Construction Of Salmonella Plasmid Virulence Gene B Mutant Strain And Its Influence On Epithelial Cell Autophagy

Objective:To investigate the influence of the Salmonella plasmid virulence gene B (spvB) on epithelial cell autophagy by cells infection model, Construct spvB defect strain, Therefore provide theoretical and experimental basis for discussing the pathogenic mechanism of the spvB, and provide tools and means for further study on the function of the gene using mouse infection model.Methods:1. Construction of Salmonella spvB defective strain(1) According to the Salmonella spvB gene sequence in the GeneBank sequence database, two pair’s specific PCR primers were designed using Primer Premier5.0, upper-and down-stream of the spvB gene to amplify two homologous DNA fragments. The homologous recombinant DNA fragment of the defective target gene was cloned into the suicide plasmid PGMB151, which was then transduced into the target cell of Salmonella typhimurium SR-11for homologous recombination. The recombination was visualized by PCR and sequencing.(2) A pair of specific primers of the spvB gene was designed according to the spvB gene sequence and used to amplify the spvB gene. The amplicon containing the total CDS region of the spvB gene was directionally inserted into the expression vector pBAD/gⅢ to form the recombinant plasmid (pBAD/spvB). The spvB deleted mutants were transformed by the positive recombinant plasmid to generate the spvB complementary strain (SR-11-c-spvB).2. The influence of spvB on epithelial cells autophagy(1) To make cell infection model, SR-11carrying spvB, SR-11-ΔspvB and SR-11-c-spvB were added to HeLa cells which were stability transfected with red and green fluorescent protein 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. This time was regarded as’0’point. Medium containing100mg/ml amikacin was added to culture plates to kill the remaining extracellular bacteria. After2h of incubation with medium containing10mg/ml amikacin to prevent the bacteria growth released from infected cells and incubation was continued for2h again. Cells were harvested at1h,3h and5h after cocultivation, then the intracellular LC3-II yellow dot structure were observed by laser confocal microscope.(2) To make cell infection model, SR-11carrying spvB, SR-11-ΔspvB and SR-11-c-spvB were added to HeLa cells 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. This time was regarded as’0’point. Medium containing100mg/ml amikacin was added to culture plates to kill the remaining extracellular bacteria. After2h of incubation with medium containing10mg/ml amikacin to prevent the bacteria growth released from infected cells and incubation was continued for2h again. Cells were harvested at1h,3h and5h after cocultivation, and then the expression of the autophagy-associated protein Beclin-1and p62were detected by Western blot, the intracellular colony forming unit (CFU) were also detected by plate counting.Results:1. Construction of Salmonella spvB defective strain(1) SR-11-ΔspvB mutants were confirmed by PCR and sequencing analysis.(2) SR-11-c-spvB rescuing strains were confirmed by PCR, sequencing analysis and WB detection.2. The influence of spvB on epithelial cell autophagy(1) Laser confocal microscope analysis showed that the expression of autophagy-associated protein LC3-II of SR-11and SR-11-c-spvB infected groups was lower than SR-11-ΔspvB infected group at1hour of cells and bacteria co-culture; and it was no significant difference at3and5h of co-culture.(2) WB detection showed that the expression of autophagy-associated protein Beclin-1of SR-11and SR-11-c-spvB infected groups was lower than SR-11-ΔspvB infected group 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 p62of SR-11and SR-11-c-spvB infected groups was higher than SR-11-ΔspvB infected group at1hour of cells and bacteria co-culture, it was no significant difference at3and5h of co-culture; CFU analysis showed that the number of active bacteria in SR-11and SR-11-c-spvB infected groups was significant more than SR-11-ΔspvB infected group (3h, P<0.05;5h, P<0.01).Conclusions:1. Suicide vector system and prokaryotic expression vector pBAD/gⅢwere successfully applied to construct the SR-11-ΔspvB mutants and SR-11-c-spvB, which provided a method to study the detail functions of spvB gene.2. The above results demonstrated that Salmonella spvB gene suppressed epithelial cell autophagy. The expression of autophagy-associated protein LC3-II and Beclin-1of SR-11and SR-11-c-spvB infected groups was lower than SR-11-ΔspvB infected group at1hour of cells and bacteria co-culture; While the expression of autophagy-associated protein p62of SR-11and SR-11-c-spvB infected groups was higher than SR-11-ΔspvB infected group1hour of cells and bacteria co-culture. The number of active bacteria in SR-11and SR-11-c-spvB infected groups was significant more than SR-11-ΔspvB infected group at3and5h of co-culture. All results showed that virulence gene spvB could enhance supression of epithelial cell autophagy in the early stage of infection, and increase Salmonella survival in cells.