Function Of Genomics And Selection Of Conditional Essential Loci In Edwardsiella Piscicida

Edwardsiella tarda is a bacterial pathogen that causes pandemics in both fresh water and marine fish,and it can also cause diseases in human beings,representing as one of the leading zoonotic pathogen.With the whole genome sequence determined,and comparative genomic analysis performed,E.tarda is yet understudied in respect of functions of most of its genes.In this thesis,transposon mutagenesis and proteomics approaches have been applied to study the functional genomics of E.tarda EIB202.Firstly,a comprehensive transposon mutant library was contructed by insertion of transposon into the EIB202 genome,massively singling out the mutants and dterminging insertion site.The transposon pMKGR,a derivative of Mariner transposon pMar2XT7,was constructed with constitutively expressed mcherry,and promoterless gfp and kanamycin resistant cassette.The transposon mutant library of EIB202 strain,composed of 20325 single mutants,and each mutant insertion site was determined by semi-arbitrary PCR and sequencing.The overall results showed that the mutant library covers 2776 coding sequences,which are 77%of all the predicted ORF.This is the first massive site-determined single colony mutant library of Edwardsiella species,and it can be further applied for functional analysis of locus under desired conditions,and gene expression study based on the examination of reporters on the transposon.Secondly,the transposon insertion sequencing technique?TIS?was applied,which combines the massive transposon mutant library and high-throughput sequencing,to determine and quantify the mutants.Therefore,the consortium of genes that required under certain condition can be tracked and selected.In this study,a massive EIB202 mutant library consisted of 300000 CFU were generated by the Mariner transposon pMar2XT7.498 genes were determined as essential genes under LB culture condition by statistical and simulation methods.The mutatnt library was selected under various in vivo and in vitro conditions.The LB continuous passage selection unraveled 221 conditional essential genes;180 conditional essential genes were determined under the DMEM culture,which mimicks the in vivo of macrophage environment and activates the T3SS and T6SS expression;82 and 130 conditional essential genes were selected in the natural sea water at 16? and 28?;the J774.1 cell model was also used to select genes required for escape the macrophage phagocytosis,86 conditional essential genes were identified.The in vivo conditional selection was conducted in the E.tarda natural infection host,turbot fish {Scophthalmus maximus L.).Overall 228 conditional essential loci required for infection in the liver were selected.The in vivo conditional essential loci include major virulence genes such as type III and type VI secretion systems?T3SS,T6SS?,and novel unreported pathogenesis related genes.They were classified into multiple functional categories and validated.Furthermore,compared with traditional TIS which only use the input and output comparisons,we extanded the selection to multiple time points.During the 14-day infection window,mutant library was recovered from 6 time points.The conditional essential genes were selected along with the procedure from invasion to systemic infection.A mothed termed as pattern analysis of conditional essential loci?PACE?was built to cluster the dynamic curves of each mutants during the in vivo selection.A significant pattern was selected and named as "Mitifits",which describes the slow in vivo clearance rates of the mutants related to most T3SS and T6SS genes.This revealed that T3SS and T6SS mutants can survive in the host for certain amount of time,while cannot cause disease as wildtype strain does.The in vivo fitness pattern was further applied to select live attenuated vaccine targets by a heuristic searching based on the known live attenuated vaccine targets.89 target loci were selected,and 5 new genes were applied,i.e.pabA,pabB,ETAE₂260,ETAE₁614,and ETAE₀023.The corresponding deletion mutants were constructed,and the in vivo survival curves were validated.The mutants were able to activate the immune response from the host.The challenge experiments showed that the relative protection ratio?RPS?were higher than 60%,and the pabB,ETAE 2260 mutant strains displayed higher RPS than known vaccine strain WED.In the third part,the protein and protein interaction?PPI?network were also constructed for all protein coding sequences in EIB202.The 3599 proteins were applied for PPI network construction by bioinformatic methods,the interolog and DDI.Overall 139041 pairs of protein protein interactions were predicted for a massive interaction network,covering 2514 proteins,which represents the first massive protein and protein interaction network for Edwardsiella species.The hub proteins in the network were applied to machine learning methods for the selection of new subunit vaccines,and 15 novel potential antigens were identified.The PPI and identified antigens will set the basis for the future subunit vaccine development to combat E.tarda infection in fish.