The Effects Of Salmonella Plasmid And Virulence Gene On Cellular Autophagy And The Molecular Mechanism

Objective:To observe the effect of Salmonella virulence plasmid pRST98and Salmonella plasmid virulence gene B (spvB) on cellular autophagy and infection outcome through cells model infected by Salmonella infection in vitro, therefore discuss the molecular mechanism and possible signaling pathway. This study can help to elucidate the virulent mechanism of pRST98in Salmonella infection; also it can provide the experimental and theoretical basis on regulating autophagic pathway, discovering new drug target sites and vaccine exploration for controlling Salmonella infection.Methods:1. The study of cellular autophagy and infection outcome influenced by Salmonella virulence plasmid. In this study, Salmonella typhi (S. typhi) strain ST8carrying a virulence plasmid pRsT98was used as an experimental strain, pRST98-deletion S. typhi strain ST8-△pRST98was used as a negative control and standard virulent strain Salmonella typhimurium (S. typhimurium) SR-11containing Salmonella plasmid virulence gene (spv) presented on a100Kb virulent plasmid was used as a positive control. Three kinds of bacteria were added to wide type mouse embryonic fibroblasts (WT-MEFs) and autophagy associated gene5deficient mouse embryonic fibroblasts (Atg5-/-MEFs) in vitro respectively at a multiplicity of infection (MOI) of100:1, meanwhile the groups of infected-WT-MEFs treated by autophagy inducer rapamycin (RAPA) were set up. After incubated at37℃for1h (0-h time point), infected cells were washed three times with sterile phosphate buffered saline (PBS); then DMEM completely containing amikacin (100μg/ml) was added to kill remaining extracellular bacteria. After2h of further incubation at37℃, medium in the plates was replaced with DMEM containing amikacin (10μg/ml) to inhibit the propagation of possible extracellular bacteria in the medium. Infected cells were collected and detected at1h,3h,5h,8h and10h time points of infection. Autophagic vacuoles of infected cells were visualized under fluorescence microscopy by monodansycadaverine (MDC) staining. The gold standard of autophagy phenomenon such as double-or multilayer membrane of autophagosome in the cytoplasma was observed by transmission electron microscopy (TEM). The expression of autophagic protein LC3-II was detected by Western Blotting (WB) assay. The apoptotic rate of infected cells was analysized by flow cytometry (FCM) through the method of Annexin.V-FITC (Ann. V)/Propidium Iodide (PI) double staining. The number of bacteria inside infected cells was quantitied by plate count method.2. The effect of spvB on cellular autophagy and its molecular mechanism discussion. WT-MEFs and Atg5-/-MEFs were co-cultured with spvB mutant of SR-11strain (SR-11-AspvB) and wide type of SR-11strain containing spvB (SR-11) in vitro. Two kinds of bacteria were added to WT-MEFs and Atg5-/-MEFs at MOI of100:1. After incubated at37℃for1h (0-h time point), infected cells were washed three times with PBS; then DMEM completely containing amikacin (100μg/ml) was added to kill remaining extracellular bacteria. After2h of further incubation at37℃, medium in the plates was replaced with DMEM containing amikacin (10μg/ml) to inhibit the propagation of possible extracellular bacteria in the medium. Infected cells were collected at1h,3h,5h,8h and10h and detected by the following methods. The expression of LC3-Ⅱ protein, phospholipase D (PLD1and PLD2) protein and Toll-ike receptor4(TLR4) protein of infected cells were identified with WB assay. PLD activity of infected cells was detected by radioactive [9,10-3H] oleic acid labeling. The expression of TLR4mRNA in infected cells was semiquantitied by reverse transcription polymerase chain reaction (RT-PCR) assay. Apoptosis of infected cells was detected by FCM through Ann. V/PI double labeling. Thl-type cytokine secretion such as interferon-γ (IFN-γ) of infected cells and Th2-type cytokines such as interleukin-4(IL-4) as well as interleukin-13(IL-13) of infected cells were examined by enzyme-linked immunosorbent assay (ELISA). The number of bacteria inside infected cells was quantitied by plate count method.Results:1. The effect of Salmonella virulence plasmid on cellular autophagy and infection outcome.(1) Results of MDC fluorescent staining under fluorescene microscopy. Only few sporadic autophagic vesicles were observed in the cytoplasm of WT-MEFs infected by and SR-11at1h, while autophagy inducer RAPA-treatment induced lots of autophagic vesicles to appear in WT-MEFs which were infected by ST8and SR-11at1h. In the cytoplasm of WT-MEFs and RAPA-treated WT-MEFs infected by ST8-△pRST98at1h, a few typical autophagic vesicles could be observed, while the number of autophagic vesicles at3h was fewer than at1h.(2) Ultrastructure of infected cells under TEM. WT-MEFs infected by ST8and SR-11did not appear typical autophagic vacuoles in the cytoplasm, however, ST8and SR-11infected-WT-MEFs treated by RAPA displayed the trypical autophagic vacuoles with double membrane at1h. WT-MEFs and RAPA-treated WT-MEFs infected with ST8-△pRST98showed typical autophagic vacuole with double-or multilayer membrane in the cytoplasm at1h.(3) Expression of autophagic protein LC3-Ⅱ in infected cells. LC3-Ⅱ protein expression in WT-MEFs infected by ST8containing pRST98and SR-11containing spv was weaker than in WT-MEFs infected by ST8-△pRST98at1h; however, this infection-mediated suppression was reversed with RAPA-treatment as shown by the LC3-II protein expression in both RAPA-treated WT-MEFs infected by ST8and SR-11. WT-MEFs infected by ST8-△pRST98showed stronger LC3-Ⅱ protein expression at1h and3h than WT-MEFs infected by ST8and SR-11, and the intensity of LC3-Ⅱ protein expression at1h was stronger than at3h. LC3-Ⅱ protein expression in RAPA-treatment WT-MEFs infected by ST8-△pRST98was higher than in RAPA-untreated WT-MEFs. The intensity of LC3-II protein expression in all infected cells at3h was weaker than at1h, and disappeared from5h to10h.(4) Punctate autophagic vesicles, LC3-Ⅱ protein expression and typical autophagic vacuole with double-or multilayer membrane could not been observed in Atg5-/-MEFs infected by three kinds of bacteria at each time points of infection.(5) Apoptotic rate of infected cells. Among cells infected by ST8containing pRsT98, the apoptotic rate in WT-MEFs had no significant difference than in Atg5-/-MEFs; while the apoptotic rate in RAPA-treatment WT-MEFs was significant lower than in RAPA untreated-WT-MEFs. The results of apoptotic rate among cells infectd by SR-11contaning spv showed similar as caused by ST8. The apoptotic rate in WT-MEFs had no significant difference than in Atg5-/-MEFs infected by SR-11; while RAPA treatment significant decreased apoptosis in WT-MEFs than in RAPA-untreated WT-MEFs infected by SR-11. The apoptotic rate in Atg5-/-MEFs was highest among cells infected by ST8-△pRST98, and the apoptotic rate in RAPA untreated-WT-MEFs was lower than in RAPA-treatment WT-MEFs infected by ST8-ApRST98.(6) The number of bacteria inside infected cells. Among cells infected by ST8, the number of intracellular bacteria in WT-MEFs was similar to the bacterial quantity in Atg5-/-MEFs, while WT-MEFs with RAPA-treatment had less intracellular bacterial quantity than RAPA-untreated WT-MEFs. Among cells infected by SR-11containing spv, the number of intracellular bacteria had no significant difference between WT-MEFs and Atg5-/-MEFs, and RAPA treatment could significant decrease bacterial quantity in WT-MEFs. The number of bacteria in Atg5-/-MEFs was highest among cells infected by ST8-△pRsT98; however, bacterial quantity in RAPA-treatment WT-MEFs had no significant difference than in WT-MEFs without RAPA treatment infected by ST8-△pRST98.2. The effect of spvB on cellular autophagy and its’possible molecular mechanism.(1) Relative quantity of LC3-Ⅱ protein expression in infected cells. Expression of LC3-Ⅱ protein in WT-MEFs infected by SR-11strain was significant weaker than in SR-11-AspvB strain-infected WT-MEFs at1h and3h. Infected cells did not express LC3-Ⅱ protein during laterly infection from5h to10h.(2) Relative quantity of PLD1and PLD2protein expression in infected cells. The expression of PLD1protein in SR-11strain-infected cells was higher than in cells stimulated by SR-11-AspvB strain, while PLD2protein expression had no significant difference between in two kinds of bacterial-infected cells.(3) Relative quantity of TLR4protein expression in infected cells. The intensity of TLR4protein expression in SR-11strain-infected cells was significant lower than in cells infected by SR-11-△spvB strain at each time points of infection.(4) PLD activity of infected cells. The activity of PLD in cells infected by SR-11and SR-11-△spvB strain was both higher than the basic PLD activity of uninfected cells. PLD activity in SR-11strain-infected cells was higher than in cells infected by SR-11-△spvB strain at each time points of infection. PLD activity of infected cells reached climax at1h and gradually declined at lately infection.(5) TLR4mRNA expression of infected cells. The expression of TLR4mRNA in SR-11strain-infected cells was weaker than in cells infected by SR-11-△spvB strain at each time points of infection.(6) The apoptotic rate of infected cells. The apoptotic rate in WT-MEFs infected by SR-11-△spvB strain was significant lower than in SR-11strain-infected WT-MEFs and SR-11-△spvB strain-infected Atg5-/-MEFs, while the apoptotic rate had no significant difference between in WT-MEFs and Atg5-/-MEFs infected by SR-11strain.(7) Cytokines secretion of infected cells. Expression of Th1type cytokine such as IFN-y in SR-11strain-infected cells was weaker than in cells infected by SR-11-△spvB strain from1h to5h, while the secretion of Th2type cytokines such as IL-4and IL-13in cells infected by SR-11strain were higher than in cells infected by SR-11-△spvB. Three cytokines had no significant difference between in cells infected by two kinds of bacteria at8h and10h.(8) Bacterial quantity of infected cells. The number of intracellular bacterial quantity in SR-11-△spvB strain-infected WT-MEFs was significant lower than in WT-MEFs infected by SR-11and Atg5-/-MEFs infected by SR-11-△spvB strain. Intracellular bacterial quantity had no significant difference between in WT-MEFs and Atg5-/-MEFs both infected by SR-11strain.Conclusions:1. Salmonella virulence plasmid pRST98can promote intracellular bacte(?)al survival or proliferation through inhibiting cellular autophagy and consequently enhance apoptosis of infected fibroblasts, a mechanism underlying pRST98-mediated virulence in S. typhi.2. The capability of autophagy inhibition by pRST98is due to the key virulent segment on its genetic sequence spvB. SpvB can inhibit cellular autophagy through TLR4expression inhibition, PLD1expression promotion and PLD activity enhancement, regulation Th1/Th2shift away through increasing Th2type cytokines IL-4and IL-13secretion and decreasing Th1type cytokine IFN-γ secretion. It is speculated that mTOR activation through serine/threonine protein kinase (AKT)-dependent pathway may be one signaling pathway of autophagy inhibition induced by spvB.3. Rapamycin (mTOR inhibitor) can attenuate autophagy inhibition induced by spv, therefore enhance the competent of intracellular bacteria elimination by autophagy and lessen the injury of host cells.4. Autophagy can act as a "double-edged sword" function in Salmonella infection. Moderate activation of autophagy has a beneficial effect on restricting Salmonella proliferation and reducing the injury of host cells, while its deficiency, inhibition or over enhancement can worse infection and result in the more serious damage of host cells.