Calyculin A Sensitive Protein Phosphatase Is Required for Bacillus anthracis Lethal Toxin Induced Cytotoxicity

Previous studies have shown that the Bacillus anthracis lethal toxin can induce both necrosis and apoptosis in mouse macrophage-like J774A.1 cells depending on both the toxin concentration and the phosphatase activity. In this study several protein kinase or phosphatase inhibitors were employed to evaluate the hypothesis that the lethal toxin induces cell death via protein phosphorylation processes. Pretreatment with a serine/threonine phosphatase inhibitor Calyculin A (300 nM) could inhibit about 78% of cell death induced by the lethal toxin, whereas inhibitors of kinases, such as H7, HA, Sphingosine, and Genestein, but other inhibitors of phosphatases, such as Okadaic acid, Tautomycin, and Cyclosporin A, did not. In addition, recent reports have demonstrated that the MEK1 protein may serve as a proteolytic target within its N-terminus for lethal factor cleavage. In this study, Calyculin A is shown to enhance the phosphorylation of the MEK1 protein. This prevents the cleavage of the MEK1 by lethal factor. These results suggest that a putative Calyculin A-sensitive protein phosphatase is involved in anthrax toxin induced cytotoxicity and that the blocking effect of Calyculin A on lethal factor cytotoxicity may be mediated through the MEK signaling pathway. Received: 27 December 2000 / Accepted: 1 June 2001     

Cellular and systemic effects of anthrax lethal toxin and edema toxin

Anthrax lethal toxin (LT) and edema toxin (ET) are the major virulence factors of anthrax and can replicate the lethality and symptoms associated with the disease. This review provides an overview of our current understanding of anthrax toxin effects in animal models and the cytotoxicity (necrosis and apoptosis) induced by LT in different cells. A brief reexamination of early historic findings on toxin in vivo effects in the context of our current knowledge is also presented.     

Lethal toxin of Bacillus anthracis causes apoptosis of macrophages

Lethal toxin is a major anthrax virulence factor, causing the rapid death of experimental animals. Lethal toxin can enter most cell types, but only certain macrophages and cell lines are susceptible to toxin-mediated cytolysis. We have shown that in murine RAW 264.7 cells, sublytic amounts of lethal toxin trigger intracellular signaling events typical for apoptosis, including changes in membrane permeability, loss of mitochondrial membrane potential, and DNA fragmentation. The cells were protected from the toxin by specific inhibitors of caspase-1, -2, -3, -4, -6, and -8. Phagocytic activity of macrophages was inhibited by sublytic concentrations of lethal toxin. Infection of cells with anthrax (Sterne) spores impaired their bactericidal capacity, which could be reversed by a lethal toxin inhibitor, bestatin. We suggest that apoptosis rather than direct lysis is biologically relevant to lethal toxin intracellular activity.     

Anthrax lethal toxin-mediated killing of human and murine dendritic cells impairs the adaptive immune response

Many pathogens have acquired strategies to combat the immune response. Bacillus anthracis interferes with host defenses by releasing anthrax lethal toxin (LT), which inactivates mitogen-activated protein kinase pathways, rendering dendritic cells (DCs) and T lymphocytes nonresponsive to immune stimulation. However, these cell types are considered resistant to killing by LT. Here we show that LT kills primary human DCs in vitro, and murine DCs in vitro and in vivo. Kinetics of LT-mediated killing of murine DCs, as well as cell death pathways induced, were dependent upon genetic background: LT triggered rapid necrosis in BALB/c-derived DCs, and slow apoptosis in C57BL/6-derived DCs. This is consistent with rapid and slow killing of LT-injected BALB/c and C57BL/6 mice, respectively. We present evidence that anthrax LT impairs adaptive immunity by specifically targeting DCs. This may represent an immune-evasion strategy of the bacterium, and contribute to anthrax disease progression. We also established that genetic background determines whether apoptosis or necrosis is induced by LT. Finally, killing of C57BL/6-derived DCs by LT mirrors that of human DCs, suggesting that C57BL/6 DCs represent a better model system for human anthrax than the prototypical BALB/c macrophages.     

Targeted Silencing of Anthrax Toxin Receptors Protects against Anthrax Toxins

Anthrax spores can be aerosolized and dispersed as a bioweapon. Current postexposure treatments are inadequate at later stages of infection, when high levels of anthrax toxins are present. Anthrax toxins enter cells via two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). We hypothesized that host cells would be protected from anthrax toxins if anthrax toxin receptor expression was effectively silenced using RNA interference (RNAi) technology. Thus, anthrax toxin receptors in mouse and human macrophages were silenced using targeted siRNAs or blocked with specific antibody prior to challenge with anthrax lethal toxin. Viability assays were used to assess protection in macrophages treated with specific siRNA or antibody as compared with untreated cells. Silencing CMG2 using targeted siRNAs provided almost complete protection against anthrax lethal toxin-induced cytotoxicity and death in murine and human macrophages. The same results were obtained by prebinding cells with specific antibody prior to treatment with anthrax lethal toxin. In addition, TEM8-targeted siRNAs also offered significant protection against lethal toxin in human macrophage-like cells. Furthermore, silencing CMG2, TEM8, or both receptors in combination was also protective against MEK2 cleavage by lethal toxin or adenylyl cyclase activity by edema toxin in human kidney cells. Thus, anthrax toxin receptor-targeted RNAi has the potential to be developed as a life-saving, postexposure therapy against anthrax.     

Intracellular calcium antagonist protects cultured peritoneal macrophages against anthrax lethal toxin-induced cytotoxicity

The lethal toxin ofBacillus anthracis is central to the pathogenesis of anthrax. Using primary cultures of mouse peritoneal macrophages, we have demonstrated that intracellular calcium release inhibitors protect against anthrax lethal toxin-induced cytotoxicity. The cytolytic effect of anthrax lethal toxin was markedly reduced by dantrolene, an inhibitor of calcium release from intracellular calcium stores. Pretreatment of macrophages with cyclosporin A, which has been shown to be a potent inhibitor of calcium release from mitochondria, also protected cells against cytotoxicity. These results indicate that calcium release from intracellular store may be an essential step for the propagation of anthrax lethal toxin-induced cell damage in macrophages. Thus our findings suggest that dantrolene, cyclosporin A, and possibly other drugs affecting intracellular calcium pools might be effectively preventing the toxicity from anthrax lethal toxin. This revised version was published online in July 2006 with corrections to the Cover Date.     

细胞自噬与炭疽致死毒素的相互作用

目的：研究炭疽致死毒素在巨噬细胞中引起细胞自噬现象以及细胞自噬对炭疽致死毒素毒性的影响。方法：采用电子显微镜观察、单丹磺酰尸胺（MDC）荧光染色、Western印迹检测研究炭疽致死毒素作用后的巨噬细胞;采用MTT法检测细胞自噬对炭疽致死毒素毒性的影响。结果：采用以上3种方法,在巨噬细胞J774A.1中均可检测到细胞自噬现象;通过诱导或抑制细胞自噬,分别提高或降低了炭疽致死毒素的半数致死浓度。结论：炭疽致死毒素在巨噬细胞内能引起细胞自噬现象;细胞自噬能减弱炭疽致死毒素对巨噬细胞的毒性。     

Immunosuppressive action of the lethal toxin of Bacillus anthracis in mice

In experiments on inbred mice infected with B. anthracis capsular strain 71/12 of Tsenkovsky's second vaccine B. anthracis lethal toxin introduced in mixture with spores has been shown to aggravate anthrax infection in CBA mice susceptible to anthrax, while producing a faint effect on the infectious process in BALB mice with hereditary resistance to anthrax. B. anthracis purified edema toxin has been found to produce a weaker aggravating effect with respect to anthrax infection than the lethal toxin. As revealed in these experiments, the capacity of the lethal toxin to suppress the activity of peritoneal macrophages in vitro is the more pronounced, the more resistant to anthrax are the mice used as the donors of these macrophages. The mechanism of hereditary immunity which may ensure resistance to infection in the presence of immunosuppression is discussed.     

Dehydroepiandrosterone and melatonin prevent Bacillus anthracis lethal toxin-induced TNF production in macrophages

The lethal toxin of Bacillus anthracis, which is composed of two separate proteinaceous exotoxins, namely protective antigen and lethal factor, is central to the pathogenesis of anthrax. Low levels of this toxin are known to induce release of cytokines such as tumor necrosis factor α (TNF-α). In the present study we investigated the effect of dehydroepiandrosterone (DHEA), melatonin (MLT), or DHEA + MLT on production of lethal toxin-induced TNF-α in mouse peritoneal macrophages. We found that treatment with DHEA significantly inhibited the TNF-α production caused by anthrax lethal toxin. Exposure of MLT to anthrax lethal toxin-treated macrophages also decreased the release of TNF-α to the extracellular medium as compared to the control. However, combined use of DHEA and MLT also inhibited TNF-α release, but not more than single therapies. These results suggest that DHEA and MLT may have a therapeutic role in reducing the increased cytokine production induced by anthrax lethal toxin. This revised version was published online in July 2006 with corrections to the Cover Date.     

A deleted variant of Bacillus anthracis protective antigen is non-toxic and blocks anthrax toxin action in vivo

Anthrax toxin is the only protein secreted by Bacillus anthracis that contributes to the virulence of this bacterium. An obligatory step in the action of anthrax toxin on eukaryotic cells is cleavage of the receptor-bound protective antigen (PA) protein (83 kilodaltons) to produce a 63-kilodalton, receptor-bound COOH-terminal fragment. A similar fragment can be obtained by limited treatment with trypsin. This proteolytic processing event exposes a site with high affinity for the other two anthrax toxin proteins, lethal factor and edema factor. Terminal sequencing of the purified fragment showed that the activating cleavage occurred in the sequence Arg164-Lys165-Lys166-Arg167. The gene encoding PA was mutagenized to delete residues 163-168, and the deleted PA was purified from a Bacillus subtilis host. The deleted PA was not cleaved by either trypsin or the cell-surface protease, and was non-toxic when administered with lethal factor. Purified, deleted PA protected rats when administered 90 min before injection of 20 minimum lethal doses of toxin. This mutant PA may be useful as a replacement for the PA that is the major active ingredient in the current human anthrax vaccine, because deleted PA is expected to have normal immunogenicity, but would not combine with trace amounts of LF and EF to cause toxicity.     

Beta-cyclodextrin derivatives that inhibit anthrax lethal toxin

Recently, we demonstrated that simultaneous blocking of bacterial growth by antibiotics and inhibition of anthrax toxin action with antibodies against protective antigen were beneficial for the treatment of anthrax. The present study examined the hypothesis that blocking the pore formed by protective antigen can inhibit the action of anthrax toxin. The potential inhibitors were chosen by a structure-based design using beta-cyclodextrin as the starting molecule. Several beta-cyclodextrin derivatives were evaluated for their ability to protect RAW 264.7 cells from the action of anthrax lethal toxin. Per-substituted aminoalkyl derivatives displayed inhibitory activity and were protective against anthrax lethal toxin action at low micromolar concentrations. These results provide the basis for a structure-based drug discovery program, with the goal of identifying new drug candidates for anthrax treatment.     

Deletion mutants of protective antigen that inhibit anthrax toxin both in vitro and in vivo

The anthrax toxin complex is primarily responsible for most of the symptoms of anthrax. This complex is composed of three proteins, anthrax protective antigen, anthrax edema factor, and anthrax lethal factor. The three proteins act in binary combination of protective antigen plus edema factor (edema toxin) and protective antigen plus lethal factor (lethal toxin) that paralyze the host defenses and eventually kill the host. Both edema factor and lethal factor are intracellularly acting proteins that require protective antigen for their delivery into the host cell. In this study, we show that deletion of certain residues of protective antigen results in variants of protective antigen that inhibit the action of anthrax toxin both in vitro and in vivo. These mutants protected mice against both lethal toxin and edema toxin challenge, even when injected at a 1:8 ratio relative to the wild-type protein. Thus, these mutant proteins are promising candidates that may be used to neutralize the action of anthrax toxin.     

CMG2-Fc 融合蛋白突变体筛选及中和炭疽毒素活性分析

目的:筛选能有效中和炭疽毒素和抵抗炭疽毒素损伤细胞的CMG2-Fc(炭疽毒素受体II-人免疫球蛋白Fc段融合蛋白)突变体。方法:运用FoldX等计算软件分析CMG2与PA晶体学结构,设计能提高CMG2-PA亲和力的突变体分子,并与人IgG1Fc片段构成融合基因,转染CHO-S细胞并通过亲和层析获得CMG2-Fc突变体蛋白,通过亲和力检测和细胞保护实验分析各突变体中和炭疽毒素能力。结果:筛选并表达了8个CMG2-Fc突变体分子,亲和力实验显示其中E117Q突变可明显提高CMG2-Fc与PA的亲和力(KD=1.35×10-11 mol/L),细胞保护实验提示E117Q突变能有效提高CMG2-Fc中和炭疽毒素能力(CMG2-Fc(E117Q)的IC50为15 ng/μL,而wt CMG2-Fc的IC50为50ng/μL)。结论:CMG2-Fc(E117Q)突变体分子可作为拮抗炭疽毒素损伤的炭疽治疗药物分子,进行进一步研究。     

Activation of phospholipase C and protein kinase C is required for expression of anthrax lethal toxin cytotoxicity in J774A.1 cells

Anthrax lethal toxin (LT) comprises two proteins: the protective antigen (PA) and the lethal factor (LF). The LT is cytotoxic to macrophage-like cell line J774A.1. Pre-treatment of these cells with neomycin, a phospholipase C inhibitor, protected them against anthrax LT cytotoxicity. Protection obtained with neomycin indicated that LT stimulates phospholipase C in these cells. It was found that levels of inositol 1,4,5-triphosphate (IP3) dramatically increased in toxin-treated cells. The rise in IP3 levels was proportional to the dose of LF that was allowed to bind to receptor-bound PA. By using protein kinase C (PKC) inhibitors, we found that the activation of PKC is required for mediating anthrax LT cytotoxicity. Activation of phospholipase C or PKC is not required for the binding of PA to the cell surface receptors or for the uptake or internalisation of the toxin. In this study, we demonstrate that the IP3 signalling cascade is initiated by anthrax lethal toxin in J774A.1 cells. The second messengers generated during the cascade aid LF in mediating lethality only after its translocation into the cytosol.     

Sensitizing anthrax lethal toxin-resistant macrophages to lethal toxin-induced killing by tumor necrosis factor-alpha

Macrophages from different inbred mouse strains exhibit striking differences in their sensitivity to anthrax lethal toxin (LeTx)-induced cytolysis. Although LeTx-induced cytolysis of macrophages plays an important role in the outcome of anthrax infection, the sensitivity of macrophages in vitro does not correlate with in vivo susceptibility to infection of Bacillus anthracis. This divergence suggests that additional factors other than LeTx are involved in the cytolysis of LeTx-resistant macrophages in vivo. We found that LeTx-resistant macrophages became sensitive to LeTx-induced cytolysis when these cells were activated by bacterial components. Tumor necrosis factor-alpha induced by bacterial components was a key factor that cooperated with LeTx in inducing LeTx-resistant macrophage death. Tumor necrosis factor-alpha/LeTx-induced death of LeTx-resistant macrophages was dependent on mTor (mammalian target of rapamycin), but independent of caspases. Our data indicate that host responses to anthrax infection contribute to cytolysis of LeTx- resistant macrophages.     

Anthrax Toxin Uptake by Primary Immune Cells as Determined with a Lethal Factor-β-Lactamase Fusion Protein

Background

To initiate infection, Bacillus anthracis needs to overcome the host innate immune system. Anthrax toxin, a major virulence factor of B. anthracis, impairs both the innate and adaptive immune systems and is important in the establishment of anthrax infections.

Methodology/Principal Findings

To measure the ability of anthrax toxin to target immune cells, studies were performed using a fusion of the anthrax toxin lethal factor (LF) N-terminal domain (LFn, aa 1–254) with β-lactamase (LFnBLA). This protein reports on the ability of the anthrax toxin protective antigen (PA) to mediate LF delivery into cells. Primary immune cells prepared from mouse spleens were used in conjunction with flow cytometry to assess cleavage and resulting FRET disruption of a fluorescent β-lactamase substrate, CCF2/AM. In spleen cell suspensions, the macrophages, dendritic cells, and B cells showed about 75% FRET disruption of CCF2/AM due to cleavage by the PA–delivered LFnBLA. LFnBLA delivery into CD4+ and CD8+ T cells was lower, with 40% FRET disruption. When the analyses were done on purified samples of individual cell types, similar results were obtained, with T cells again having lower LFnBLA delivery than macrophages, dendritic cells, and B cells. Relative expression levels of the toxin receptors CMG2 and TEM8 on these cells were determined by real-time PCR. Expression of CMG2 was about 1.5-fold higher in CD8+ cells than in CD4+ and B cells, and 2.5-fold higher than in macrophages.

Conclusions/Significance

Anthrax toxin entry and activity differs among immune cells. Macrophages, dendritic cells, and B cells displayed higher LFnBLA activity than CD4+ and CD8+ T cells in both spleen cell suspension and the purified samples of individual cell types. Expression of anthrax toxin receptor CMG2 is higher in CD4+ and CD8+ T cells, which is not correlated to the intracellular LFnBLA activity.     

Neutrophil elastase mediates pathogenic effects of anthrax lethal toxin in the murine intestinal tract

Neutrophils isolated from BALB/c or C57BL/6 mice and treated in vitro with anthrax lethal toxin release bioactive neutrophil elastase, a proinflammatory mediator of tissue destruction. Similarly, neutrophils isolated from mice treated with anthrax lethal toxin in vivo and cultured ex vivo release greater amounts of elastase than neutrophils from vehicle-treated controls. Direct measurements from murine intestinal tissue samples demonstrate an anthrax lethal toxin-dependent increase in neutrophil elastase activity in vivo as well. These findings correlate with marked lethal toxin-induced intestinal ulceration and bleeding in neutrophil elastase(+/+) animals, but not in neutrophil elastase(-/-) animals. Moreover, neutrophil elastase(-/-) mice have a significant survival advantage over neutrophil elastase(+/+) animals following exposure to anthrax lethal toxin, thereby establishing a key role for neutrophil elastase in mediating the deleterious effects of anthrax lethal toxin.     

Anthrax toxin targeting of myeloid cells through the CMG2 receptor is essential for establishment of Bacillus anthracis infections in mice

Bacillus anthracis kills through a combination of bacterial infection and toxemia. Anthrax toxin working via the CMG2 receptor mediates lethality late in infection, but its roles early in infection remain unclear. We generated myeloid-lineage specific CMG2-deficient mice to examine the roles of macrophages, neutrophils, and other myeloid cells in anthrax pathogenesis. Macrophages and neutrophils isolated from these mice were resistant to anthrax toxin. However, the myeloid-specific CMG2-deficient mice remained fully sensitive to both anthrax lethal and edema toxins, demonstrating that targeting of myeloid cells is not responsible for anthrax toxin-induced lethality. Surprisingly, the myeloid-specific CMG2-deficient mice were completely resistant to B. anthracis infection. Neutrophil depletion experiments suggest that B. anthracis relies on anthrax toxin secretion to evade the scavenging functions of neutrophils to successfully establish infection. This work demonstrates that anthrax toxin uptake through CMG2 and the resulting impairment of myeloid cells are essential to anthrax infection.     

Preparation of cyanobacterial peptide toxin as a biopesticide against cotton pests

Glycine-rich peptide toxin of cyanobacterium Scytonema MKU 106 was purified. UV spectral analysis showed an absorption maximum at 228 nm and the molecular mass was less than 12 kDa. The mortality rate of American boll worms (Helicoverpa armigera) was about 80% and 40% 84 h after treatment with 0.001% crude and purified peptide toxins respectively; 100% mortality was observed after 108 h treatment with both purified and crude peptide toxins. The LC₅₀ (lethal concentration to 50% of the population) for Heliothis larvae after 96 h was 8.3 μg/ml purified peptide toxin and 6.2 μg/ml crude peptide toxin. Observations also show that the peptide toxin at 0.01% concentration acts as a biopesticide and at high (0.1%) concentrations it will act as an anti-feeding compound for Stylepta derogata (leaf-roller) larvae of the cotton crop. Received: 22 May 1996 / Accepted: 8 July 1996     

Cytolethal distending toxin B as a cell-killing component of tumor-targeted anthrax toxin fusion proteins

Cytolethal distending toxin (Cdt) is produced by Gram-negative bacteria of several species. It is composed of three subunits, CdtA, CdtB, and CdtC, with CdtB being the catalytic subunit. We fused CdtB from Haemophilus ducreyi to the N-terminal 255 amino acids of Bacillus anthracis toxin lethal factor (LFn) to design a novel, potentially potent antitumor drug. As a result of this fusion, CdtB was transported into the cytosol of targeted cells via the efficient delivery mechanism of anthrax toxin. The fusion protein efficiently killed various human tumor cell lines by first inducing a complete cell cycle arrest in the G2/M phase, followed by induction of apoptosis. The fusion protein showed very low toxicity in mouse experiments and impressive antitumor effects in a Lewis Lung carcinoma model, with a 90% cure rate. This study demonstrates that efficient drug delivery by a modified anthrax toxin system combined with the enzymatic activity of CdtB has great potential as anticancer treatment and should be considered for the development of novel anticancer drugs.