| Objective:Salmonella Typhi(Salmonella enterica serovar Typhi,S.Typhi)infection is still a global public health problem,and the study of its pathogenic mechanism is crucial for the treatment of infection.The type VI secretion system(T6SS)of S.Typhi has the ability to directly deliver bacterial effector proteins to the eukaryotic host or extracellular environment.It is considered an important virulence factor in bacterial survival and pathogenesis.In our previous study,we used proteomic techniques to compare the secreted protein levels of S.Typhi wild-type strain(WT)and T6 SSdeficient strain(Δsci G)under T6 SS expression condition,and suggested that the secretion of several flagellin-related proteins changed significantly.As the main structural device of bacterial motility,flagella is involved in bacterial adhesion,colonization and invasion,and is also closely related to bacterial survival ability and pathogenic mechanism.However,the relationship between T6 SS and flagellar system is not yet known.This thesis focuses on the effect and mechanism of S.Typhi T6 SS on flagellin secretion.It is of great significance to understand the function of T6 SS and the interaction between T6 SS and flagellar secretion system.Methods:1.Effect of T6 SS on flagellar synthesis and secretion(1)The flagella recombinant vectors carrying Flag protein tag were constructed and imported into S.Typhi to construct control strains respectively.(2)The RNA of WT and Δsci G were collected under T6 SS expression condition and incubated for 12 h,and flagellin-related genes transcriptional levels were detected by q RT-PCR.(3)The secreted and cell proteins of WT,Δsci G and C-Δsci G were collected under T6 SS expression conditions and incubated for 12 h,and the flagellin-related proteins Fli C,Flg D,Flg E and Fli D levels were detected by western blot.(4)The WT,Δsci G and C-Δsci G cells cultured for 12 h under T6 SS expression conditions were collected and injected into 0.3% LPM semi-solid medium,seeded dropwise on the surface of 0.6% LPM semi-solid medium,and incubated at 37℃ to observe the size of the circle formed by each strain.2.Whether T6 SS is involved in flagellin secretion(1)Screening of flagellin defective strains: the gene responsible for the secretion function of the flagellar system was screened by q RT-PCR,western blot and motility experiment under routine culture conditions,and the autonomous flagellar protein secretion function of partial flagellar system was turned off by the mutant of the gene(mot A).(2)The double-deficient strain Δmot AΔsci G was constructed by using the recombinant plasmid.(3)The RNA of WT,Δmot A,Δsci G and Δmot AΔsci G were collected after 12 h of culture under the condition of T6 SS expression,and flagellin-related genes transcriptional levels were determined by q RT-PCR.(4)The secreted and cell proteins of WT,Δmot A,Δsci G and Δmot AΔsci G under T6 SS expression conditions were collected and cultured for 12 h,and the content of Fli C was detected by western blot.(5)The WT,Δmot A,Δsci G and Δmot AΔsci G cultured for 12 h under the condition of T6 SS expression were collected and injected into 0.3% LPM semi-solid medium,and then seeded on the surface of 0.6% LPM semi-solid medium.The size of motility circle formed by each strain was observed by incubating at 37℃.(6)The WT,Δmot A,Δsci G and Δmot AΔsci G cultured for 12 h under the condition of T6 SS expression were collected for transmission electron microscopy to observe the bacterial flagellar structure,mainly the number,length and position of flagella.3.Whether T6 SS Sci G can replace the function of ATPase in the flagellar system(1)The flagellar ATPase deficient strain Δfli I was constructed by the recombinant plasmid.(2)The sci G+p BAD/Myc-His A recombinant vector was constructed and electroporated into Δfli I to obtain Sci G expression strain and its control strain.(3)The RNA of WT+p BAD/Myc-His A,Δfli I+p BAD/Myc-His A andΔfli I+sci G+p BAD/Myc-His A cultured for 12 h under T6 SS expression condition was collected,and the expression level of flagellin-related genes were detected by q RTPCR.(4)The secreted and cell proteins of WT+p BAD/Myc-His A,Δfli I+p BAD/MycHis A and Δfli I+sci G+p BAD/Myc-His A cultured under T6 SS expression condition for12 h were collected,and the content of Fli C was detected by western blot.(5)The WT+p BAD/Myc-His A,Δfli I+p BAD/Myc-His A andΔfli I+sci G+p BAD/Myc-His A strains cultured for 12 h under the condition of T6 SS expression were collected and injected into 0.3% LPM semi-solid medium,and then seeded on the surface of 0.6% LPM semi-solid medium.The size of motility circle formed by each strain was observed by incubating at 37℃.(6)The WT+p BAD/Myc-His A,Δfli I+p BAD/Myc-His A andΔfli I+sci G+p BAD/Myc-His A strains cultured for 12 h under the condition of T6 SS expression were collected for transmission electron microscopy to observe the bacterial flagellar structure,mainly the number,length and position of flagella.Results:1.Effect of T6 SS on flagellin synthesis and secretion(1)flg D::Flag,flg E::Flag,fli D::Flag recombinant vectors were successfully constructed and introduced into S.Typhi to obtain their control strains:flg D::Flag+WT+p BAD33,flg D::Flag+Δsci G+p BAD33,flg D::Flag+ C-Δsci G,flg E::Flag+WT+p BAD33,flg E::Flag+Δsci G+p BAD33,flg E::Flag+ C-Δsci G,fli D::Flag+WT+p BAD33,fli D::Flag+Δsci G+p BAD33,fli D::Flag+ C-Δsci G.(2)q RT-PCR results show that,compared with WT,Δsci G up-regulated the transcriptional expression levels of flh D and fli A,while down-regulating the transcriptional expression levels of flg D,flg E,and fli D,with no significant differences in the effects on the expression of flj B and fli C.(3)In western blot experiment,compared with WT,there was no significant difference in the expression level of Δsci G cell flagellin-related proteins,while the secretion of flagellin-related proteins in the supernatants of Fli C,Flg D,Flg E,and Fli D was significantly reduced.(4)In the motility experiment,the radius of Δsci G motility circle was smaller and the bacterial motility level was weakened compared with the WT.2.T6 SS is involved in the secretion of flagellin(1)Screening of flagellar deficient strains: under conventional culture conditions,compared with wild strains,Δmot A affected flh D and fli A transcriptional levels downregulated,fli C and fli D transcriptional levels up-regulated,and had no significant effect on flg G and flj B expression;western blot results showed that compared with WT,the flagellin Fli C content in the supernatant of Δmot A;Compared with the WT,the Δmot A showed reduced motility,suggesting that the mot A could shut down a part of the flagellar system autonomous flagellin secretion.(2)The dual-deficient strain Δmot AΔsci G was successfully constructed.(3)q RT-PCR results,under T6 SS expression conditions,Δmot A down-regulated the transcriptional expression levels of flh D,fli A,flg G,flj B and fli C compared to WT,while up-regulating the transcriptional expression levels of flg D,flg E and fli D,for fli K,there was no significant difference in expression;compared to WT,Δmot AΔsci G upregulated the transcriptional levels of flg D,flg E,fli A,fli K,and fli D,while downregulating the transcriptional levels of flg G as well as flj B and fli C,with no significant difference in the effect on the expression of flh D compared to WT.(4)Under T6 SS expression conditions,compared with WT,the Fli C content in the cell protein of Δsci G,Δmot A and Δmot AΔsci G were basically the same,while the secretion of flagellin in the supernatant of Δmot A and Δsci G was significantly reduced compared to WT,while compared to Δmot A and Δsci G,the secretion of flagellin in the supernatant of the Δmot AΔsci G supernatants showed a further decrease in the secretion of flagellin.X(5)The motility results showed that Δsci G,Δmot A and Δmot AΔsci G were significantly less motility compared with WT.(6)Transmission electron microscopy results showed that the number of Δsci G,Δmot A and Δmot AΔsci G flagella was significantly reduced compared to WT.3.T6 SS Sci G replaces the ATPase function of the flagellar system partly(1)Δfli I was successfully constructed.(2)Construction of sci G+p BAD/Myc-His A recombinant vector,Δfli I+sci G+p BAD/Myc-His A and its control strains: WT+p BAD/Myc-His A,Δfli I+p BAD/Myc-His A.(3)Under T6 SS expression conditions,q RT-PCR results showed thatΔfli I+p BAD/Myc-His A down-regulated the expression of flh D,fli A,flg D,flj B,fli C and fli D compared to WT+p BAD/Myc-His A,with no significant difference on the expression of fli K;compared to Δfli I+p BAD/Myc-His A,Δfli I+sci G+p BAD/MycHis A up-regulated the expression of flg D,fli A,and flj B,while down-regμLating the expression of fli K as well as fli D,and no significant difference on the expression of flh D as well as fli C,compared with Δfli I+p BAD/Myc-His A.(4)In western blot analysis,there was no significant difference in the expression levels of Δfli I+p BAD/Myc-His A and Δfli I+sci G+p BAD/Myc-His A with cell Fli C compared with WT+p BAD/Myc-His A,while Δfli I+p BAD/Myc-His A supernatant showed a significant decrease in the secretion of flagellin Fli C,and the secretion level of flagellin Fli C in Δfli I+sci G+p BAD/Myc-His A supernatant was upregulated compared with Δfli I+p BAD/Myc-His A.(5)Compared with WT+p BAD/Myc-His A,both Δfli I+p BAD/Myc-His A andΔfli I+sci G+p BAD/Myc-His A motility were weakened,and the difference in the motility circle between them was not significant.(6)Transmission electron microscopy results showed that the bacterial flagella number of Δfli I+p BAD/Myc-His A and Δfli I+sci G+p BAD/Myc-His A was reduced compared to WT+p BAD/Myc-His A.Conclusions:1.Under the condition of T6 SS expression,the contents of flagellin Flg D,Flg E,Fli D and Fli C in the Δsci G supernatant decreased,which was consistent with the results suggested by proteomics,and there were no significant difference in cell proteins.T6 SS mainly affects the secretion of flagellin.2.Under the expression of T6 SS,T6SS is involved in the secretion of flagellin-related proteins,affects S.Typhi motility level and flagellum structure.3.Sci G of T6 SS can partially replace the flagellar system ATPase Fli I to function and help flagellar protein secretion to the extracellular. |
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