Structural And Functional Studies On Pseudomonas Aeruginosa Transcriptional Factor PA4203 And PA3721

Pseudomonas aeruginosa is a Gram-nagetive bacterium that is also known as one of the most commom opportunistic pathogenic.P.aeruginosa mainly infects immunocompromised people and can cause postburn infections,wound infections and serious pneumonias.P.aeruginosa is able to adapt to a range of environments and is highly resistant to multiple antibiotics.Therefore,the treatment of P,aeruginosa infections are gaining more and more attentions.P.aeruginosa can cause infections in different environments is inseparable from the system containing multiple transcription factors and complex virulence factors.And multidrug efflux pumps is one of the important reasons for the antibiotic resistance of P.aeruginosa.Therefore,it is crucial to study that the regulation mechanism of transcription factors on multidrug efflux pumps and the function of virulence factors in this mechanism.Recent researches shown that Adding the virulence factor pyocyanin can result in the up-regulation of the efflux pump MexGHI-OpmD related genes and the transcription factor PA3721,which can upregulate the efflux pump MexAB-OprM.In this paper,we focus on the effect of pyocyanin on transcription factor-regulated efflux pumps and the mechanism of action of PA4203 and PA3721 by studying the transcription factor PA4203 located in upstream of the MexGHI-OpmD and the transcription factor PA3721.Our work is divided into two parts.The first part of our work is the structure and function of PA4203.PA4203 belongs to the LysR-type transcriptional regulator(LTTR)family with a conserved N-terminal HTH DNA binding domain and a variable C-terminal domain.LTTRs is structurally diverse and has a wide range of functions,and is involved in various regulatory pathways such as cell division,quorum sensing,and toxin production.In our study,we successfully achieved the structure of PA4203 with a resolution of 2.4 A by anomalous scattering.Acroding to the analysis of the structure,PA4203 contains four protein molecules per asymmetric unit and was verified by analysis of the ultra-dissociation experiment.And adding the pyocyanin could result in the up-regulation of PA4203,and it was verified that PA4203 was not a receptor protein of pyocyanin,but the mechanism of PA4203 regulating efflux pump MexGHI-OpmD had not been studied.The second part of our work is the structure and function of PA3721.PA3721 is a tetR-type transcription factor that inhibits the repressor ArmR of the transcription factor MexR,thereby indirectly inhibiting the multidrug efflux pump MexAB-OprM in Pseudomonas aeruginosa.In our study,we successfully resolved the structure of PA3721 with a resolution of 2.1 A.Acroding to the analysis of the structure,PA3721 had a protein molecule in each asymmetric unit.But it was proved to be dimer in solution state by pisa calculation and gel chromatography.It was found that PYO had no significant effect on the DNA binding ability of PA3721.When the PA3721 gene was knocked out,the resistance of the cells to abiotics such as azithromycin,chloramphenicol,carbenicillin,norfloxacin,ofloxacin,enoxacin,and ciprofloxacin was enhanced.In conclusion,we mainly studied the structure and function of two transcription factors PA4203 and PA3721 of Pseudomonas aeruginosa,and explored the effect of PYO on their function,and explored their function in the mechanism of multidrug efflux pumps.In view of multi-drug efflux pumps and pyocyanin playing a important role in the pathogenicity and antibiotic resistance of P.aeruginosa,our research needs further study.