Salmonella typhimurium invades macrophages RAW264.7 and induces Cuproptosis in cells
Objective To discuss the phenomenon of Cuproptosis in macrophages induced by pathogen infection and analyze the effect of this cellular response on bacterial intracellular survival,and to explore the mechanism of Cuproptosis and its effect on the intracellular survival and pathogenicity of Salmonella typhimurium.Methods 24 hours after the invasion of Salmonella Typhimurium and the addition of copper ion carrier Elesclomol(ES)and copper chloride,the cells were collected and set up as untreated cell control group,ES control group and WT infection group,and the changes in intracellular copper ion concentration were detected by inductively coupled plasma mass spectrometry;after intracellular copper staining,the distribution of copper ions was observed using a confocal microscope;48 hours after cell invasion,different concentrations of copper ions(1 μmol/L,2.5 μmol/L,5 μmol/L)were added to the untreated cell control group,cells and infected cells,and the difference in the mortality rate of infected cells before and after the addition of copper ions was detected by the lactate dehydrogenase method;24 hours after cell invasion,immunofluorescence and Western blot methods were used to detect the production and distribution of lipoyl-modified DLAT protein oligomers,a key indicator of Cuproptosis.Results There was no difference in the content of copper in RAW264.7 cells infected and treated with drugs.The content of copper in cells was 0.06533±0.008327 in NC group,0.1580±0.002646 in ES group,and 0.07367±0.007572 in WT group.Confocal microscopy showed that the distribution of copper ions in cells changed after infection,and copper ions aggregated into dots.After 48 hours of cell infection,the cell death rate in Cell group was 19.60±9.758,that in Cell+1μmol/L CuCl2 group was 17.27±7.130,that in Cell+2.5 μmol/L CuCl2 group was 40.13±5.650,and that in Cell+5 μmol/L CuCl2 group was 65.50±1.241.The cell death rate of the cells in the WT+1 μmol/L CuCl2 group was 65.44±1.329,that in the WT+2.5 μmol/L CuCl2 group was 80.56±4.543,that in the WT+5 μmol/L CuCl2 group was 76.42±1.316.After the invasion of Salmonella typhimurium,the cell death rate increased with the increase of copper ion concentration added to the culture medium.Confocal microscopy and Western blot showed that the key protein of Cuproptosis,pyruvate dehydrogenase DLAT subunit,oligomerized to form polymers after the invasion of Salmonella typhimurium.Conclusion After infection,RAW264.7 cells initiated Cuproptosis and inhibited the intracellular survival of Salmonella typhimurium,providing a theoretical basis for the study of the mechanism of Cuproptosis.
Salmonella Typhimuriummacrophage RAW264.7Cuproptosiscopper ionsDLAT protein
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