A Study On The Pathogenic Mechanism Of Pseudomonas Fluorescens And The Effect Of Host Anti-bacterial Immune Factors

Mariculture fish are often infected by pathogenic microorganisms,resulting in reduced yields and economic losses.However,there is no effective measures at present.One of the reasons is that the pathogenic mechanism of pathogen and the immune defense mechanism of the host are not elucidated clearly.In this study,we have investigated the pathogenic mechanism of Pseudomonas fluorescens,which is a common pathogen of fish.At the same time,we have studied the anti-bacterial function of the immune factors(bactericidal/permeability increasing protein and B-type mannose-specific lectins)in a fish host half-smooth tongue sole(Cynoglossus semilaevis).In this study,we performed proteomic analysis firstly to compare the global protein profiles of P.fluorescens strain TSS cultured under iron-replete and iron-deplete conditions.Twenty two differentially expressed proteins were identified of global proteins.The genes encoding four of the 22 proteins,i.e.Hem O(heme oxygenase),Psp B(serine protease),Sod(superoxide dismutase),and Tfe R(Ton B-dependent outermembrane ferric enterobactin receptor),were knocked out,and the pathogenicity of the mutants was examined.The results showed that compared to the wild type,the hem O,psp B,and tfe R knockouts were significantly impaired in the ability to survive in host serum,to invade host tissues,and to cause host mortality.Immunization of turbot with recombinant Tfe R and Psp B(r Tfe R and r Psp B)induced production of specific serum antibodies and significant protections against lethal TSS challenge.Further analysis showed that r Tfe R antibodies recognized and bound to TSS,and that treatment of TSS with r Tfe R antibodies significantly impaired the infectivity of TSS to fish cells.Taken together,these results indicate for the first time that in pathogenic P.fluorescens,iron affects the expression of a large number of proteins including those that are involved in host infection.Then we compared the secreted protein profiles of TSS cultured under iron-replete V and iron-deplete conditions by proteomic analysis.Fifteen differentially expressed proteins were identified.In this study,we identified filamentous hemagglutinin(FHA)as an iron-responsive protein secreted by TSS,furthermore,the fha mutant TSSfha was constructed.In vitro study,compared to the wild type,the fha mutant(i)exhibited a largely similar vegetative growth profile but significantly retarded in the ability of biofilm growth and producing extracellular matrix,(ii)displayed no apparent flagella and motility,(iii)was defective in the attachment to host cells and unable to form selfaggregation,(iv)displayed markedly reduced capacity of hemagglutination and surviving in host serum.In vivo infection analysis revealed that mutant was significantly attenuated in the ability of dissemination in fish tissues and inducing host mortality,and that antibody blocking of the natural FHA produced by the wild type TSS impaired the infectivity of the pathogen.Furthermore,when introduced into turbot as a subunit vaccine,recombinant FHA elicited a significant protection against lethal TSS challenge.Taken together,these results indicate for the first time that P.fluorescens FHA is a key virulence factor essential to multiple biological processes associated with pathogenicity.We investigated the role of two antibacterial factors of tongue sole in the process of resistancing to TSS.Bactericidal/permeability-increasing protein(BPI)is an important factor of innate immunity that in mammals is known to take part in the clearance of invading Gram-negative bacteria.In teleost,the function of BPI is unknown.In the present work,we studied the function in resistancing to TSS of tongue sole BPI,Cs BPI.We found that Cs BPI was produced extracellularly by peripheral blood leukocytes(PBL).Recombinant Cs BPI(r Cs BPI)was able to bind to a number of Gram-negative bacteria but not Gram-positive bacteria.Binding to TSS led to bacterial death through membrane permeabilization and structural destruction,and the bound TSS were more readily taken up by PBL.In vivo,r Cs BPI augmented the expression of a wide arrange of genes involved in antibacterial and antiviral immunity.Furthermore,r Cs BPI enhanced the resistance of tongue sole against TSS as well as viral infection.These results indicate for the first time that a teleost BPI possesses immunoregulatory effect and plays a significant role in antibacterial and antiviral defense.Lectins are a group of sugar-binding proteins that are important factors of the innate immune system.In this study,we examined,in a comparative manner,the expression and function of three Bulb-type(B-type)mannose-specific lectins(named Cs BML1,Cs BML2,and Cs BML3)from tongue sole.All three lectins possess three repeats of the conserved mannose binding motif QXDXNXVXY.Expression of Cs BML1,Cs BML2,and Cs BML3 was most abundant in liver and upregulated by bacterial infection.Recombinant(r)Cs BML1,Cs BML2,and Cs BML3 bound to a wide arrange of bacteria including TSS in a dose-dependent manner and with different affinities.All three lectins displayed mannose-specific and calcium-dependent agglutinating capacities but differed in agglutinating profiles.r Cs BML1 and r Cs BML2,but not r Cs BML3,killed target bacteria in vitro and inhibited bacterial dissemination in fish tissues in vivo.These results indicate for the first time that in teleost,different members of B-type mannosespecific lectins likely play different roles in antibacterial immunity.