The Research Of Autophagy Was Induced By SARS-CoV 3a And Anthrax Lethal Toxin

We used electron microscopy, Western blotting, GFP-LC3 monitor and monodansylcadaverin (MDC) staining to detect the autophagy in vivo, which was induced by the severe acute respiratory syndrome (SARS) coronavirus 3a protein and anthrax lethal toxin. We investigated the signaling pathway of autophagy which was induced by SARS-CoV 3a protein and the effect of autophagy on the intoxication of anthrax lethal toxin.Autophagy is one of the major pathways involved in the lysosomal degradation of cellular components in eukaryote. This process has an important role in various biological events such as eliminate intracellular pathogen. As autophagy is evolutionarily conserved in eukaryotes, some pathogen was able to escape autophagy, moreover some pathogen subvert the cellular autophagy pathway to form advantage on replication and release through evolution.Electron microscopy, Western blotting, MDC staining and GFP-LC3 monitor have been used to monitor autophagy in vivo. Microtuble?associated protein 1 light chain 3 (LC3), a mammalian homolog of yeast Atg8, has been used as a specific marker to monitor autophagy. Upon induction of autophagy, LC3 is conjugated to phosphatidylethanolamine and targeted to autophagic membranes. We constructed the expression plasmid encoding GFP-LC3, cDNA of HeLa cell was used as template and LC3 gene was amplified by PCR, then the gene was cloned into pEGFP-C1 vector, to generated the pEGFP-LC3 plasid. HeLa cells was transiently transfected with pEGFP-LC3 plasmid, the expression of GFP-LC3 protein in vivo was measured by fluorescence microscopy and Western blotting. We observed the GFP-LC3 puncta in HeLa cells when autophagy was induced. So we can use the pEGFP-LC3 plasmid to monitor the autophagy in cell.The non-structural proteins of SARS-CoV vary widely among different coronavirus species, which may be the reason of SARS-CoV was more lethal than other coronavirus species. 3a protein is the biggest non-structural protein of SARS-CoV. We speculated the SARS-CoV 3a protein could induce autophagy in vivo by references. Firstly, we transiently transfected pCI-3a plasmid into Vero E6 cells to monitor the autophagy in vivo, but we couldn't measure the autophagy in Vero E6 cells because the expression of SARS-CoV 3 protein in Vero E6 cells was very low. To improve the expression of SARS-CoV 3a protein in vivo, we transfected pcDNA5/FRT-3a plasmid into Flp-InTM CHO cells and screened the stable expression of SARS-CoV 3a transformant named CHO-3a. We detected autophagic vacuoles in CHO-3a cells. After transfection with pEGFP-LC3 plasid, we observed the percentage of cells display fluorescent punctate GFP-LC3 in CHO-3a cells much more than in CHO cells. We observed the blue fluorescence in CHO-3a cells through by fluorescence microscopy that was stained by MDC, but the phenomenon in CHO cells was very low. Western blotting show the conversion of LC3-I to LC3-II was increased in CHO-3a cells relative to CHO cells. The SARS-CoV 3a protein induced autophagy in CHO cells by these experimentsSARS-CoV 3a protein could bind to calcium-modulating cyclophilin ligand (CAML), as revealed by yeast two-hybrid analysis. The interaction of SARS-CoV 3a protein and CAML was identified by GST pull-down assay. The autophagy could be inhibited by siRNA design for CAML measured by western blotting and GFP-LC3 monitor. CAML expression elevates the cytosolic Ca2+ concentration and the rise in the cytosolic Ca2+ concentration is a potent inducer of autophagy. The autophagy was inhibited by BAPTA/AM -a Ca2+ chelator, measured by western blotting and GFP-LC3 mintor. The Ca2+ induced Ca2+-CaMKK-β-AMPK signaling pathway and AMPK could induce autophagy by the negative regulation of mTOR. The autophagy was inhibited by compound C which was the AMPK inhibitor, measured by western blotting and GFP-LC3 monitor. According to above results, the interaction of SARS-CoV 3a and CAML could induce autophagy through a Ca2+ signaling pathway.Anthrax toxin contains three subunits: protective antigen (PA), lethal factor (LF) and edema factor (EF).Anthrax lethal factor was the most important toxin factor, but the research was less than EF or PA. In this research, we detected autophagic vacuoles in J774A.1 cells which was induced by anthrax lethal toxin through by Electron microscopy, Western blotting, MDC staining and a proportional relationship between with LT treating time and the degree of autophagy. Lethal toxin consists of PA and LF, we used PA, LF, PA plus LF, PA plus LFn to treated J774A..1 cells. The results showed only that the PA plus LF could induce autophagy, so the cytosolic LF was the functional factor to induce autophagy. Lethal toxin was reported to induce the production of reactive oxygen species (ROS) in macrophages, glutathione (GSH) is an antioxidant that is widespread in the cells and it can eliminate ROS. The result of Western blotting showed autophagy was inhibited by GSH and it's suggested that the ROS was the sitmulating facter of the autophagy. The median lethal concentration (LC50) of LT was measured by MTT assay. The LC50 of anthrax lethal toxin was influenced by autophagy, Autophagy was induced in murine macrophage by anthrax lethal toxin and was used as a means of weaken anthrax lethal toxin in murine macrophage.