Analysis to the relationship between peroxisomes and Salmonella typhimurium in macrophages / 解放军医学杂志

ABSTRACT

Objective To understand the role of peroxisomes on the intracellular survival of Salmonella typhimurium in macrophages. Methods Vesicles from infected or uninfected RAW264.7 macrophage-like cells were isolated by stepwise centrifugation after cells were broken by nitrogen cavitation and purified with magnetic beads containing polyclonal antibodies to TassC (Target for Salmonella secreted protein SpiC). Western-blot was used to detect the characters of the binding vesicles. Three-dimensional (xyz) fluorescence microscopy was used to determine the recruitment of peroxisomes tagged with pDsRed2-Perxi to the SCV after infection of RAW264.7 cells with Salmonella typhimurium mutant strains spiCkan producing GFP. Volocity software was employed for image analysis. Overlap of individual fluorescence pixels from separated channels for each optical plane was determined with the Volocity 4.1 colocalization module. Results It was shown by western-blot that the anti-TassC magnetic beads could bind TassC and peroxisomal marker catalase in infected or uninfected RAW264.7 cells. Inducible nitric oxide synthase (iNOS) could be detected in infected samples, but couldn’t in uninfected samples. Lysosome associated membrane protein (LAMP1), one of the lysosome markers, and a bacterium marker RecA could not be detected from the elution of anti-TassC magnetic beads. It was determined by a fluorescence microscopy that the recruitment or overlapping of peroxisomes tagged with pDsRed2-Perxi to the Salmonella-containing vacuoles after infection of RAW264.7 cells with Salmonella mutant strains producing GFP. Calculation by Volocity 4.1 showed that peroxisome abundance increased by 1.2- or 1.3-fold, respectively, at 1h and 24h time point of infection in the infected macrophages than in uninfected cells while the bacteria abundance decreased with the infection time. Conclusion It is suggested that TassC is localized with peroxisomes, but not with lysosomes. Inducible nitric oxide synthase might be localized to peroxisomes and recruited to the SCV during infection with Salmonella mutant strains for killing the bacterium.