Molecular Mechanism Of Global Regulator Esrb Regulating The Expression Of Virulence Determinants In Edwardsiella Piscicida

Edwardsiella piscicida is a Gram-negative broad-host-range pathogen that causes hemorrhagic septicemia in many commercially important fish species.A better understanding of E.piscicida pathogenesis may help to produce safe and effective vaccine to prevent edwardsiellosis.Several virulence determinants have been reported to be involved in the pathogenesis in this bacterium,including T3SS,which enables it to escape phagocytic killing and to replicate intracellularly in phagocytes,and T6SS,which also contributes to establish colonization inside the host.The two-component system?TCS?EsrA-EsrB has been shown to be essential to regulate the expression of T3SS and T6SS.However,the overall regulatory roles of EsrA-EsrB on the virulence and physiology in Edwardseilla bacterium is still unknown.In this study,we compared the transcriptomes of E.piscicida wild type and ?esrB strains cultured in different conditions,analysed differently expressed genes and provided insights into host adaptation and virulence genes regulated by EsrB.We identified 6 novel T3SS-dependent effectors translocated into host cells and required for in vivo colonization and virulence.Furthermore,we identified two genes with conserved binding box regulated directly by EsrB,and also solved the structure of the C-terminal DNA binding domain of EsrB by X-ray.7 extracellular proteins of T3SS and T6SS were detected when EIB202 WT was cultured in DMEM,but were absent in WT grown in LB or ?esrB in DMEM,which indicated that EsrB activates the expression or secresion of T3SS/T6SS in DMEM.Then we performed RNA-seq analysis with WT and ?esrB RNA isolated from LB and DMEM cultures.There were 104 co-upregulated genes and 191 co-downregulated genes in these comparisons,30 of 35 T3SS cluster genes?ETAE₀854-ETAE₀888?and the whole 16 T6SS cluster genes?ETAE₂428-ETAE₂443?were significantly upregulated more than 4-fold,which comprise over 40%of the co-upregulated genes.295 differentially expressed genes were classified via COG categories.Genes associated with intracellular trafficking and secretion were highly induced,including T3SS,T6SS and iron acquisition system.Genes associated with energy production,metabolism,protein biosynthesis and cell motility were significantly repressed,including hemolysins,adhesin and outer membrane proteins.Interestingly,plasmid pEIB202 genes associated with antibiotic resistance and T4SS were induced by EsrB and LB condition,although the plasmid is not involved in the bacterial virulence and colonization in fish.These data indicated that EsrB plays important role in adaptation and virulence of EIB202.We identified a total of 9 candidates,among the 104 upregulated genes,showed positive extracellular secretion and intracellular translocation in HeLa cells based on extracellular protein?ECP?profiles and TEM-1 reporter system.EseG?ETAE₀866?is a known Edwardsiella T3SS effector,which mediates microtubule disassembly.EseJ?ETAE₀888?is a newly identified T3SS effector,which inhibits bacterial adhesin to EPC cells and facilitates intracellular replication.EseH?ETAE₁757?was recently identified as a T3SS effector which is located into host nucleus and inhibits phosphorylation of JNK/MAPK pathways in host cells.ETAE₁586 has a papain fold toxin domain and may inhibit host inflammation,ETAE₁604 has an exosort_VPLPA motif and may involve in secretion and translocation of proteins,ETAE₂186 is a putative thioredoxin,ETAE₂188 has a conserved DUF1471 domain,EvpJ?ETAE₂438?has a PAAR like motif and may facilitate T6SS-mediated secretion,ETAE₃282 is an unknown protein without any conserved motif.All 9 candidates were translocated into HeLa cells in a T3SS-dependent manner,while T6SS appeared to modulate the EvpJ and ETAE₂188 translocation.9 effector genes were highly upregulated by EsrB when EIB202 infected J774A.1 and turbot.Cl-seq indicated that AeseJ showed marginal reduction in CI at 3 DPI,but significant decrease at 7 DPI,demonstrating the essential roles of EseJ in in vivo colonization.The mutant with absence of all the 9 putative effectors?9??displayed dramatic colonization deficiency as that of AesrB,?T3SS and ?T6SS mutants.Furthermore,9A showed attenuated virulence,defective colonization and high protection towards wild-type challenge.EMSA analysis indicated that EsrB and EsrBc?142?214 aa?directly binding to the promoter regions of esrC and esaM,but not to esaB and eseJ.The binding box identified by footprinting was highly similar to SsrB DNA recognition motif which is 7'-4-7" tail-to-tail palindrome sequences.X-ray crystal structure showed EsrBc consists of 3 ?-helix.However,all of NarL homologues contain four ?-helices.The a-helix 1 and a-helix 2 of EsrBc are similar to NarL family proteins while a-helix 3 is much longer.Thus we proposed that there should be a domain reorganization when EsrBc bound to DNA to form a-helix 4 dimerization interface.Based on the crystal structures of NarLc-DNA and DosRc-DNA,we predicted that residues Lys181,Glu184,and Leu188 are important for EsrBc binding to DNA and Thr182 makes nonspecific contact with the phosphate backbone.