The Pathogenic Mechanism Of Two Effector Proteins Of Vibrio Alginolyticus T3SS And The Antibacterial Effect Of Innate Immune Factors Of Trachinotus Ovatus

T3SS is an essential system for Vibrio alginolyticus to infect hosts.Previous studies in our laboratory have identified the T3SS effector proteins(HopPmaJ and Va1686),the transporter protein(Type3hy322),and the molecular chaperone proteins(VscO and VscX)in V.alginolyticus HY9901 strain.However,the structure of the T3SS effector coding region of strain HY9901,the chaperone proteins corresponding to the effector proteins VopR and VopQ,and the function of the critical effector protein VopR remain unclear.It has been shown that fliR,a structural protein of T3SS and flagellum,is a key gene for bacterial infection of the host,but its mechanism of role in V.alginolyticus is unknown.Golden pompano(Trachinotus ovatus)is considered as an excellent mariculture species and suffers from Vibrio infection during extensive farming.The innate immune system plays an essential role in responding to pathogenic invasion,and it is important to investigate the molecular mechanism of innate immune response to Vibrio infection in golden pompano to promote the healthy and sustainable development of this industry.This present study used bioinformatics analysis,yeast two-hybrid and protein pull down to study the structure of the T3SS effector coding region of V.alginolyticus HY9901 and the cognate partner proteins corresponding to the effector proteins VopR and VopQ;Meanwhile,gene knockout,biological phenotyping,and transcriptomic analysis were conducted to explore the role of the effector vopR on V.alginolyticus.Additionally,subcellular localization,overexpression,transcriptomic and proteomic analyses were performed to explore the effects of vopR on the host.A combination of genome-wide screening and molecular biology approaches was used to identify immune-related genes in golden pompano and to investigate their role in resistance to V.alginolyticus infection.Finally,fliR was knocked out and the feasibility of ΔfliR as an attenuated live vaccine was evaluated;the results were as follows:1.Analysis of the effect coding region of V.alginolyticus HY9901 T3SSThe T3SS effector region of both V.parahaemolyticus RIMD2210633 strain and V.alginolyticus HY9901 strain contained the same effector proteins,both containing VopS,VopQ and VopR,with 81%,81% and 82% similarity in the three effector genes,respectively.Differently,T3SS effector region of V.harveyi QT520 strain contains vopQ and vopR,while only vopQ is identified in T3SS effector region of V.campbellii ATCC BAA-1116 strain.A phylogenetic study was conducted based on the nucleotide sequences of the effector coding regions.The evolutionary tree showed that V.alginolyticus HY9901 was clustered with other serotypes.The chaperones of the V.alginolyticus effector proteins VopR and VopQ were confirmed,VAGM003377 and VAGM003375,respectively.2.Effect of vopR deficiency on V.alginolyticusThe vopR gene is 954 bp long that encoding 317 amino acids and shows significant similarity to its homologs of other Vibrio species.Knock down of vopR resulted in 275 genes down-regulated and 254 genes up-regulated in comparasion with wild strains.These differentially expressed genes were mainly enriched into biological processes including cellular processes,localisation,biological regulation.These DEGs were also involved in molecular functions including catalytic activity,binding,and transporter activity,and cellular composition including membranes,cellular fractions,etc.There are mainly associated with carbohydrate metabolism,amino acid metabolism,nucleotide metabolism,energy metabolism,cell motility,membrane transport,and signal transduction.There were no significant differences between the mutant and wild strains for in vitro cell infection and in vitro infection did not cause apoptosis but stimulated an inflammatory response in cells.3.Functional characterisation of vopRSubcellular localization showed that VopR protein localized on the host cell membrane,and only full-length vopR could localize on the membrane and round the cell.The VopR protein binds to phosphatidic acid(PA),Ptd Ins(3,5)P2 and Ptd Ins(4,5)P2 on the host cell membrane.The experiment of overexpressing vopR in grouper splenocytes(EAGS)showed that the ubiquitination system of host cells was activated by vopR.Meanwhile,vopR can induce apoptosis through the activation of the MKK pathway.The results of the EAGS overexpression vopR transcriptome showed that a total of 410 genes were upregulated and 207 genes were downregulated.These significantly different genes KEGG analysis was mainly enriched to metabolic pathways,including cancer pathways,MAPK signaling pathway,TNF signaling pathway,and TGF-β signaling pathway.The EAGS overexpression vopR proteome results showed that a total of 659 proteins were upregulated and 1189 proteins were downregulated.Significantly different genes KEGG analysis was mainly enriched to metabolic pathways,including glycerophospholipid metabolism,cell adhesion molecules,drug metabolism-cytochrome P450,complement and coagulation cascade reactions,and steroid biosynthesis.4.Identification of innate immunity-related genes in golden pompano and their function against V.alginolyticusIn this study,we identified 2 tlr13 and 1 tlr23 genes in golden pompano by whole-genome screening,and their transcription patterns in response to various immune challenges were investigated.The open reading frames(ORFs)of,Totlr13-1,Totlr13-2 and Totlr23 are 2868,2844 and 2802 bp in length,encoding 955,947 and 933 amino acids,respectively.Multiple sequences alignment and protein structure analysis showed that these TLR receptors share high sequence homology.Phylogenetic analysis showed that Tlr13 comprises 2 subclades including Tlr13-1 and Tlr13-2.These three tlrs are extensively distributed and show a consistent pattern,with high expression levels in brain,kidney and spleen.The results showed that Totlr13 s and Totlr23 were involved in the immune response of various tissues against Vibrio alginolyticus.we report two caspase8 paralogs,termed as ToCas8 and ToCas8-like,from golden pompano.The two proteins encoded by ToCas8 and ToCas8-like were characterized by multiple sequence alignment and protein structure analysis showing similarity to their counterparts in vertebrates and exhibiting typical features of the Caspase8 protein family.Gene synteny analysis further confirmed that two isoforms of caspase8 genes exactly existed in golden pompano.Phylogenetic analysis showed that the Caspase8 proteins of the selected species in the study were clustered together,and Cas8 and Cas8-like were clustered into two different subgroups.In addition,ToCas8 and ToCas8-like were widely distributed and both were expressed at high levels in gill,kidney and intestinal tissues.Transcript levels of ToCas8 and ToCas8-like were significantly upregulated in response to V.alginolyticus challenge,suggesting that caspase-8 genes are involved in the immune response to pathogens.These findings will expand the understanding of the molecular mechanism of the TLR11 superfamily and Caspase8 in responding to the stimulation of V.alginolyticus,and contribute to the future healthy breeding of golden pompano.5.Characterization of fliR deletion mutants and evaluation as live attenuated vaccinesThe fliR gene of V.alginolyticus was 783 bp in length,encoding 260 amino acids,and showing significant similarity to homologs of other Vibrio species.The fliR-deletion mutantΔfliR of V.alginolyticus was successfully constructed,and its biological phenotype analysis showed no significant differences in growth capacity and extracellular enzyme activity compared to the wild-type.However,a substantial reduction of motility ability was detected in ΔfliR.Transcriptomic analysis revealed that the absence of fliR gene is responsible for a significantly decreased expression of flagellar genes,including flaA,flaB,fliS,flh B and fliM.The fliR-deletion mainly affects the related pathways involved in cell motility,membrane transport,signal transduction,carbohydrate metabolism,and amino acid metabolism in V.alginolyticus.The efficacy of ΔfliR as a candidate of live attenuated vaccine were evaluated by intraperitoneal injection in golden pompano.The results showed ΔfliR effectively improved the immunity of inoculated fish.The results suggest that ΔfliR is an effective live attenuated vaccine against vibriosis in golden pompano.Therefore,the structure of the effector coding region of V.alginolyticus type Ⅲ secretion system and its chaperone proteins were investigated.In addition,the innate immunity-related genes of Trachinotus ovatus and their response to Vibrio stimulation were identified;finally,the essential virulence factor fliR deletion mutant of V.alginolyticus was constructed,its biological phenotype was analyzed and its effectiveness as a live attenuated vaccine was evaluated.The study results provide further insights into the pathogenesis of V.alginolyticus,a critical fish pathogen,and are of great importance for the development of fish vaccines.