The Signal Pathways Of Catecholamine Hormones Mediated By Two-component System NarQ/P In Actinobacillus Pleuropneumoniae

Microbial endocrinology is an emerging research field in recent years,which aim to study the interaction between bacterias and the host hormones.Actinobacillus pleuropneumoniae(APP)is an important pathogen that caused economic losses in the pig production by inducing porcine pleuropneumonia in pig.APP takes five two-components systems inside,including YgiY/YgiX,NarQ/NarP,CpxA/CpxR,ArcB/ArcA,PhoR/PhoB,involved in many progress of a wide range of aspects in APP.While except ArcA/ArcB,these TCSs is still not clearabout their functions.The previous study of our group have found that there are two TCSs of APP,YgiY/YgiX and NarQ/NarP,regulated by the catecholamine hormones epinephrine(Epi)and norepinephrine(NE).In this work,we have studied the NarQ/NarP of the two TCSs.By researching its functions and mechanism to sense hormone signals,we can have a better understand to the pathogenesis of APP and it can contribute to the exploration of new drug targets.In this work,we have studied the cross phosphorylation between NarQ and five response regulators of APP by labellinng the phosphate group with?-³²P,and identify the target genes of NarP by Electrophoretic Mobility Shift Assay(EMSA)and fluorescent report vectors.We have studied the difference in growth between APP mutant?narP and wild type,too,by measuring the growth curve.This study obtained the following main results:1.Identifiction of target genes of NarP by EMSABased on the genes screening by DAP-Seq that can bind to NarP,we choose 18genes involved in metabolism,virulence,motility and so on.By EMSA,we make a further verification to these 18 genes.The results have showed that 14 of the 18 genes can bind to NarP,and the majority of the 14 genes involved in iron metabolism.2.Construction and application of reporter vector pBT-promoter-GFPFor verifying NarP regulate expression of genes in vivo,fluorescent report vectors of 18 genes above have been constructed.Clone the reporter gene GFP to the pBT vector at first,and then insert the promoter of genes to the upstream of GFP,finally get the pBT-promoter-GFP reporter vector.And then,transform both of the fluorescent report vectors and the prokaryotic expression vector of NarP to E.coli BL21 competent cell,through the function of reporter gene,we studied the regulation of NarP to genes in vivo.The results showed that 6 of the 18 reporter vectors can express GFP protein successfully,and 5 of the 6 genes were inhibition by NarP and the fluorescence of bacterias receded significantly.These 5 genes include appser1?11590(hgbA),appser1?14210,appser1?16100(ftpA),appser1?16110 and appser1?20850(mutL).3.Measuring of the growth curve of APP mutant?narP and wild typeAdditionally,we have measured the growth curve of APP mutant?narP and wild type under different conditions,and study the impact of hormones to APP in growth under different conditions.The reasults have showed that mutant?narP grow faster than wild type under iron-limited condition,while there's no obvious difference between them under common,anaerobic or hyperthermy conditions.Moreover,hormones could promote the growth of APP,both to?narP and wild type.According to all the results of the research above,we find that NarP mostly involved in iron metabolism in APP.In the research of NarP by reporter vector,NarP inhibit the expression of genes involved in iron metabolism,suggesting that NarP may inhibit the iron uptake of APP,and the following phenotypic experiments verified this suppose.