Regulation Mechanism Of Response Regulator EsrB In Edwardsiella Piscicida

As a kind of broad-host-range gram-negative pathogen,Edwardsiella piscicida causes hemorrhagic septicemia in many kinds of fish species and leads to huge loss in aquaculture.Thus,it becomes more and more important to have a full understanding on the pathogenicity mechanism of E.piscicida.At present,the investigation on the pathogenesis of E.piscicida is mainly focusing on the type ? secretion system(T3SS),type VI secretion system(T6SS),and the important virulence regulation,including a two-component system EsrA-EsrB.We previously found that the function of EsrB is not only to regulate virulence,but also to take part in other important processes such as growth,metabolism and environmental adaptation.The investigation of the regulation network of EsrB under various environments and growth conditions will help us to deeply and comprehensively understand the virulence mechanism of the bacterium.In this work,when E.piscicida EIB202 was cultured in DMEM,the expressions of EsrB and T3SS/T6SS proteins were high,while T3SS/T6SS proteins were undetectable when cells grew in LB.The transcription of esrB gene in LB culture was demonstrated with the transcriptional fusion fluorescence assays.Using comparative proteomics,we analyzed the difference of the proteoms of wild-type strain and AesrB under DMEM or LB culture conditions,respectively.In summary,we found 28 proteins that might be regulated by EsrB.Under the condition of DMEM,7 proteins were upregulated by EsrB,and the expression of 12 protein was inhibited by EsrB.While cultured in LB,the expression of 5 proteins depended on EsrB,and the expressions of genes encoding other 4 proteins were repressed by EsrB.The 28 proteins were demonstrated their roles in various pathways,include reactive oxygen species(ROS)related protein(SodB)and glutamine synthetase(GlnA).It was also demonstrated in further experiments that EsrB could participate in the regulations of ROS resistance pathways and glutamine synthesis pathway.A genome-wide screen of upstream regulators of EsrB network was conducted.The technologies of transposon insertion site sequencing(TIS)and pull-down assays with DNA fragments as bait were employed for this purpose.According to the results of TIS screening,there were 39 putative regulators of the EsrB in E.piscicida EIB202,23 of which might be activators,and the rest 16 be repressors.Interestingly,RpoS,as a sigma subunit of RNA polymerase related to the general stress response,acted as a repressor of EsrB.The pull-down screening results indicated that a leucyl aminopeptidase PepA could directly bind to the promoter of esrB.In-frame deletion mutation,qRT-PCR,ECPs,transcriptional fusion fluorescence detection and self-aggregation experiments proved that RpoS and PepA acted as inhibitors in the expression of EsrB.Meanwhile,RpoS also mediated the regulation of environmental stresses on the expression of EsrB.The molecular mechanism of RpoS to control EsrB expression was studied by the combination of multi-omics methods.The binding sites of RpoS on the promoter of esrB were uncovered according to DNase I footprinting and RNA-seq,while the antagonism relationship between RpoS and RpoD in the binding of esrB promoter was found by point mutation and in vitro transcription analysis.The-6G key base in the discriminator sequence on esrB promoter and R99 residue constituted the essential interaction pair for RpoS repression of esrB transcription.Finally,ChIP-seq and RNA-seq data sets were combined along with statistical analysis to identy a general mechanism for the RpoS repression of gene expression in EIB202 or possibly in other bacteria.Most important of all,our findings supported the hypothesis that RpoS is involved in the balance of fine-tuning self-protection and nutrional competence(SPANC)balance,and that this trade-off mechanism also involves in the virulence repression in response to environmental stresses.In this study,iron uptake systems of EIB202 were comprehensively identified by screening the transposon insertion mutant library.Combined with RNA-seq data,it was proved that there was direct regulatory relationship between EsrB and iron uptake system.By analyzing ChIP-seq data and phenotypic identification of Fur overexpressing strain,it was proved that Fur could regulate and control the expression of T3SS/T6SS related proteins through EsrB and ultimately affect the virulence.With the help of EMSA,in vitro transcription and intracellular cAMP assays,it was found that Fur could regulate intracellular cAMP level by directly regulating Icc,and further regulate the pathways dependent on the cAMP.Collectively,these studies elucidated a complicated regulation network of EsrB and would facilitate the deepened understanding of the pathogenesis of Edwardsiella bacterium.