The Functions And Mechanisms Of Novel Pseudomonas Aeruginosa Two-component Regulatory Systems In Skin Infection

Pseudomonas aeruginosa is a Gram-negative bacterial pathogen existing in extensively natural environment including soil,water,plants,animals,as well as domestic and clinical environments.P.aeruginosa is associated with acute and chronic infections because of its considerable nutritional and metabolic versatility.Two-component systems（TCSs）of P.aeruginosa are responsive for their adaptive response to changes in the surrounding environments both in nature and host.TCS is the dominant signal transduction pathway of prokaryotic cells,which enables bacteria to sense extracellular signals,thus regulating the expression of related genes,which play an important regulatory role in the process of bacterial adaptation to the environment.However,little is known about whether unknown TCS in P.aeruginosa are associated with skin wound infections and how these TCSs play roles in skin infections.In this study,four unkown TCSs were selected through high-throughput sequencing of the transposon mutation library（Tn-seq）of P.aeruginosa UCBPP-PA14 based on results from our previous studies,suggesting their important roles in skin wound infection.In this study,functional mechanisms of four histidine kinase receptors or response regulator genes including PA14₄9420,PA14₄5870,PA14₆4570,PA14₃2580（named nioR,preliminary studies of the pathogenic mechanism on nioR have been completed）were investigated.In order to study the specific functional roles of the three TCSs including PA14₄9420,PA14₄5870,PA14₆4570 in the skin wound infection,those TCSs gene knockout strains were constructed.Next,mouse skin wound infection established with PA14 WT,△PA14₄9420,△PA14₆4570,△PA14₄5870.The bactial load results demonstrated that the ability of skin wound infection of three knockout strains was significantly impaired,suggesting three TCSs play important roles in P.aeruginosa skin infection.To investigate how those TCSs promoted P.aeruginosa skin wound infection,RNA-seq analysis of these TCSs deletion mutants were performed.RNA-seq results showed that PA14₄5870 regulated the expression of virulence genes including quorum sensing,bacterial chemoattractivity,secretory system;PA14₄9420 regulated the expression of virulence genes including molybdenopterin and secretory system;and PA14₆4570 regulated the expression of virulence genes including iron absorption,quorum sensing,DNA replication,homologous recombination,and secretory system.Those results suggest the downregulation of these virulence genes may lead to the decrease of the infection ability of P.aeruginosa.Our preliminary results showed that NioSR could regulate the denitrification system genes.Among these,the nitric oxide reductase genes norCBD,which plays an important role in the denitrification system,were the most significantly regulated by NioSR.In this study,the molecular mechanism by which NioSR regulates denitrification system gene norCBD to promote skin wound infection were further studied.The qPCR results demonstrated that nitric oxide reductase subunit gene norC was significantly down-regulated in △nioSR strain under both aerobic and anaerobic conditions.Furthermore,in vitro nitric oxide killing assay showed that the resistance of △nioS,△nioR and △nioSR to nitric oxide killing was significantly reduced compared with WT,indicating that NioSR play an important role in resistance to nitric oxide killing by regulating the nitric oxide reductase genes norC.The △norCBD strain showed significantly impaired ability of skin wound infection,indicating that norCBD play an important role in skin wound infection.In summary,NioSR can degrade nitric oxide through regulating the nitric oxide reductase genes norCBD to resist nitric oxide killing and improve the ability to infect skin wounds.Finally,to determine whether NioSR could interact with other regulators of denitrification pathways,anr,dnr,atvR,narXL genes deletion mutants of the WT and △nioSR strains were constructed.Then,Transcriptional expression levels of different genes in different knockout strains under anaerobic conditions were detected.The results showed that ANR regulated the expression of dnr and narXL,and inhibited the expression of atvR.Dnr and NarXL slightly interacted with each other.Those results were consistent with the previous reports.While NioSR inhibited expression of atvR and narXL,but ANR and DNR had no effect on the expression of nioSR.Those results indicate that NioSR is a novel denitrification regulation system which is not regulated by other regulators of denitrification system.Taken together,this study demonstrates that three new two-component systems of P.aeruginosa regulate the expression of virulence factors to improve the ability of skin infection.Furthermore,the molecular mechanism by which NioSR imprve the degradation of nitric oxide by regulating the expression of nitric oxide reductase norCBD during skin wounds infection was clarified.This study provides new insights into the treatment clinical burn and skin wound infected by P.aeruginosa.