| Vibrio harveyi is an important pathogen of aquaculture animal vibriosis,which can infect a variety of fish,shrimp,etc.and cause a large number of deaths,causing huge economic losses and causing widespread concern.However,the pathogenic mechanism of V.harveyi is currently unclear.Therefore,studying the functions of virulence-related genes of V.harveyi is important to find out the pathogenic mechanism.In this thesis,V.harveyi 345 was used as the research object,and 16 kinds of mutants of hypothetical virulence genes were constructed using homologous recombination technology.The biological characteristics were analyzed,and the mutants with significant differences in biological characteristics were analyzed.Construct corresponding gene complementary strains,analyze the expression differences of virulence-regulating genes and virulence genes,and further evaluate the virulence,colonization ability and host immune gene expression of each strain on pearl gentian(♀ Epinephelus fuscoguttatus ×♂Epinephelus lanceolatu)The effects of the virulence gene of V.harveyi in its pathogenesis were explored.The specific research results are as follows:1.Construction of mutant and complementary strains of V.harveyiHomologous recombination technology was used to construct hypothetical virulence gene deletion mutants ΔflaA,ΔflaB,ΔflrBC,ΔflhF,ΔvscCD,ΔluxS,ΔcqsA,ΔluxR,ΔaphA,ΔexsA,ΔtoxR,ΔhlyⅢ,Δvhh,Δna1,Δna2 and Ana3.ΔflhF/pMMB207-flhF,ΔvscCD/pMMB207-vscCD,ΔluxS/pMMB207-luxS,ΔcqsA/pMMB207-cqsA,ΔtoxR/pMMB207-toxR,and Δna3/pMMB207-na3 were also constructed ascorresponding genes complementary strains of ΔflhF,ΔvscCD,ΔluxS,ΔcqsA,ΔtoxR,and Ana3 with significant differences in biological characteristics2.Biological characteristics of wild strains of V.harveyi,hypothetical virulence mutants and complementary strainsFor the growth,mobility and surging of wild strains of V.harveyi and mutants of various genes,secretion of extracellular proteases,resistance to HCl,H2O2,Cu2+,absorption of iron ions,resistance to antibiotics and biofilms,etc.The test shows thatΔflaA,ΔflaB,ΔflrBC,ΔluxR,ΔaphA,ΔexsA,ΔhlyⅢ,Δna1,and Δna2 have no difference in the above biological characteristics compared with their wild strains,but ΔflhF,ΔvscCD,ΔluxS,ΔcqsA,ΔtoxR,Δvhh,and Δna3 show significant differences in some of these biological characteristics.(1)ΔflhF significantly reduced resistance to the antibiotics flufenicol and chloramphenicol(P<0.05),indicating that flhF is involved in the antibiotic resistance mechanism of V.harveyi and is regulating the antibiotic resistance of V.harveyi features.The biofilm formation capacity of ΔflhF was significantly enhanced(P<0.001),indicating that flhF negatively regulates the expression of biofilm-related genes.(2)The survival rate of vscCD gene mutant in the environment of pH=3.1 HCl and 0.2%H2O2 gradually decreased,and its survival rate was significantly lower than that of wild strains during 0.5 h-2 h,indicating that vscCD gene mutant is resistant to strong acids and strong oxidizing environments.It is speculated that vscCD positively regulates the expression of stress-resistant genes of V.harveyi.The resistance of vscCD gene mutant to fluphenicol and chloramphenicol was significantly reduced(P<0.05),indicating that vscCD is regulating the antibiotic resistance function of V.harveyi.(3)The swimming and surging diameters of ΔluxS significantly increased,and the difference was extremely significant(P<0.01),indicating that luxS negatively regulates the motor function of V.harveyi.The extracellular protease secretion capacity of ΔluxS was significantly reduced(P<0.01),indicating that the secretion function of extracellular proteases requires the participation of luxS.It is speculated that luxS is regulating the secretion of extracellular proteases.(4)The swimming and surging diameters of ΔcqsA significantly increased,and the difference was extremely significant(P<0.01),indicating that cqsA negatively regulated the motor function of V.harveyi.The extracellular protease secretion capacity of ΔcqsA was significantly reduced(P<0.05),indicating that cqsA is regulating the secretory function of extracellular proteases.The survival rate of cqsA gene mutant was gradually decreased under the environment of 30 mM DIP and 0.2%H2O2.After stimulation for 0.5 h,the survival rate was significantly higher than that of wild strain.Increased,it is speculated that cqsA negatively regulates the expression of stress-related genes of V.harveyi.The biofilm formation ability of ΔcqsA was significantly enhanced(P<0.001),indicating that cqsA negatively regulates the expression of biofilm related genes.(5)The biofilm formation ability of ΔtoxR was significantly enhanced(P<0.001),indicating that toxR negatively regulates the expression of biofilm-related genes.(6)The swimming and surging diameters of Δvhh are significantly larger,and the difference is extremely significant(P<0.01),suggesting that vhh negatively regulates the motor function of V.harveyi.Since Δvhh has not been tested for complementation,more experimental evidence is needed for the role of vhh in athletic ability.(7)The resistance of Δna3 to fluphenicol and chloramphenicol was significantly reduced(P<0.05),indicating that na3 is regulating the antibiotic resistance function of V.harveyi.3.Effect of the deletion of cqsA and vscCD genes of V.harveyi on host virulence,colonization ability,host immune genes,and the expression of virulence-regulated genes and virulence genes of V.harveyi(1)The fish challenge test showed that the virulence of cqsA gene mutant was significantly enhanced,indicating that cqsA negatively regulated the virulence of V.harveyi.In terms of colonization ability,the bacterial load of cqsA gene mutant in the spleen and liver was always higher than that of wild strains after prolonged infection with fish.Among them,the bacterial load in spleen reached the highest value 24 hours after infection.(P<0.001);the bacterial load reached the highest value in the liver 12 hours after infection,and the difference was extremely significant(P<0.01).In response to the host immune gene,the expression of immunoglobulin gene IgM and interleukin gene IL16 in the spleen and head kidney was significantly increased 12 hours after infection(P<0.01);tumor necrosis factor gene TNF-α,major histocompatibility complex genes MHC2 and MHC-1α,Toll-like receptor 3 gene TLR3,IL-1β interleukin gene and CD8α,different levels of up-regulation indicate that the cqsA gene mutant has the ability to improve the immunity of fish.In terms of its effects on virulence-regulating genes and virulence genes,the expression of quorum-sensing transcription factor genes aphA and flagellar movement genes flaA,flaC,fliA,fliS,cheR and lafA,and fur of cqsA gene mutant were significantly up-regulated(P<0.05)However,the down-regulation of the expression of the hemolysin gene vhh and the quorum-sensing regulatory gene hapR indicates that cqsA is involved in the regulation of virulence-regulating genes and virulence genes.(2)A similar fish challenge experiment was performed with vscCD gene mutant.The results showed that its virulence was significantly reduced,indicating that vscCD was regulating the virulence of V.harveyi.After the fish was infected,the bacterial load of vscCD gene mutant in the spleen and liver was always lower than that of the wild strain with time.The bacterial load in the spleen reached 48 hours after infection,and the difference was significant(P<0.001).;After 24 hours of infection,the bacterial load in the liver was significantly lower than that in the wild strain(P<0.001).In response to host immune genes,IL-16 expression in the spleen and head kidney was significantly upregulated during infection(P<0.001);the other host immune genes were activated to varying degrees,indicating that vscCD gene mutant has the ability to improve the immunity of fish.In terms of its effects on its own virulence regulatory genes and virulence genes,the transcriptional regulator protein gene luxR of vscCD gene mutant was significantly up-regulated(P<0.05),indicating that vscCD was involved in the expression of virulence-regulated genes;the expression of type III secretion system ATPase gene vcrH and the chaperone gene vp1668 was significantly down-regulated(P<0.05),indicating that vscCD is regulating the expression of vp1668 and vcrH.To sum up,cqsA negatively regulates the virulence of V.harveyi,which can regulate the expression level of some virulence-regulated genes significantly up or down,while negatively regulating the expression of some motor genes and positively regulating the expression of some virulence genes.vscCD is regulating the virulence of V.harveyi,which can downregulate the expression level of some virulence-regulated genes,and it is also regulating the expression of some type Ⅲ secretion system genes. |
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