Investigation Of The Interaction Between Metabolism And Virulence Expression In Edwardseilla Piscicida With A Defined Transposon Mutant Library

Transposon is a DNA fragment that can jump on chromosome and has been designed to be a tool for transposon insertion mutant library construction.We designed a transposon pMKGR based on Mariner Himarl transposon pMar2xT7.pMKGR,which was labelled with gentamicin resistant and mCherry,and carried promoterless genes for kanamycin resistant and EGFP.Using pMKGR,a defined transposon mutant library of E.piscicida EIB202 was successfully constructed,which contains 20,668 mutants.The defined transposon mutant library disrupted 2,806 genes and the rate of coverage of EIB202 genome?3,559 genes?was 78.0%.According to the analysis of the library and neutral-base model,it was predicted that there were 464 essential genes on the chromosome of EIB202.Five different subset libraries were separated from the parent library for various screens.The analysis of in vivo and in vitro screening revealed that energy metabolism,e.g.mannose and lipid metabolism,modulates virulence in E.piscicida.In terms of mannose metabolism,after its phosphorylation to yield mannose-6P by ManXYZ,the mannose was transported into cells.Subsequently,mannose,6P was converted into fructose-6P and entered into glycolysis pathway.Further experiments revealed that mannose-6P could enhance the expression of type ? and type VI secretion system?T3/T6SS?via directly binding to ETAE₂071?EvrA?,a DeoR family protein.EvrAR141 and EvrAR221 were experimentally affirmed to be the essential amino acids for the interaction of EvrA and mannose-6P.Mutation in these two residues abolished the binding and regulation of esrB by EvrA.In addition,we discovered that T3/T6SS expression was modulated by lipid metabolism,and unsaturated fatty acids?UFAs?level were negatively correlated with T3SS expression level?R2=0.981?.Free long chain unsaturation fatty acids directly interacted with EsrC and abolished the function of EsrC binding to DNA,which lead to turn off T3/T6SS.EsrCR38 were confirmed to be the key site interacted with UFA to control T3 SS and T6SS expression.UFAs could make some important contribution more than regulating T3/T6SS expression during E.pis cicida invasion and colonization.After E.piscicida wild type?WT?infection,the number of LDs in host cells was significantly decreased.Evidently,the SCD1 expression and the LDs formation in host cells were activated during E.piscicida ?T3SS infection.Furthermore,inhibition of E.piscicida T3SS to LD formation in host cells was dependent on specific T3SS effectors but not T3SS needle structure.It was experimentally found that UFAs significantly inhibited the colonization of E.piscicida WT and AesrC in HeLa cells,and the decrease of E.piscicida colonization resulted in UFAs might be independent on the interaction of EsrC and UFAs.The colonization level of E.piscicida in SCD1-/-cells was significantly higher than in wild type HeLa cells,and this further confirmed the inhibition of UFAs to E.piscicida colonization in host cells.Furthermore.it was confirmed that oleic acid helped zebrafish to be protected from E.piscicida infection and the SCD1 knocked out zebrafish showed more sensitive to E.piscicida infection.Overall,UFAs could enhance the ability of host both to resist pathogens and to regulate down the expression of T3/T6SS of E.piscicida.Collectively,pathogens use the metabolites as molecular signal to modulate virulence expression as their important strategy to adpate various environments.This study comprehensively investigates the interaction between metabolism and viruelence expression in E.piscicida,which provides important guidance to aquaculture industry.