Preliminary Study On The Expression And Biological Function Of Salmonella Enterica Serovar Typhi Type Ⅵ Secretion System

Objective:The type VI secretion system is widely present in Gram-negative bacteria and considered as an important determinant of virulence.It plays an important role in the survival and pathogenicity of bacteria.The previous study has confirmed the existence of type VI secretion system in S.typhi and it is functional.This work will further study the expression conditions and biological functions of T6SS.It is of great significance to understand the functions of T6SS in the survival,environmental stress and pathogenic processes of S.typhi.Methods:1.Strain constructions（1）sciK::Flag-tagged strain:3×Flag gene sequence is added to the 3’end of sciK（hcp）gene of S.typhi by homologous recombination of suicide plasmid to produce a Flag-tagged protein.（2）sciG complement strain（C-ΔsciG）:The coding region of the sciG gene was cloned into the pBAD33 plasmid and electrotransformated intoΔsciG,a mutant strain with dysfunctional type VI secretion system,to prepareΔsciG complement strain.The expression of sciG was induced by L-arabinose.the empty vector pBAD33 was introduced into wild-type strain andΔsciG as control strains,respectively（WT+pBAD33 andΔsciG+pBAD33）.2.Expression of T6SSWestern blot was used to detect the expression of SciK and analyze the effects of different conditions on the expression of type VI secretion system.The conditions are as follows:（1）Culture medium:Bacteria was cultivated in different medium（LB medium,LPM medium that imitates the intracellular environment of macrophages）;（2）Growth period:Bacteria was cultivated to different growth periods such as lag phase,early logarithmic phase,middle logarithmic phase,late logarithmic phase and stationary phase;（3）Stress conditions:Including anaerobic environment,micro-aerobic environment,aerobic environment,acid stress,reactive oxygen stress,hyperosmotic stress,low temperature,high temperature,etc.3.Preliminary study on T6SS biological functionThe biological functions of T6SS were analyzed by comparing WT+pBAD33,ΔsciG+pBAD33 and C-ΔsciG.Including:（1）The growth curve:Growth curves were drawn by using time as the abscissa and value of OD₆₀₀ as the ordinate.In LB liquid medium,growth curve of WT andΔsciG under the conditions of no stress,acid stress,reactive oxygen stress,hyperosmotic stress,low temperature and high temperature,are plotted to analyze the effect of T6SS on bacterial growth;（2）The motility assay:2 ul of each bacterial suspension was stab inoculated through 0.2%semi-solid medium（Swimming motility）,and spotted on the 0.6%semi-solid medium（Swarming motility）.The diameter of the circular zones were measured to evaluate the motility;（3）Bactericidal activity assay:After T6SS was induced to expression in LPM medium,WT andΔsciG as predator strains was mixed with prey strain E.coli DH5α/pet28a at a ratio of 4:1.The mixture was spotted on the LPM agar plates and incubated for 4 h.The bacterial spots were resuspended in PBS and diluted,then spotted on the LB agar plates containing kanamycin to compare the density of surviving prey strain bacteria and analyze the bactericidal activity of each predator strains;（4）Analysis of biofilm formation:Each strain was transferred into a 96-well plate,and stained after 4 days incubation in TSB at 30°C.30%glacial acetic acid was used to dissolve the crystal violet and the amount of biofilm formation was quantified at 570 nm.The ability of biofilm formation was compared.The total RNA of each strain was extracted for reverse transcription and qRT-PCR was carried out to detect the mRNA levels of biofilm-related genes;（5）Epithelial cell adhesion assay:Each strain was cultivated to the early logarithmic phase（OD600≈0.4）and infects HeLa cells at an MOI of 20 for 90 min after 1 h’induction by L-arabinose.After the cells were lysed,the bacteria were diluted and incubated on the plates.the colonies were counted to compare the adhesion level of each strain.;（6）The THP-1 intracellular survival assay:After cultivated and induced,each strain infects macrophages at an MOI of 10 for 1 h.After adding gentamicin for another 1 h and washed with PBS,1/3 of cells were lysed and incubated on LB plate to count the colonies（T0）.Other cells were incubated for a further 12 or 24 h and incubated on LB plate.The colonies were counted as T12 or T24.The value of T12/T0and T24/T0 were used to compare the bacteria intracellular survival ability.Results:1.sciK::Flag-tagged strain,ΔsciG complement strain,WT andΔsciG with empty vector were successfully constructed,which laid the foundation for further research on the function of T6SS;2.Western blot showed that no SciK::Flag protein was detected in each growth period（including:lag phase,early logarithmic phase,middle logarithmic phase,late logarithmic phase and stationary phase）in LB or LPM liquid medium under the aerobic environment.No SciK::Flag protein was detected in the stress conditions,including:acid stress,reactive oxygen stress,hyperosmotic stress,high temperature and low temperature.The SciK::Flag protein was expressed at a high level from the eighth hour in LPM medium under microaerobic environment,while the expression of SciK::Flag protein was significantly decreased under the anaerobic environment.No SciK::Flag protein was detected under the aerobic environment;3.The growth curve showed that the growth ofΔsciG was significantly slower than WT under reactive oxygen stress.However,there was no difference between WT andΔsciG under the conditions of no stress and acid,hypertonic,high temperature,low temperature stress;4.The results of swimming and swarming test showed that there was no significant difference in motility between WT+pBAD33 andΔsciG+pBAD33;5.Bactericidal activity assay showed that there was no significant difference between WT andΔsciG in bactericidal activity;6.The biofilm formation ability ofΔsciG+pBAD33 significantly declined compared to WT+pBAD33（p<0.05）,while complementary strain partially recovered;7.qRT-PCR analysis showed that the mRNA level of fimA inΔsciG+pBAD33was significantly lower than that in WT+pBAD33.While the mRNA levels of bapA,flhD,fliA,fljA,fljB,bcsA,rfaD,wcaA,yihP,csgA,and csgD in WT+pBAD33 andΔsciG+pBAD33 had no significant difference;8.Epithelial cell adhesion assay showed that there was no significant difference in adhesion ability between WT+pBAD33 andΔsciG+pBAD33;9.The THP-1 intracellular survival assay showed that the intracellular survival ability ofΔsciG+pBAD33 was decreased than WT+pBAD33,while complementary strain partially recovered.Conclusions:In LPM medium under microaerobic conditions,SciK::Flag protein was expressed at a high level.The type VI secretion system has no effect on the growth of S.typhi under conditions such as no stress,acid stress,hyperosmotic stress,high temperature stress,low temperature stress.While the growth of bacteria can be affected under reactive oxygen stress.Type VI secretion system does not affect the dynamics,bactericidal activity and adhesion ability of epithelial cells.However,it affects the biofilm formation by regulating the expression of pili-related genes fimA and the THP-1 intracellular survival ability.