The Influence Of Salmonella Plasmid Virulence Gene SpvB On The Delayed Pyroptosis Of Macrophage And The Underlying Mechanisms

Objective:To investigate the mechanisms of the delayed pyroptosis of macrophages induced by Salmonella plasmid virulence gene spvB and provide new ideas for the prevention and treatment of Salmonella infection,RAW264.7 macrophages infected with Salmonella enterica serovar Typhimurium(S.typhimurium,STM)were established to explore the effects of spvB on the inflammasomes NLRP3 and NLRC4,and nlrp3-/-RAW264.7 macrophages constructed by CRISPR/Cas9 technique were used to study the influence of NLRP3 on the activation of NLRC4 and the delayed pyroptosis.Methods:Part One.Effect of Salmonella plasmid virulence gene spvB on NLRP3 and NLRC4Wild-type S.typhimurium(SL1344),S.typhimurium mutant with plasmid virulence gene spvB knock-out(SL1344-?spvB)and spvB complement strain(SL1344-c-?spvB)were used to infect RAW264.7 cells at an multiplicity of infection(MOI)of 10:1.The infected macrophages were collected at different time points for experiments.1.To observe the ultrastructure of the infected macrophages Ultra-thin sections were made to observe the ultrastructure of cells in different infection groups under transmission electron microscope.2.To analyze the transcription level of nlrp3 and nlrc4To extract cellular RNA and detect the level of nlrp3 and nlrc4 by qPCR.3.To detect the expression levels of proteins related to pyroptosis The expression levels of NLRP3,NLRC4,GSDMD,GSDMD-NT and Caspase-1 were detected by Western blot.4.To determine the level of reactive oxygen(ROS)in cells The level of ROS in infected cells was detected using ROS Test Kit.ROS can oxidize DCFH to generate fluorescent DCF.The level of ROS can be determined by measuring the fluorescence of DCF.Part Two.Effects of NLRP3 on the pyroptosis of macrophages SL1344,SL1344-?spvB and SL1344-c-?spvB were used for infection.Wild-type and nlhh3-/-RAW264.7 cells were infected with STM at an multiplicity of infection(MOI)of 10:1.The infected macrophages were collected at different time points for experiments.1.To analyze the transcription level of nlrc4RNA of both Wild-type and nlrp3-/-RAW264.7 cells infected by STM was extracted and the level of nlrc4 was determined by qPCR.2.To determine the level of GSDMD and GSDMD-NTThe expression level of GSDMD and GSDMD-NT were determined by Western blot.3.To detect Caspase-1 activityCaspase-1 activity test kit and colorimetry were used to detect the activity of Caspase-1 in different infection groups.4.To determine the level of IL-1?IL-1? secretion in the cell culture supernatants of different infection groups was detected by ELISA method.Results:Part One.Effect of Salmonella plasmid virulence gene spvB on NLRP3 and NLRC41.Cellular ultrastructure of STM infected macrophagesAt 18-24 h post infection(hpi),the cytoplasms of SL1344 and SL1344-c-?spvB infected group were loose and destroyed,mitochondrial swollen and more intracellular bacteria were observed.The SL1344-?spvB infected group had better cell status,relatively normal mitochondrial shape,and relatively less intracellular bacteria.2.The level of nlrp3 and nlrc4 in STM infected macrophages qPCR results showed that nlrp3 played a role in early stage of cell infection(1-4 h).The level of nlrp3 of macrophages infected with SL1344 and SL1344-c-?spvB was lower than that infected with SL1344-?spvB,the difference was most significant at 1 hpi and 2 hpi;while nlrc4 was evident at 18 hpi and 24 hpi.The level of nlrc4 of macrophages infected with SL1344 and SL1344-c-?spvB was lower than that infected with SL1344-?spvB.3.The expression of NLRP3,NLRC4 and proteins related to pyroptosisWestern blot results showed that NLRP3 played a role in early stage of cell infection.The expression of NLRP3 of macrophages infected with SL1344 and SL1344-c-AspvB was lower than that infected with SL1344-?spvB,the difference was most significant at 4 hpi.While NLRC4 was evident at late stage of cell infection,the expression of NLRC4 of macrophages infected with SL1344 and SL1344-c-?spvB was lower than that infected with SL1344-?spvB,the difference was most significant at 24 hpi.The expression of GSDMD-NT in macrophages infected with SL1344 and SL1344-c-?spvB was lower than that in SL1344-?spvB infected macrophages from 4 hpi to 12 hpi,while the expression of GSDMD-NT infected with SL1344 increased significantly at 18 hpi.The trend of level of p20,mature Caspase-1 was similar to GSDMD-NT.4.The level of ROS in STM infected macrophagesThe content of ROS in macrophages infected with SL1344 and SL1344-c-?spvB was lower than that of SL1344-?spvB infected macrophages at 2 hpi.Part Two.Effects of NLRP3 on the pyroptosis of macrophages1.The transcription level of nlrc4The qPCR results showed that nlrc4 played a role at the later stage of cell infection(18-24 hpi).In both wild-type and nlrp3-/-RAW264.7 infected group,the expression of nlrc4 was lower in the SL1344 and SL1344-c-?spvB infected cells than that of SL1344-?spvB infected cells.While the difference of nlrc4 level between spvB carrying Salmonella(SL1344,SL1344-c-?spvB)and spvB free Salmonella(SL1344-?spvB)was significantly decreased in nlrp3-/-RAW264.7 cells infected group in comparision with wild-type RAW264.7 infected group.2.The level of GSDMD and GSDMD-NTWestern blot results showed that at 4 hpi,8 hpi,18 hpi,and 24 hpi,the level of GSDMD-NT was significantly decreased in nlrp3-/-RAW264.7 infected group in comparision with wild-type RAW264.7 infected group.3.Caspase-1 activity and the detection of IL-1?The colorimetric assay of Caspase-1 activity and determination of IL-1? by ELISA showed both increased with infection time prolonged in wild-type RAW264.7 group,the different Caspase-1 activity and IL-1? serection level of macrophages infected by various bacteria were evident at 18 hpi and 24 hpi.While in nlrp3-/-RAW264.7 infected group,there were no significant difference.Conclusions:1.The delayed pyroptosis of macrophages induced by Salmonella plasmid virulence gene spvB was associated with the influence of inflammasome NLRP3 and NLRC4,spvB could inhibit the production of ROS and the activation of NLRP3 at the early stage of infection,and then inhibited the activation of NLRP4.2.The inflammasome NLRP3 was a key element that could affect the function of Salmonella plasmid virulence gene spvB on inflammasome NLRC4,and was significant for the outcome of infection.