Research Of Mechanism Of Murine Macrophage Lysis By Anthrax Lethal Toxin

Anthrax lethal toxin rapidly kills macrophages from certain mouse strains. Lethal factor is a Zn²⁺-dependent metalloprotease that cleaves N terminal of several MAPK kinases block the MAPK signaling pathway and induce the formation of the Nalp1b inflammasome. The reason of how the LT lysis the macrophages are unknown.Various expression hosts have been used for the purification of PA and LF, including Escherichia coli,Bacillus subtilis,B. anthracis. The activity of the purified LF is not the same, even a 3 fold change. The stability of LF in cytosol of cells depends on identity of the N-terminal residue. For receive LF with impact N terminal, we amplify the LF gene from the Bullilus anthrax A16R with PCR, construct the plasmid pAS22-LF and transform the plasmid into the host E. coli DH5α. Induced by IPTG for expression the LF for several hours and release the LF with high penetrability. Obtain the LF with the purity exceed 90% through 4 step purification. The molecular weight and the N terminal amino acid sequence are consistent with natural LF. The purity of LF and its mutation LF E687A determined by HPLC is exceed 90%. The content of LPS is lower than 0.5EU/ml. Cytotoxicity assay in the mouse macrophage J774A.1 cell line were performed, and the LD50 beneath 1ng/ml with 100ng/ml PA. This work establishes the foundation for the next study.LF induced the murine macrophages contain a Nalp1bS gene rapidly lysis. Events that occur following intoxication include cleavage of MEKs and/ or an unidentified target(s), proteasome cleavage of unknown target(s), mitochondrial dysfunction, potassium efflux, caspase-1 inflammasome activation, plasma membrane permeability and cellular lysis with release of IL-1βand IL-18. Some inhibitors of caspase and proteasome can protect the macrophage from the attack of LT, and proteasome inhibitor can control the activity of caspase-1. The relationship between caspase signaling pathway and proteasome in the event that macrophage rapidly lysis is less known. To study the substrate of proteasome that is essential for activation of caspase-1, is significance to understand the LF lethal activity. We chose the LT-sensitive J774A.1 cell line as a model, detect the caspase-1,3/7 activities after LT attack. We found that the activity of caspase-1 increase at 15 min after addition LT, and maintain until 75 min. The activity of caspase-3/7 was no statistics significance compare with the group add PA+LF E687A. The staining with Annexin V and IP and the flow cytometry confirm the results. So we think that LT lysis macrophage by a caspase–independent way, but the activity of caspase-1 is needed. We found that the proteasome inhibitors MG132 and Lactacystin often used to protect macrophages, can also inhibite the activity of NF-κB signaling pathway. So, as a consequence, we test the changes of families of the NF-κB pathway. Through the Western blot test, the activity of NF-κB can be induced as the degration of IκB-α.The J774A.1 cell line lysis by the LT in a rapid way, to know what happened in the cell when they are dying is our purposes. Mitochondrial impairment is a critical event in anthrax lethal toxin-induced cytolysis of murine macrophages, and the changes in the nuclei is unknown. In order to analysis the changes after the attack of LF, we extract soluble/membrane protein, mitochondrial protein and nuclear protein subject to two dimensional electrophoresis. Proteome analysis of LT-treated and control macrophages revealed 32 differentially expressed protein spots. Analysis of up- and down-regulated proteins revealed that primarily the stress response and energy generation protein play an important role in the LT-mediated macrophage cell death. In addition, we find two proteins have potential relationship with NF-κB signaling pathway. The first one is valosin containing protein p97(VCP), which is up-regulate and the another one named HMGB1(high mobility group B1) is down-regulate in the nuclei. VCP can form a complex with IκB-αand facilitate its degradation by proteasome, resulting in activation of NF-κB. HMGB1 is a high conserved no histone DNA-binding protein, and it is also an endogenesis dangerous signal. When the cell stress or necrosis, the HMGB1 release into extracellular and activated NF-κB by interaction with RAGE and TLR.In summary, our findings suggested that LT attack murine macrophages activate the NF-κB signaling pathway, combined with caspase-1 inflammasome result in bursting of cytokine release and the inhibitory mechanisms do not have enough time to counteract the potentially deadly effects.