Preliminary Analysis Of The Interaction Between Peroxisomes And Salmonella Typhimurium In The Infected Macrophages

Salmonella typhimurium belongs to enterobacteriaceae. It is an important enteric pathogenic bacteria, which can cause a spectrum of diseases ranging from self-limiting gastroenteritis to life-threatening systemic diseases. Salmonella are facultative intracellular bacteria which can invasion a variety of phagocytic and non-phagocytic cells. It can survival and reproduction in host cells. Macrophages are important hosts of salmonella typhimurium which play an important role in the development of salmonellosis. The host cells infected with salmonella typhimurium will synthesize inducible nitric oxide synthase which can produce nitric oxide and reactive nitrogen species. They can inhibit and kill the intracellular bacteria.INOS can localize in cytoplasm vesicles and peroxisomes in various cells. In this study,we infected macrophages and Kunming mice with living or inactived salmonella typhimurium. The expression of iNOS and the 70-kDa Peroxisomal Membrane(PMP70) and the number of intracellar salmonella was determined in infected macrophages . 1400W(N-(3-[Aminomethyl]benzyl)acetamidine) was added in 10 %DMEM to handle macrophages. The concentration of nitric oxide in culture medium and the bacterial number in the macrophages infected with living bacteria were detected. The concentration of nitric oxide in blood and liver tissue of mice infected with salmonella were determined. The characteristic of salmonella infection was discuss by the difference between living and inactived bacteria and by the change with the infection time. The results of the infected macrophages handled with 1400W were compared with the results of macrophages were not handled with 1400W. The function of iNOS was invested by the results. To investigate the orientation relationship among iNOS peroxisome and salmonella typhimurium in the infected macrophages. To explore the antimicrobial function of peroxisomes in macrophages, which provides theoretical basis of biological treatment to salmonellosis.1,The expression of iNOS in macrophages infected with salmonella typhimuriumRAW264.7 macrophages were infected at multiplicity of infection(MOI)of 20 with living or inactivated salmonella typhimurium for 1h .Then the free bacteria were washed out with PBS and the infected cells were cultered for the special time (1h,4h,8h,12h,24h). The follow detections were did in this study . Total RNA was extracted from the infected cells at the given time point. Real-time PCR was performed using reference gene GAPDH and target gene iNOS after the total RNA being reverse transcribed into cDNA. Griess reagent system was used to determine the concentration of nitric oxide in the host cells culture medium at the given time. The results demonstrated living or inactivated salmonella typhimurium could promote the the expression of iNOS and the concentration of nitric oxide in culture medium of infected macrophages. However inactivated bacteria could induce the the expression more effective than the living .The expression of iNOS continued to increase within 12h and reach the maximum. The values were 21.723 and 22.051 times the control respectively induced by living and inactivated salmonella at 12h. There were no significant difference between the two values. However the values of the expression of iNOS in infected macrophages with inactivated salmonella significantly higher than that of the living bacteria at 1h 4h 8h 24h. The concentration of nitric oxide in culture medium of macrophages infected with living or inactivated bacteria increase at the beginning and then decrease at 12h within 24h. The concentration of nitric oxide in culture medium of macrophages infected with living bacteria is significantly lower than that of inactivated bacteria at the same given time. These results demonstrated that salmonella typhimurium in host cells could significantly induce the expression of iNOS and living salmonella typhimurium could inhibit the expression of iNOS and the elevation of concentration of nitric oxide.2,Test of counting the intracellular salmonella typhimurium in infected macrophages at the given timeRAW264.7 macrophages were infected at multiplicity of infection of 20 with living or inactivated salmonella typhimurium for 1h. The infected cells were cultered for the special time (1h,4h,8h,12h,24h). The infected cells grew on cover glass were fixed by 4% paraformaldehyde. Immunofluorescence stain was employed to count the bacterial numbers in infected RAW264.7 macrophages. 0.1%Triton X-100 PBS was used to lysis RAW264.7 macrophages infected with living bacteria at the given time point, then count the living bacterial numbers in the macrophages by quantitative bacterial culture in LB plate. There was insignificant difference between the two counting methods used in the test of infection with living bacteria .The reason was part of intracellular living bacteria was killed. The bacterial number counted by immunofluorescence or schizolysis display the bacteria number continued to increase by 12h in the infectd macrophages. The maximum emerge at 12h counted by the two methods. There were insignificant difference between the two counting methods at the same time by 8h. There were significant difference between the two counting methods at the same time at 12h and 24h。The number of bacterial was 13.857counted by immunofluorescence and 7.940 counted by schizolysis at 24h. There is significant difference between the two counting methods at 24h. There is significant difference among the number of bacterial in test of infection with inactived bacteria. That means the inactivated salmonella was digested by the host cell and became smaller.The photos of macrophages infected with inactived bacteria showed that the intracellular bacteria gradually shrink and became fine grain. These results demonstrated that salmonella typhimurium can survival and reproduction in these host cells.They could induce the expression of iNOS and promote the production of NO. These results and the results of the expression of iNOS demonstrated that macrophages could kill and digest the intracellular salmonella at a certain level of nitric oxide.3,Test of the concentration of NO in culture medium and the intracellular bacterial number of the macrophages handled with 1400WThe macrophages were cultured in six well plate with 10%DMEM added 1400W to 50umol/L for 24h. The macrophages were infected at multiplicity of infection of 20 with living salmonella typhimurium for 1h. The infected cells were cultered for the special time (1h,4h,8h,12h,24h). 10%DMEM added 1400W to 50umol/L was used in the whole process. The concentration of nitric oxide in culture medium of infected macrophages was determined by the Griess reagent. Immunofluorescence stain was employed to count the bacterial numbers in infected macrophages. 0.1%Triton X-100 PBS was used to lysis macrophages infected with living bacteria at the given time point, then count the living bacterial numbers in the macrophages by quantitative bacterial culture in LB plate. There were insignificant difference among the concentration of nitric oxide in culture medium at different time. The concentration of nitric oxide in culture medium of macrophages handled with 1400W was significantly lower than that of macrophages handled without 1400W. The results demonstrated that the activity of iNOS was suppressed by 1400W. The intracellar bacterial numbers in the macrophages handled with 1400W were significantly higher than that of macrophages handled without 1400W. There were insignificant difference between the two counting methods at the same time. The results above all demonstrated that the activity of iNOS was suppressed by 1400W, the production of bactericidal NO significantly decreased . The survival and proliferation of salmonella in macrophages handled with 1400W significantly higher than that of macrophages not handled with 1400W.4,Test of the concentration of NO in blood and liver tissue of Kunming mice infected with salmonella typhimuriumKunming mice were inoculated intraperitoneally with living or inactivated salmonella typhimurium.Each mice was infected with1.75×107 bacteria.The blood and liver tissue were collected at the special time(1h,4h,8h,12h,24h). To determine the concentration of nitric oxide in mice blood and liver tissue. The concentration of nitric oxide in blood and liver tissue of mice infected with living salmonella is significantly higher than the inactived bacteria. The concentration of nitric oxide in blood of mice infected with living salmonella is significantly higher than the control. The concentration of nitric oxide increased to the maximum at 12h,which is 5.823 fold over that of the control. The concentration of nitric oxide in liver tissue of Kunming mice infected with living salmonella typhimurium increased and were continuously in high level between 8h and 12h . The concentration of nitric oxide in blood and liver tissue of Kunming mice infected with inactivated salmonella typhimurium increased but changed flat. There was insignificant difference between the two values of the concentration of NO in blood of mice infected with living and inactived bacteria at all given time. There was insignificant difference between the two values of the concentration of NO in liver tissue of mice infected with living and inactived bacteria at all given time besides 1h. These results demonstrated that living salmonella could induce significantly higher NO level in blood of infected mice than inactivated salmonella. Salmonella transferred to liver could induce the expression of NO.5,The expression of the 70-kDa Peroxisomal Membrane of RAW264.7 infected with salmonella typhimuriumRAW264.7 macrophages were infected at MOI of 20 with living or inactivated salmonella typhimurium for 1h .Then the infected cells were cultered for the special time (1h,4h,8h,12h,24h). Total RNA was extracted from the infected cells at the given time point. Real-time PCR was performed using reference gene GAPDH and target gene PMP70 after the total RNA being reverse transcribed into cDNA. The result show there was insignificant difference bttween the expression of PMP70 induced by living and inactivated salmonella typhimurium.The maximum value in inactived bacteria group at 4h was significantly higher than the control. It is the only on in inactived bacteria group which were significantly higher than the control. The expression of PMP70 in macrophages infected with living bacteria increased at the beginning and then decreased. The value of PMP70 at 8h and 12h are significantly higher than the other time point and significantly higher than the inactived group at the same time. The results demonstrated living bacteria infected in macrophages could significantly induce the expression of peroxisome than the inactived bacteria.6,orientation relationship among iNOS peroxisome and salmonella typhimurium in the infected macrophagesRAW264.7 were infected at MOI of 20 with salmonella typhimurium for 1h and cultered for 12h. Immunofluorescence stain was employed to mark iNOS peroxisome and Salmonella typhimurium in the infected macrophages. The orientation relationship among iNOS peroxisome and Salmonella typhimurium in the infected macrophages were examined with fluorescence microscope . RAW264.7 were infected at MOI of 50 with salmonella typhimurium for 1h and cultered for 12h.Colloidal gold was used to mark the peroxisome in the infected macrophages . The orientation relationship betweem peroxisome and salmonella typhimurium in the infected macrophages were examined with scanning electron microscopes. Peroxisome iNOS and salmonella typhimurium co-localization eachother in the infected macrophages . The immune colloidal gold electron microscopy images show that peroxisome and salmonella typhimurium co-localization in the infected macrophages. peroxisomes carry iNOS and take part in the killing of salmonella typhimurium infected in the macrophages.The results of the study demonstrated salmonella typhimurium in host cells could significantly induce the expression of iNOS mRNA and NO. When the concentration of bactericidal material reached certain level , part of the intracellar salmonella were killed . Living bacteria infected in macrophages could inhibit the expression of iNOS . The ability to survive and proliferate of intracellar salmonella was improved when the activity of iNOS was inhibited by 1400W. Living bacteria infected in macrophages could significantly induce the expression of PMP70 mRNA.Peroxisome and iNOS co-localization in the infected macrophages. Peroxisome and iNOS co-localization with salmonella in the infected macrophages. We concluded Living bacteria infected in macrophages could significantly induce the expression of iNOS . INOS co-localization in peroxisome had the function of killing living bacteria . We propose peroxisome had the function of killing living bacteria, which provides theoretical basis of biological treatment to salmonellosis.