RbsB,vgrG,hcp Gene Of Type Ⅵ Secretion System(T6SS) In Vibrio Harveyi:Expression And Functional Analysis

Vibrio harveyi,an important pathogen of marine aquaculture,can infect a variety of fish,shrimps,crabs and cause the death of a large amount of marine animals.The Type VI Secretion System（T6SS）which enhances biofilm formation and recognizes non-self is widely found in Gram-negative bacteria,also involved in bacterial interactions and exhibits complex biological functions.To understand the function of T6SS of V.harveyi,Three genes of T6SS were selected to analysis their sequence characteristics and their evolutionary relationships,included the needle-like structure,valine glycine repeat protein（VgrG）,effect factors and molecular chaperone hemolysin synergistic protein（Hcp）and ribosomal binding protein（RbsB）.The prokaryotic expression vectors for vgrG,hcp and rbsB was constructed,then the purified recombinant protein was prepared for polyclonal antibodies.Then the qPCR technique was used to investigate the expression of three genes under different cultured conditions.Finally,by knocking out vgrG,rbsB,hcp,and other T6SS-related genes,the role of three genes with their regulatory genes in T6SS and T6SS in the entire bacterial pathogenic mechanism was studied.The main results were as follows:1.The lengths of rbsB,vgrG and hcp gene sequences were 879bp,1836bp and 480bp,which can be coded 292aa,611aa and 159aa respectively.RbsB protein was a secreted peripherin protein,playing a role in the carrier combination.VgrG protein was homologous to the T4bacteriophage gp27/gp5 and helped bacteria anchor host cells to secrete effectors.Hcp protein was homologous to the T4 bacteriophage gp19/gp5,which could form a hexagonal channel for small molecule protein secretion and self-transport.The similarity rates in harveyi clade were 97-100%,99-100%and 70-99%respectively.The similarity rates in Vibrio were 90%,84%and 40%respectively,which was consistent with their biological function.2.To explore the role of T6SS in bacterial adaptation to the environment,V.harveyi strain 354 was cultured for 4,8,12,24,and 48h under different culture conditions（temperature,salinity,pH,copper ion,and iron ion）,and the expression of rbsB,vgrG,and hcp mRNA was investigated.The expression of rbsB mRNA showed a concave pattern at 28℃,1%NaCl,pH 7.5,no Fe³⁺,no Cu²⁺cultivation conditions.The expression level in the medium and late stage was obviously improved in the condiontion of rasing the cultured temperature only.Changing the salinity,the expression of rbsB was showed a high salt-inducing effect in the early stage.Regardless of acidity and the partial alkali,its expression was increased in the later stage.In the early stage,the expression of rbsB was inhibited by high concentration of Fe³⁺.There was no obvious inductive effect which caused by the concentration of Cu²⁺.The expression of vgrG mRNA was inhibited under high temperature.Hypotonic conditions（0.5%NaCl）significantly induced a high expression of the gene,while the influence of hyperosmotic condition was not significant.In the partial acid or alkalinity conditions,there was no effect on the expression of vgrG in the early stage of bacterial growth,and the expression level of vgrG was increased in the middle and late culture stage.For the experimental group with Fe³⁺addition,high concentration of Fe³⁺significantly induced high expression of vgrG.Appropriate concentration of Cu²⁺（1 mM,2 mM）could stimulate the expression of vgrG,which was increased significantly in the middle and later periods of culture,while the high concentration of Cu²⁺inhibited the expression of vgrG to some extent.The expression of hcp mRNA was not significantly different from that of the control group that showed a concave model when cultured temperature was increasd,but it was decreased in the later cultured period.The expression of hcp was stimulated at the logarithmic phase of bacterial growth when 3%Nacl was added,as the salinity continued to increase,the expression level of hcp was inhibited in the early stage but it was obviously improved in the later period of bacterial growth.Regardless of the acidity and the partial alkali,expression level of hcp in the later stage of bacterial growth was increased.The additon of iron ions could promote the expression of hcp to a certain extent,while copper ions could significantly stimulate the expression of hcp in the incubation stage of bacteria（P<0.05）.3.To further explore the functions of vgrG,hcp and rbsB in T6SS,rbsB,luxp,luxO,vgrG,vasC deletion strains were constructed sucessfully.Difference in hcp expression,capacity of biofilm formation,motility,and antagonism between bacteria under the co-cultured were also studied The results showed that compared with the wild-type strains,the expression level of hcp in the genes knock-out strains was increased to different degrees,indicating that the hcp has a negative regulatory function in T6SS.The motility ofΔrbsB andΔluxP strains on the soft agar plates was stronger than that of the wild-type strains or other mutant strains.In the bacterial biofilm formation ability,all the deleted strains were weaker than the wild strains,indicating that the formation of biofilm requires the participation of T6SS.In an antagonistic experiment co-cultivated with Escherichia coli,all strains showed different degrees of antagonism against E.coli compared to the control group,except for the vasC mutant.It was implied that vasC was mainly involved in the antagonism of exogenous bacteria in T6SS.