Influence Of Salmonella Plasmid Virulence Gene On Autophagy In Hela Cells And Infection Process In Vivo

Objective：Salmonella plasmid virulence gene (spv) is closely related to bacterial virulencephenotype. In this experiment, to investigate the influence of spv gene on autophagy andinfection process, optimize the electransformantion processing and construct stablebioliminence Salmonella typhimurium strains, build cells and mice models to study theinfluence of spv gene on autophagy and the infection process in vitro and vivo. Providetheoretical and experimental basis for further study of Salmonella pathogenesis and themolecular mechanisms of autophagy, and provide an effective tool for real-time trackingSalmonella infection process.Methods：1. Construction of stable bioliminence strainsTo study factors of electransformation efficiency, a small plasmid vector DNApDMT-GFP was electrotransformed into S.typhimurium under various conditionsincluding growth stage, medium, concentration of competent cells and pulse strength.The transformation efficiency changes were discovered and the optimized conditionswere evaluated according to them. The large plasmid pBEN276was electrotransformedinto S.typhimurium χ3306and UF110in the optimized condition. Induce recombinationof transformants by arabinose. The recombination was selected by bioluminescencedetected. Then visualized the stability of the recombination and detected the quantitativerelation of bioluminescence indensity and CFU.2. The influence of spv on autophagy of HeLa cells(1) To make cell infection model, S.typhimurium χ3306lux and UF110lux wereadded to HeLa cells which were stability transfected with red and green fluorescentprotein markers microtubule-associated protein1light chain3(LC3)(mRFP-GFP-LC3)in vitro. Using the logarithmic phase bacteria infect cells by multiplicity of infection(MOI)100:1. Cells and bacteria were co-incubated for1h in culture plates, and then the medium was removed. Medium containing100mg/ml amikacin was added to cultureplates to kill the remaining extracellular bacteria. After2h of incubation with mediumcontaining10mg/ml amikacin to prevent the bacteria growth released from infectedcells and incubation was continued for2h again. Cells were harvested at1h,3h and5h after cocultivation, then the intracellular LC3-Ⅱ yellow dot structure were observedby laser confocal microscope.(2) To make cell infection model, S.typhimurium χ3306lux and UF110lux wereadded to HeLa cells in vitro. Using the logarithmic phase bacteria infect cells bymultiplicity of infection (MOI)100:1. Cells and bacteria were co-incubated for1h inculture plates, and then the medium was removed. This time was regarded as ‘0’ point.Medium containing100mg/ml amikacin was added to culture plates to kill theremaining extracellular bacteria. After2h of incubation with medium containing10mg/ml amikacin to prevent the bacteria growth released from infected cells andincubation was continued for2h again. Cells were harvested at1h,3h and5h aftercocultivation, and then the expression of the autophagy-associated protein LC3-Ⅱ,Beclin-1and P62were detected by Western blot, the bioluminescence indensity ofintracellular bacterials was detected and the intracellular colony forming unit (CFU)were also detected by plate counting.3. influence of spv on infection progress in vivoThe in vivo experiment using a mouse model. S.typhimurium χ3306lux andUF110lux were used in the intraperitoneal injection of mouse, and the control grouponly received an injection of same amount of saline. At different time points, thecommon condition of mouse was obsereved, the growth and dissemination of bacterialswas visualized by in vivo imaging, then the visiual pathological change of livers andspleens were compared after sacrifice of mice, and the viable bacterial amounts in liversand spleens were detected by plate count method, Beclin-1and p62expression in liversand spleens were detected by western bloting.Results:1. Construction of Salmonella typhimurium stable bioluminescence strainsThe optimal electrotransformation effect can be obtained when OD600is0.3, themedium is hypertonic LB contained15g/L NaCl, concentration of competent cells is1010cfu/mL and the electroporation parameters is2.5kV,400,50μF. And Salmonellatyphimurium stable bioluminescence strains χ3306lux and UF110lux were confirmed by PCR and bioluminescence detection.2. The influence of spv on HeLa cell autophagy(1) Laser confocal microscope analysis showed that the expression of autophagy-associated protein LC3-Ⅱ of χ3306lux infected group was lower than UF110luxinfected group at1hour of cells and bacteria co-culture，and it was no significantdifference at3and5h of co-culture.(2) WB detection showed that the expression of autophagy-associated proteinLC3-Ⅱ and Beclin-1of χ3306lux infected group was lower than UF110lux infectedgroup at1hour of cells and bacteria co-culture, and it was no significant difference at3and5h of co-culture; and that the expression of autophagy-associated protein p62ofχ3306lux infected group was higher than UF110lux infected group at1hour of cells andbacteria co-culture, it was no significant difference at3and5h of co-culture; CFUanalysis showed that the number of active bacteria in χ3306lux infected group wassignificant more than UF110lux infected group (3h, p <0.05;5h, p <0.01).3. influence of spv on infection progress in vivo(1) The in vivo results showed that in1-5days after infection, the wild type andmutant strains infected mice were fur chaotic, less active, poor mental state, diarrhea,weight loss and other phenomena, the wild-type strain infection group were moreserious and of lower survival rate. Control group of mice were activite, mental state andthe weight essentially unchanged.(2) The results of in vivo imaging showed that wild type and mutant strains in vivocan be detected2days after infection, in the same time bioluminescence intensity ofwild type strains were higher than mutants. With the extension of infection, the rate ofproliferation and dissemination of the wild type strains in mice were faster, which haddisseminated to the whole body to the fifth day, while the proliferation anddissemination of mutants infected group were slower. Anatomized mice to observe thechange in appearance of the liver, spleen, and cecum. Compared with the mutant strainsinfected group, liver and spleen enlargement were more serious, the cecum shortenedmore with more serious blister-like swelling in the wild type strain infected group. Thebioluminescence test results showed that the luminescence intensity in the organs of thewild type strains infected group were higher than the group infected with mutant strains.(3)The results of the viable count in the liver and spleen intracellular wild strain infected group intracellulare of the liver and spleen were higher than the number ofviable mutant infected group (p <0.05). WB test results showed that compared with themutant infected group, Beclin-1protein expression of liver and spleen was significantlyhigher (p <0.05) and p62expression was significantly lower (p <0.05) in wild typestrain infected mice p.i.6h,1d and2d, no significant difference (p>0.05) in the twogroups p.i.3d to5d.Conclusions:1. Optimized the processing of electransformation and successfully applied it toconstruct the Salmonella bioluminescence strans, which provided a tool and method toreal-time tracking Salmonella infection process in vitro and in vivo.2. Salmonella virulence gene spv could regulate the level of HeLa cell autophagy,and increase Salmonella survival in cells.3. The spv gene could increase the pathogenicity of the host bacteria, promote thereproduction and dissemination in vivo, and exacerbate infection process.