Structural Analyses And Metabolomic Regulatory Roles Of Response Regulator EsrB In Edwardsiella Piscicida

As a kind of facultative anaerobic Gram-negative bacteria,Edwardsiella piscicida can infect a wide range of economically cultured fish and cause hemorrhagic sepsis,which often leads to serious economic losses.As one of the important virulence regulators in E.piscicida,EsrB regulates the Type Ⅲ and Type Ⅵ secretion systems（T3/T6SS）in response to a variety of environmental signals,facilitating bacterial colonization in macrophages and leading to systemic infection in fish.Current researches mainly focus on EsrB’s response to environmental signals,while the sophisticated molecular mechanisms of DNA binding profiles of EsrB remain unclear,neither of the regulatory roles of EsrB on various metabolic processes.In this study,the crystal structure of the C-terminal of EsrB（EsrBc）determined at 2.20-A resolution was firstly solved after the failure of attempts to resolve the full length EsrB due to its prone to precipitation.Based on the electrophoretic mobility shift assays（EMSAs）,we confirmed that the crucial DNA-binding residues of Lys181,Glu184,and Leu188 enable the DNA recognition process.Moreover,Cys49 was involved in the dimerization of EsrB through a disulfide bond,while Leu202 was involved in the formation of EsrBC dimerization interface with DNA.Next,according to the metabolomics data analysis,EMSA,qRT-PCR,and pyruvate dehydrogenase activity assay,we found that EsrB played an important role in regulating the basal metabolism of E.piscicida,including pyruvate cycle（P cycle）,tricarboxylic acid cycle（TCA）,glutamic acid metabolism,and fatty acid anabolism.Based on EMSA and qRT-PCR analyses,the genes including ETAE₀293,ETAE₂868,and ETAE₂047,were upregulated,while the genes,including ETAE₂586,ETAE₂826,and ETAE₂587,were downregulated inΔesrB.Moreover,as one of the key enzymes,pyruvate dehydrogenase（PDH）in P cycle was found to be significantly inhibited in ΔesrB,indicating an activation role of EsrB on this metabolic process.Moreover,the binding ability of FabR to its target DNA was activated by oleoyl-CoA and inhibited by stearoyl-CoA.Analyses of EMSA and DNase I footprinting with wild-type and a null mutant（F131A）of FabR indicated that FabR also bound to the promoter region of esrB for its activation and Phe131 was the key amino acid site FabR binding to fatty acyl-CoA.Moreover,FabR modulated the expression of esrB in coordination with RpoS as revealed by analyses of extracellular protein patterns,qRT-PCR and bacterial survival in turbot.In conclusion,we unveiled the specific molecular mechanism of EsrB regulation of virulence factors from molecule structure determination followed by analyses of metabolic processes modulated by EsrB.The results demonstrated that virulence regulation will usually accompany the reprograming of metabolic processes in bacterial pathogen.These findings will improve our understanding of EsrB and its key regulatory roles in Enterobacteriaceae family and facilitate the development of novel live attenuated vaccine and strategies to combat the bacterial infection diseases.