to the host cell and there is no self-activation phenomenon;(2)The geneomic library with high-quality and representative of S.paratyphiA was construced,which can be used to screen interaction proteins;(3)After the first screening there are more than 300 blue single clones were gained,and more than 200 positive clones were obtained by rescreening on selection plates M63.The mixed plasmids were extracted and transformed into the reporter strain BTH101 for initial verification and more than 60 positive clones were obtained.Purified plasmid library plasmids were co-transformed with the bait plasmid to BTH101 for reply authentication,and more than 30 positive clones were selected.(4)The reply verification positive library plasmids were sequenced,alignmented and reading frame analysed.The right gene was ybjZ,which encode macrolide ABC transporter permease/ATP binding protein MacB.Validation interaction of bait and target protein and function of the target protein results showed that:(1)The bait His tag fusion expression vectors and target c-Myc tag fusion expression vector were constructed.SDS-PAGE and Western-bolt were used to detect the protein expression after induced by EPTG.Both protein can exist as soluble state.(2)The mixture of bacterial lysates were used for Co-IP.The results show that protein SdiA interaction with YbjZ protein.(3)ybjZ knockout of host does not affect its resisitance to erythromycin.In this study,function of SdiA in S.paratyphi A were studied.The results indicated that sdiA might plays an important role in anti-phage of host.Macrolide ABC transporter permease/ATP binding protein MacB showed interaction with SdiA by screening with bacterial two-hybrid system.This protein was previously proofed to efflux 14 and 15 yuan macrolide antibiotics in E.coli.But in this study ybjZ deletion did not affect resistance to erythromycin.