| Vibrio parahaemolyticus(VP)widely exists in sea water and other environments,and is one of the important food-borne pathogens.Eating raw or uncooked seafood such as fish,shrimp and crabs can be infected by V.parahaemolyticus,causing inflammation and even sepsis.V.parahaemolyticus can also infect aquatic products such as shrimp and endanger the development of related aquaculture.Hemolysin,adhesion factors,Type Ⅲ Secretion Systems(T3SS),etc.are important pathogenic factors.T3SS is a multi-subunit needle device used to transport bacterial effector proteins into the host cytoplasm.The contact between the bacteria and the host triggers the release of effector proteins.Effector proteins change the biological activity of host cells by interfering with signal pathways or modifying protein functions,thereby exerting virulence.The device is assembled from 20-30 structural proteins,including a matrix structure embedded in the cell membrane,an external needle protruding from the surface of the bacteria,and a tip complex covering the needle.When in contact with the host cell,the transporter assembles between the needle tip complex and the host cell,transporting the effector protein through the formation of pores in the host cell membrane.Chaperone protein has the function of regulating protein secretion and maintaining protein stability.The genome sequence of V.parahaemolyticus showed the existence of two sets of T3 SS systems.T3SS1 was obtained from ancestors and exists in environmental strains and clinical strains,it mainly exerts cytotoxicity.T3SS2 is obtained through horizontal transfer and exerts enterictoxicity.Analysis of protein sequence found that the VscJ protein in V.parahaemolyticus T3SS1 belongs to the YscJ protein family,which is a structural protein that assembles the inner membrane loop in the T3 SS device.The VscG protein is a member of the YscG protein family and acts as the needle-like protein YscF in T3SS1 chaperone protein.Studies have shown that T3SS1 mainly affects the biofilm,motility and cytotoxicity of bacteria.In this study,the vscJ and vscG genes were knocked out on the V.parahaemolyticus RIMD2210633 POR-1 strain,and the corresponding deletion strains and complement strains were constructed to study their effects on the biological characteristics and cytotoxicity.1.Construction of T3SS1 structural protein vscJ gene deletion strain and analysis of its biological characteristicsThe homologous recombination method was used to construct the deletion strain and the complement strain of the vscJ gene,which were named ΔvscJ and CΔvscJ respectively.Through phenotypic test and fluorescence quantitative PCR test,the differences of POR-1,ΔvscJ and CΔvscJ in growth rate,motility,biofilm formation ability,hemolytic activity,etc.were analyzed.The test results showed that the vscJ gene did not affect the growth rate of V.parahaemolyticus and the hemolysis of rabbit red blood cells;the motility of the ΔvscJ strain was significantly enhanced compared with POR-1(P<0.001),and the biofilm formation ability was significantly reduced(P<0.001),the ability of CΔvscJ strain to produce biofilm was partially restored(P<0.01).The locomotor ability of V.parahaemolyticus is conferred by lateral flagella and polar flagella.The q RT-PCR experiment found that the vscJ gene down-regulated the expression of flagella-related genes mot Y,fli M,flg B,laf A and fli D,and up-regulated the expression of biofilm syp G and scr A genes,which can promote the formation of V.parahaemolyticus biofilm.In order to explore the role of vscJ gene in the pathogenic process of the bacteria on host cells,adhesion tests and cytotoxicity tests were carried out.The results showed that the deletion of vscJ gene significantly reduced the adhesion ability(P<0.01)and cytotoxicity(P<0.0001)of V.parahaemolyticus to He La cells,the effect was restored in the complement plant CΔvscJ.In summary,the vscJ gene is involved in the formation and movement of biofilms,so that the bacteria can better adapt to different living environments,and play an important role in the adhesion and toxicity of V.parahaemolyticus to host cells.2.Construction of T3SS1 chaperone protein vscG gene deletion strain and analysis of its biological characteristicsThe suicide plasmid p YAK1 was used to construct the deletion strain ΔvscG and the complementing strain CΔvscG of V.parahaemolyticus POR-1.Detect the expression of vscG gene in the above-mentioned strains by q RT-PCR,it was found that vscG gene can be expressed normally in POR-1 strain and CΔvscG,the deletion strain did not express vscG gene,which proved that the deletion strain and the complement strain were successfully constructed.Next,we explored the effect of vscG gene on the biological characteristics of V.parahaemolyticus.The growth rate of POR-1,the deletion strain ΔvscG and the complementing strain CΔvscG in LB medium showed no significant difference.Each strain showed a translucent circular track on a 0.5% agar LB plate.Compared with the POR-1strain,the movement diameter of the ΔvscG strain was significantly increased(P<0.0001),and the production of biofilm was significantly reduced(P<0.01).The target gene was selected through q RT-PCR test to further explore the molecular mechanism of the gene affecting the formation and motility of VP biofilm.The test found that the transcription levels of flagella-related genes flg B,laf A and fli D in the ΔvscG strain were significantly higher than POR-1(P<0.01).In addition,the expression levels of the biofilm-related genes syp G and scr A of the ΔvscG strain were significantly lower than those of the POR-1 strain(P<0.0001),and the expression of target genes in the complement strain CΔvscG was restored.During the infection process,bacteria secrete adhesion factors to come into contact with host cells and infect them.This study explored the adhesion and toxicity effects of vscG gene on He La cells,and found that the deletion of vscG gene significantly reduced the adhesion ability of V.parahaemolyticus(P<0.001)and significantly reduced cytotoxicity(P<0.0001),indicating that vscG gene is beneficial to bacteria to the host Pathogenesis of cells.VscG protein forms a heterotrimer with VscF and VscE in V.parahaemolyticus.In order to explore the effect of vscG gene on the expression of vscF and vscE genes,a fluorescent quantitative PCR test was performed.The results showed that the deletion of vscG gene down-regulated vscE and vscF genes The expression of vscG gene promotes the expression of vscF and vscE genes.The test results show that the vscG gene in the T3SS1 gene cluster of V.parahaemolyticus affects the formation and movement of the bacteria’s biofilm and participates in the pathogenic process of host cells.The vscG gene is also necessary for the stable expression of vscF and vscE. |
PDF Full Text Request