Molecular Mechanism Of Anthrax Edema Toxin’s Inhibition On Macrophage Function

Bacillus anthracis isthe pathogen of anthrax,an acute,rapidly progressing infectious disease that affects bothanimals and humans.Anthrax spores have repeatedly been used for bioterrorism attacks,and,are also a potential biological warfare agent.Therefore,in-depth understanding of the mechanism of infection and pathogenicity of Bacillus anthracis is helpful to the discovery of new methods for the prevention,control and treatment of anthrax infection,and is conducive to the promotion of biosafety.The virulence factors of Bacillus anthracis mainly include capsule and anthrax toxins.Anthrax toxins include three components:protective antigen（PA）,lethal factor（LF）,and edema factor（EF）.The lethal toxin（LT）,which is consists of LF and PA,mainly inactivatesthe relevant pathway through cleavage of mitogen-activated protein kinase kinases（MAPKK）1-4,6,and 7.Edema toxin（ET）is composed of EF and PA.The EF that enters cells with PA can dramatically increase intracellular cAMP levels and affect the body’s signal transduction.Previous studies of anthrax toxins have focused on LT,and the role of ET in anthrax infection and death has not been fully understood.Anthrax spores can infect the host through the respiratory tract,skin,or digestive tract.The spores of anthrax entering the body through the respiratory tract enter the local lymph nodes after being phagocytosed by alveolar macrophages and germinate into propagule bacteria.The Bacillus anthracis that germinates in the local tissues can also enter the local lymph nodes by destroying the innate immune barrier by anthrax toxins.Macrophages are one of the key members of the innate immune system,so we have studied the inhibitory effects and molecular mechanisms of ET on macrophage function in this study.Firstly,we expressed and purified the wild-type anthrax edema factor EF in E.coli,and EF（H351R）and EF（H351A）,which mutated a key amino acid residue of adenylyl cyclase activity.In in vitro cell-free reaction systems and CHO-k1 cells,wild-type EF showed extremely high cAMP production efficiency,while EF（H351R）and EF（H351A）produced cAMP with a greatly reduced efficiency.In mice,ET,ET（H351R）and ET（H351A）consisting of wild-type or mutant EF and PA caused varying degrees of organ damage and local edema.The severity of injury was positively correlated with cAMP levels in the tissues.25-50μg of wild-type ET couldcause mice to die,while the same dose of ET（H351R）or ET（H351A）didnot kill mice.We used mouse mononuclear macrophage cell line RAW264.7 cells as a cell model to evaluate the effect of ET.Monocytes can differentiate into two distinct phenotypes upon maturation.RAW264.7 surface molecular markersdetected by Flow cytometryassay showed that these cells exhibited a tissue-resident phenotype of CD14⁺CD16/32⁺when untreated or treated with LPS alone,and these cells were converted into CD14^hiCD16/32^-inflammatory phenotype after co-stimulation of ET with LPS.E.coli expressing GFP protein ican be phagocytosed by macrophages.The GFP positive rate of macrophages detected by flow cytometry and the averagefluorescent intensity of GFP assessed in each cell can reflect the phagocytosis and microbicidalactivity of them.The results showed that the phagocytic function of macrophages after 3h of ET pretreatment did not decrease,but the killing function to intracellular bacteria was significantly weakened.The detection of cytokines secreted by macrophages showed that ET treatment downregulated the production of TNF-αand MCP-1 and upregulated the production of IL-6 and IL-10.ET treatment makes RAW264.7 cells with surface markers of inflammatory cells but does not have true pro-inflammatory function and is a pseudo-inflammatory phenotype.We further analyzed the transcriptomes of RAW264.7 cells treated with ET,ET（H351A）,adenylate cyclase agonist Forskolin,or PA using next-generation sequencing technology.Compared with PA treatment group,ET,ET（H351A）and Forskolin treatment resulted in the up-regulation or down-regulation of 1228,264,and 211 genes in RAW264.7 cells,respectively.Examination of intracellular cAMP levels showed that elevated cAMP levels were positively correlated with transcriptome differences.Thirty-nine cAMP-dependent genes were screened from differential genes produced by ET processing.The functions of these genes are mainly related to macrophage immune responses,while the remaining non-cAMP-dependent genes are mainly involved in cellular material and energy metabolism functions.The KEGG signaling pathway was used to cluster the differentially expressed genes produced by ET processing.It was found thatmacrophage activation mediated by MAPK and NF-kB signaling enriched in theTNF-αpathway.ET treatment couldinhibit the activation of the NF-kB binding site reporter gene and downregulate the activation of NF-kB by TNF-α.The NF-kB inhibitor BAY 11-7082also down-regulatedthe activation of the TNF-αpromoter.P65 is the most important NF-kB subunit.ET treatment inhibited the phosphorylation of p65 and reduced the localization of p65 in the nucleus.cAMP exerts its activity primarily by activating PKA.PKA inhibitor H89 couldreverse ET inhibition of NF-kB transcriptional activity.These results indicate that ET may inhibit p65-mediated transcriptional activation of TNF through the PKA pathway.At the same time,western-blot detection revealed that ET treatment also inhibited the phosphorylation of ERK,a member of the MAPK protein family.The treatment of RAW264.7 cells with the ERK phosphorylation inhibitor PD3125901 also inhibited the production of TNFa.This suggests that ET downregulation of macrophage TNF-αproduction may be related to inhibition of ERK phosphorylation.Combining these results,ET may reduce macrophage TNF-αproduction by inhibiting NF-kB and ERK activation.In summary,firstly,wild-type EF and its mutants EF（H351A）and EF（H351R）,of which the adenylate cyclase activity was reduced,were preparedin this study;secondenly,thesurface molecular markers alteration and the pro-inflammatory functionof macrophageswere inhibited by ET treatment;thirdly,through transcriptome sequencing,it was revealed that the regulation of macrophage immune function by ET was mainly related to its adenylate cyclase activity;further experimental validation showed that ET may inhibit the activation of NF-kB and ERK through the PKA pathway and downregulate the production of macrophage inflammatory factor TNF-α.