Structure Analysis Of Key Virulence Regulators EnrR,FabR And EvrA In Edwardsiella Piscicida

Edwardsiella piscicida,a Gram-negative bacterial pathogen,can cause edwardsiellosis,which severly inflicts the global aquaculture industries and results in huge economic losses.Understanding of the virulence mechanisms of E.piscicida will facilitate the development of novel vaccines against the pathogen.Type ? secrection system(T3SS)and type ?secrection system(T6SS)are the key virulence determinants in the bacterium.E.piscicida encodes several key virulence regulators including the recently identified novel regulatory factors,FabR,EnrR and EvrA with our transposon insertion sequencing platform.Among them,FabR binds to unsaturated fatty acids and allosterically regulates the biosynthesis of fatty acids and the expression of T3SS.EnrR is a novel nucleoid-associated protein(NAP),binding to the low G+C loci in the chromosome to globally regulate the viruelnce of E.piscicida.EvrA might be able to bind to mannose-6-phosphate(Man-6P),which can exert feedback regulatory roles in the transportation of mannose from host cells and meanwhile control the expression of T3SS/T6SS.In this study,we constructed the Escherichia coli strains to express FabR,EnrR and EvrA proteins,respectively.We used various fusion tags to express these proteins,and optimized the expression plasmids,incubation temperatures and purification proecesses to be able to obtain highly purified FabR,EnrR and EvrA proteins.As for FabR,we purified the proteins with His6-MBP tag,and found out the conditions used for the crystalization of the protein.The resolution we obtained for the His6-MBP-FabR is only of 4.5 A through X-ray crystal diffraction.EnrR was purified by fusing the His6 tag in the N-terminal,which facilitated the affinity chromatography purification of the protein and the following crystalization.X-ray crystal diffraction analysis revealed its resolution of 2.60 A.We tried two methods to resolve the atomic coordination,i)selenomethionine replacing methionine to obtain the 2.60 A EnrR of selenomethionine protein,and ?)brominated DNA to successfully resolve the resolution of EnrR and DNA complex is 1.70 A.Importantly,we obtained the structure of EnrR and complex structure of EnrR packing DNA,elucidated the ineraction between EnrR and DNA,and obtained the potential binding key residues,R47 and R55.As for EvrA,because of the formation of highly oligomerization and inclusion body,EvrA was fused with His6-SUMO tag to functionally express truncated protein,which is the functional binding domain for small moleculers in the C-terminal.Our results showed that truncated EvrA was still in a high-polymerization state.Our results also confirmed the direct interaction between EvrA and Man-6P through isothermal titration calorimetry(ITC).This project explored the expression,purification and crystallization structure of these three regulatory factors,which will facilitate the elucidation of molecular mechanisms of key regulators in gene expression in E.pisicicida.