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1.  Metalloproteinase Inhibitors, Nonantimicrobial Chemically Modified Tetracyclines, and Ilomastat Block Bacillus anthracis Lethal Factor Activity in Viable Cells  
Infection and Immunity  2005;73(11):7548-7557.
Lethal toxin, produced by the bacterium Bacillus anthracis, is a major contributor to morbidity and mortality in animals and humans who have contracted anthrax. One component of this toxin, lethal factor (LF), proteolytically inactivates members of the mitogen-activated protein kinase kinase (MAPKK or MEK) family. In this study we show that CMT-300, CMT-308, and Ilomastat, agents initially characterized as matrix metalloproteinase inhibitors which are in early stages of development as pharmaceuticals, effectively inhibit the zinc metalloproteinase activity of LF. All three inhibitors, CMT-300, CMT-308, and Ilomastat, inhibit LF-mediated cleavage of a synthetic peptide substrate based on the N-terminal domain of MEKs. Inhibition of LF-mediated MEK proteolysis by all three agents was also achieved using lysates of the human monocytoid line MonoMac 6 as sources of MAPKKs and visualization of the extent of cleavage after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by detection by Western blotting. Finally, we have demonstrated inhibition of intracellular MEKs in viable human monocytes and MonoMac 6 cells by these agents after incubation of the cells with a reconstituted preparation of recombinant lethal toxin. All three agents are effective inhibitors when incubated with LF prior to exposure to cells, while the CMTs, but not Ilomastat, are also effective when added after LF has already entered the viable cell targets. These results offer promise for strategies to combat effects of the lethal toxin of B. anthracis.
doi:10.1128/IAI.73.11.7548-7557.2005
PMCID: PMC1273843  PMID: 16239558
2.  An anthrax lethal factor mutant that is defective at causing pyroptosis retains pro-apoptotic activity 
The FEBS journal  2009;277(1):119-127.
Summary
Anthrax lethal toxin triggers death in some cell types, such as macrophages, and causes a variety of cellular dysfunctions in others. Collectively, these effects dampen the innate and adaptive immune systems to allow Bacillus anthracis to survive and proliferate in the mammalian host. The diverse effects caused by the toxin have in part been attributed to its interference with signaling pathways in target cells. Lethal factor (LF) is the proteolytic component of the toxin, which cleaves six members of the mitogen activated protein kinase kinase (MAPKK) family after being delivered to the cytosol by the cell-binding component of the toxin, protective antigen. The effect of cleaving these MAPKKs is to interfere with ERK, p38 and JNK signaling. Here we characterize an LF mutant, LF-K518E/E682G, that is defective at causing pyroptosis in RAW 264.7 cells and at activating the Nlrp1b inflammasome in a heterologous expression system. LF-K518E/E682G does not exhibit an overall impairment of function, however, because it is able to downregulate the ERK pathway, but not the p38 or JNK pathways. Furthermore, LF-K518E/E682G efficiently killed melanoma cells, which were shown previously to undergo apoptosis in response to lethal toxin or to pharmacological inhibition of the ERK pathway. Our results suggest that LF-K518E/E682G is defective at cleaving a substrate involved in the activation of the Nlrp1b inflammasome.
doi:10.1111/j.1742-4658.2009.07458.x
PMCID: PMC2805769  PMID: 19922472
lethal toxin; anthrax; MAPKK; Nlrp1b
3.  Bacillus anthracis Lethal Toxin Reduces Human Alveolar Epithelial Barrier Function 
Infection and Immunity  2012;80(12):4374-4387.
The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal-toxin-induced cleavage of multiple MEKs. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness.
doi:10.1128/IAI.01011-12
PMCID: PMC3497415  PMID: 23027535
4.  Erythropoiesis Suppression Is Associated with Anthrax Lethal Toxin-Mediated Pathogenic Progression 
PLoS ONE  2013;8(8):e71718.
Anthrax is a disease caused by the bacterium Bacillus anthracis, which results in high mortality in animals and humans. Although some of the mechanisms are already known such as asphyxia, extensive knowledge of molecular pathogenesis of this disease is deficient and remains to be further investigated. Lethal toxin (LT) is a major virulence factor of B. anthracis and a specific inhibitor/protease of mitogen-activated protein kinase kinases (MAPKKs). Anthrax LT causes lethality and induces certain anthrax-like symptoms, such as anemia and hypoxia, in experimental mice. Mitogen-activated protein kinases (MAPKs) are the downstream pathways of MAPKKs, and are important for erythropoiesis. This prompted us to hypothesize that anemia and hypoxia may in part be exacerbated by erythropoietic dysfunction. As revealed by colony-forming cell assays in this study, LT challenges significantly reduced mouse erythroid progenitor cells. In addition, in a proteolytic activity-dependent manner, LT suppressed cell survival and differentiation of cord blood CD34+-derived erythroblasts in vitro. Suppression of cell numbers and the percentage of erythroblasts in the bone marrow were detected in LT-challenged C57BL/6J mice. In contrast, erythropoiesis was provoked through treatments of erythropoietin, significantly ameliorating the anemia and reducing the mortality of LT-treated mice. These data suggested that suppressed erythropoiesis is part of the pathophysiology of LT-mediated intoxication. Because specific treatments to overcome LT-mediated pathogenesis are still lacking, these efforts may help the development of effective treatments against anthrax.
doi:10.1371/journal.pone.0071718
PMCID: PMC3747219  PMID: 23977125
5.  Direct Inhibition of T-Lymphocyte Activation by Anthrax Toxins In Vivo  
Infection and Immunity  2005;73(12):8275-8281.
The causative agent of anthrax, Bacillus anthracis, produces two toxins that contribute in part to its virulence. Lethal toxin is a metalloprotease that cleaves upstream mitogen-activated protein kinase kinases. Edema toxin is a calmodulin-dependent adenylate cyclase. Previous studies demonstrated that the anthrax toxins are important immunomodulators that promote immune evasion of the bacterium by suppressing activation of macrophages and dendritic cells. Here we showed that injection of sublethal doses of either lethal or edema toxin into mice directly inhibited the subsequent activation of T lymphocytes by T-cell receptor-mediated stimulation. Lymphocytes were isolated from toxin-injected mice after 1 or 4 days and stimulated with antibodies against CD3 and CD28. Treatment with either toxin inhibited the proliferation of T cells. Injection of lethal toxin also potently inhibited cytokine secretion by stimulated T cells. The effects of edema toxin on cytokine secretion were more complex and were dependent on the length of time between the injection of edema toxin and the isolation of lymphocytes. Treatment with lethal toxin blocked multiple kinase signaling pathways important for T-cell receptor-mediated activation of T cells. Phosphorylation of the extracellular signal-regulated kinase and the stress-activated kinase p38 was significantly decreased. In addition, phosphorylation of the serine/threonine kinase AKT and of glycogen synthase kinase 3 was inhibited in T cells from lethal toxin-injected mice. Thus, anthrax toxins directly act on T lymphocytes in a mouse model. These findings are important for future anthrax vaccine development and treatment.
doi:10.1128/IAI.73.12.8275-8281.2005
PMCID: PMC1307061  PMID: 16299324
6.  Auranofin Protects against Anthrax Lethal Toxin-Induced Activation of the Nlrp1b Inflammasome ▿  
Anthrax lethal toxin (LT) is the major virulence factor for Bacillus anthracis. The lethal factor (LF) component of this bipartite toxin is a protease which, when transported into the cellular cytoplasm, cleaves mitogen-activated protein kinase kinase (MEK) family proteins and induces rapid toxicity in mouse macrophages through activation of the Nlrp1b inflammasome. A high-throughput screen was performed to identify synergistic LT-inhibitory drug combinations from within a library of approved drugs and molecular probes. From this screen we discovered that auranofin, an organogold compound with anti-inflammatory activity, strongly inhibited LT-mediated toxicity in mouse macrophages. Auranofin did not inhibit toxin transport into cells or MEK cleavage but inhibited both LT-mediated caspase-1 activation and caspase-1 catalytic activity. Thus, auranofin inhibited LT-mediated toxicity by preventing activation of the Nlrp1b inflammasome and the downstream actions that occur in response to the toxin. Idebenone, an analog of coenzyme Q, synergized with auranofin to increase its protective effect. We found that idebenone functions as an inhibitor of voltage-gated potassium channels and thus likely mediates synergy through inhibition of the potassium fluxes which have been shown to be required for Nlrp1b inflammasome activation.
doi:10.1128/AAC.00772-10
PMCID: PMC3067098  PMID: 21149629
7.  Anthrax Lethal Toxin Promotes Dephosphorylation of TTP and Formation of Processing bodies 
Cellular microbiology  2009;12(4):557-568.
Anthrax lethal toxin (LeTx) is composed of protective antigen (PA) and lethal factor (LF) – PA is the receptor-binding moiety and LF is a protease that cleaves mitogen-activated protein kinase kinases (MAPKKs). LeTx subverts the immune response to B. anthracis in several ways, such as downregulating interleukin-8 (IL-8) by increasing the rate of IL-8 mRNA degradation. Many transcripts are regulated through cis-acting elements that bind proteins that either impede or promote degradation. Some of these RNA binding proteins are regulated by MAPKs and previous work has demonstrated that interfering with MAPK signaling decreases the half-life of IL-8 mRNA. Here, we have localized a segment within the IL-8 3′ untranslated region responsible for LeTx-induced transcript destabilization and show that this is caused by inhibition of the p38, ERK, and JNK pathways. TTP, an RNA binding protein involved in IL-8 mRNA decay, became hypophosphorylated in LeTx-treated cells and knock-down of TTP prevented LeTx from destabilizing the IL-8 transcript. Cells that were treated with LeTx exhibited increased localization of TTP to Processing-bodies, which are structures that accumulate transcripts targeted for degradation. We furthermore observed that LeTx promoted the formation of Processing-bodies, revealing a link between the toxin and a major mRNA decay pathway.
doi:10.1111/j.1462-5822.2009.01418.x
PMCID: PMC2859114  PMID: 19995385
anthrax; lethal toxin; TTP; IL-8; P-body
8.  Toxin Inhibition of Antimicrobial Factors Induced by Bacillus anthracis Peptidoglycan in Human Blood 
Infection and Immunity  2013;81(10):3693-3702.
Here, we describe the capacity of Bacillus anthracis peptidoglycan (BaPGN) to trigger an antimicrobial response in human white blood cells (WBCs). Analysis of freshly isolated human blood cells found that monocytes and neutrophils, but not B and T cells, were highly responsive to BaPGN and produced a variety of cytokines and chemokines. This BaPGN-induced response was suppressed by anthrax lethal toxin (LT) and edema toxin (ET), with the most pronounced effect on human monocytes, and this corresponded with the higher levels of anthrax toxin receptor 1 (ANTXR1) in these cells than in neutrophils. The supernatant from BaPGN-treated cells altered the growth of B. anthracis Sterne, and this effect was blocked by LT, but not by ET. An FtsX mutant of B. anthracis known to be resistant to the antimicrobial effects of interferon-inducible Glu-Leu-Arg (ELR)-negative CXC chemokines was not affected by the BaPGN-induced antimicrobial effects. Collectively, these findings describe a system in which BaPGN triggers expression of antimicrobial factors in human WBCs and reveal a distinctive role, not shared with ET, in LT's capacity to suppress this response.
doi:10.1128/IAI.00709-13
PMCID: PMC3811742  PMID: 23876807
9.  MATRIX METALLOPROTEINASE-ACTIVATED ANTHRAX LETHAL TOXIN DEMONSTRATES HIGH POTENCY IN TARGETING TUMOR VASCULATURE 
The Journal of biological chemistry  2007;283(1):529-540.
Anthrax lethal toxin (LT), a virulence factor secreted by Bacillus anthracis, is selectively toxic to human melanomas with the BRAF V600E activating mutation due to its proteolytic activities toward the mitogen-activated protein kinase kinases (MEKs). To develop LT variants with lower in vivo toxicity and high tumor specificity, and therefore greater potential for clinical use, we generated a mutated LT that requires activation by matrix metalloproteinases (MMPs). This engineered toxin was less toxic than wild-type LT to mice due to the limited expression of MMPs by normal cells. Moreover, the systemically administered toxin produced greater anti-tumor effects than wild-type LT towards human xenografted tumors. This was shown to result from its greater bioavailability, a consequence of the limited uptake and clearance of the modified toxin by normal cells. Furthermore, the MMP-activated LT had very potent anti-tumor activity not only to human melanomas containing the BRAF mutation, but also to other tumor types including lung and colon carcinomas regardless of their BRAF status. Tumor histology and in vivo angiogenesis assays showed that this anti-tumor activity is due largely to the indirect targeting of tumor vasculature and angiogenic processes. Thus, even tumors genetically deficient in anthrax toxin receptors were still susceptible to the toxin therapy in vivo. Moreover, the modified toxin also displayed lower immunogenicity compared to the wild-type toxin. All these properties suggest that this MMP-activated anti-tumor toxin has a potential for use incancer therapy.
doi:10.1074/jbc.M707419200
PMCID: PMC2394502  PMID: 17974567
10.  Effective antiprotease-antibiotic treatment of experimental anthrax 
Background
Inhalation anthrax is characterized by a systemic spread of the challenge agent, Bacillus anthracis. It causes severe damage, including multiple hemorrhagic lesions, to host tissues and organs. It is widely believed that anthrax lethal toxin secreted by proliferating bacteria is a major cause of death, however, the pathology of intoxication in experimental animals is drastically different from that found during the infectious process. In order to close a gap between our understanding of anthrax molecular pathology and the most prominent clinical features of the infectious process we undertook bioinformatic and experimental analyses of potential proteolytic virulence factors of B. anthracis distinct from lethal toxin.
Methods
Secreted proteins (other than lethal and edema toxins) produced by B. anthracis were tested for tissue-damaging activity and toxicity in mice. Chemical protease inhibitors and rabbit immune sera raised against B. anthracis proteases were used to treat mice challenged with B. anthracis (Sterne) spores.
Results
B. anthracis strain delta Ames (pXO1-, pXO2-) producing no lethal and edema toxins secrets a number of metalloprotease virulence factors upon cultivation under aerobic conditions, including those with hemorrhagic, caseinolytic and collagenolytic activities, belonging to M4 and M9 thermolysin and bacterial collagenase families, respectively.
These factors are directly toxic to DBA/2 mice upon intratracheal administration at 0.5 mg/kg and higher doses. Chemical protease inhibitors (phosphoramidon and 1, 10-phenanthroline), as well as immune sera against M4 and M9 proteases of B. anthracis, were used to treat mice challenged with B. anthracis (Sterne) spores. These substances demonstrate a substantial protective efficacy in combination with ciprofloxacin therapy initiated as late as 48 h post spore challenge, compared to the antibiotic alone.
Conclusion
Secreted proteolytic enzymes are important pathogenic factors of B. anthrasis, which can be considered as effective therapeutic targets in the development of anthrax treatment and prophylactic approaches complementing anti-lethal toxin therapy.
doi:10.1186/1471-2334-5-25
PMCID: PMC1090577  PMID: 15819985
11.  Inhibition of Anthrax Lethal Toxin-Induced Cytolysis of RAW264.7 Cells by Celastrol 
PLoS ONE  2008;3(1):e1421.
Background
Bacillus anthracis is the bacterium responsible for causing anthrax. The ability of B. anthracis to cause disease is dependent on a secreted virulence factor, lethal toxin, that promotes survival of the bacteria in the host by impairing the immune response. A well-studied effect of lethal toxin is the killing of macrophages, although the molecular mechanisms involved have not been fully characterized.
Methodology/Principal Findings
Here, we demonstrate that celastrol, a quinone methide triterpene derived from a plant extract used in herbal medicine, inhibits lethal toxin-induced death of RAW264.7 murine macrophages. Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin. Surprisingly, celastrol conferred almost complete protection when it was added up to 1.5 h after intoxication, indicating that it could rescue cells in the late stages of intoxication. Since the activity of the proteasome has been implicated in intoxication using other pharmacological agents, we tested whether celastrol blocked proteasome activity. We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro. Furthermore, celastrol blocked stimulation of IL-18 processing, indicating that celastrol acted upstream of inflammasome activation.
Conclusions/Significance
This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway.
doi:10.1371/journal.pone.0001421
PMCID: PMC2170518  PMID: 18183301
12.  Cisplatin Inhibition of Anthrax Lethal Toxin 
Bacillus anthracis lethal toxin (LT) produces symptoms of anthrax in mice and induces rapid lysis of macrophages derived from certain inbred strains. LT is comprised of a receptor binding component, protective antigen (PA), which delivers the enzymatic component, lethal factor (LF), into cells. We found that mouse macrophages were protected from toxin by the antitumor drug cis-diammineplatinum (II) dichloride (cisplatin). Cisplatin was shown to inhibit LT-mediated cleavage of cellular mitogen-activated protein kinases (MEKs) without inhibiting LF's in vitro proteolytic activity. Cisplatin-treated PA lost 100% of its ability to function in toxicity assays when paired with untreated LF, despite maintaining the ability to bind to cells. Cisplatin-treated PA was unable to form heptameric oligomers required for LF binding and translocation. The drug was shown to modify PA in a reversible noncovalent manner. Not surprisingly, cisplatin also blocked the actions of anthrax edema toxin and of LF-Pseudomonas aeruginosa exotoxin A fusion peptide (FP59), both of which require PA for translocation. Treatment of BALB/cJ mice or Fischer F344 rats with cisplatin at biologically relevant concentrations completely protected the animals from a coadministered lethal dose of LT. However, treatment with cisplatin 2 hours before or after animals received a lethal bolus of toxin did not protect them.
doi:10.1128/AAC.01412-05
PMCID: PMC1538677  PMID: 16870755
13.  Consequences and Utility of the Zinc-Dependent Metalloprotease Activity of Anthrax Lethal Toxin  
Toxins  2010;2(5):1038-1053.
Anthrax is caused by the gram-positive bacterium Bacillus anthracis. The pathogenesis of this disease is dependent on the presence of two binary toxins, edema toxin (EdTx) and lethal toxin (LeTx). LeTx, the major virulence factor contributing to anthrax, contains the effector moiety lethal factor (LF), a zinc-dependent metalloprotease specific for targeting mitogen-activated protein kinase kinases. This review will focus on the protease-specific activity and function of LF, and will include a discussion on the implications and consequences of this activity, both in terms of anthrax disease, and how this activity can be exploited to gain insight into other pathologic conditions.
doi:10.3390/toxins2051038
PMCID: PMC3153234  PMID: 22069624
anthrax; lethal factor; mitogen-activated protein kinase kinase; pathogenesis; metalloprotease; tumorigenesis; retinal neovascularization
14.  Lung Epithelial Injury by B. Anthracis Lethal Toxin Is Caused by MKK-Dependent Loss of Cytoskeletal Integrity 
PLoS ONE  2009;4(3):e4755.
Bacillus anthracis lethal toxin (LT) is a key virulence factor of anthrax and contributes significantly to the in vivo pathology. The enzymatically active component is a Zn2+-dependent metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MKKs). Using ex vivo differentiated human lung epithelium we report that LT destroys lung epithelial barrier function and wound healing responses by immobilizing the actin and microtubule network. Long-term exposure to the toxin generated a unique cellular phenotype characterized by increased actin filament assembly, microtubule stabilization, and changes in junction complexes and focal adhesions. LT-exposed cells displayed randomly oriented, highly dynamic protrusions, polarization defects and impaired cell migration. Reconstitution of MAPK pathways revealed that this LT-induced phenotype was primarily dependent on the coordinated loss of MKK1 and MKK2 signaling. Thus, MKKs control fundamental aspects of cytoskeletal dynamics and cell motility. Even though LT disabled repair mechanisms, agents such as keratinocyte growth factor or dexamethasone improved epithelial barrier integrity by reducing cell death. These results suggest that co-administration of anti-cytotoxic drugs may be of benefit when treating inhalational anthrax.
doi:10.1371/journal.pone.0004755
PMCID: PMC2649448  PMID: 19270742
15.  Antibacterial Role for Natural Killer Cells in Host Defense to Bacillus anthracis 
Infection and Immunity  2012;80(1):234-242.
Natural killer (NK) cells have innate antibacterial activity that could be targeted for clinical interventions for infectious disease caused by naturally occurring or weaponized bacterial pathogens. To determine a potential role for NK cells in immunity to Bacillus anthracis, we utilized primary human and murine NK cells, in vitro assays, and in vivo NK cell depletion in a murine model of inhalational anthrax. Our results demonstrate potent antibacterial activity by human NK cells against B. anthracis bacilli within infected autologous monocytes. Surprisingly, NK cells also mediate moderate antibacterial effects on extracellular vegetative bacilli but do not have activity against extracellular or intracellular spores. The immunosuppressive anthrax lethal toxin impairs NK gamma interferon (IFN-γ) expression, but neither lethal nor edema toxin significantly alters the viability or cytotoxic effector function of NK cells. Compared to human NK cells, murine NK cells have a similar, though less potent, activity against intracellular and extracellular B. anthracis. The in vivo depletion of murine NK cells does not alter animal survival following intranasal infection with B. anthracis spores in our studies but significantly increases the bacterial load in the blood of infected animals. Our studies demonstrate that NK cells participate in the innate immune response against B. anthracis and suggest that immune modulation to augment NK cell function in early stages of anthrax should be further explored in animal models as a clinical intervention strategy.
doi:10.1128/IAI.05439-11
PMCID: PMC3255656  PMID: 22006566
16.  Anthrax Toxin Uptake by Primary Immune Cells as Determined with a Lethal Factor-β-Lactamase Fusion Protein 
PLoS ONE  2009;4(11):e7946.
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.
doi:10.1371/journal.pone.0007946
PMCID: PMC2775957  PMID: 19956758
17.  Proteasome Activity Is Required for Anthrax Lethal Toxin To Kill Macrophages 
Infection and Immunity  1999;67(6):3055-3060.
Anthrax lethal toxin (LeTx), consisting of protective antigen (PA) and lethal factor (LF), rapidly kills primary mouse macrophages and macrophage-like cell lines such as RAW 264.7. LF is translocated by PA into the cytosol of target cells, where it acts as a metalloprotease to cleave mitogen-activated protein kinase kinase 1 (MEK1) and possibly other proteins. In this study, we show that proteasome inhibitors such as acetyl-Leu-Leu-norleucinal, MG132, and lactacystin efficiently block LeTx cytotoxicity, whereas other protease inhibitors do not. The inhibitor concentrations that block LF cytotoxicity are similar to those that inhibit the proteasome-dependent IκB-α degradation induced by lipopolysaccharide. The inhibitors did not interfere with the proteolytic cleavage of MEK1 in LeTx-treated cells, indicating that they do not directly block the proteolytic activity of LF. However, the proteasome inhibitors did prevent ATP depletion, an early effect of LeTx. No overall activation of the proteasome by LeTx was detected, as shown by the cleavage of fluorogenic substrates of the proteasome. All of these results suggest that the proteasome mediates a toxic process initiated by LF in the cell cytosol. This process probably involves degradation of unidentified molecules that are essential for macrophage homeostasis. Moreover, this proteasome-dependent process is an early step in LeTx intoxication, but it is downstream of the cleavage by LF of MEK1 or other putative substrates.
PMCID: PMC96621  PMID: 10338520
18.  Anthrax Lethal Toxin Induces Human Endothelial Cell Apoptosis  
Infection and Immunity  2004;72(1):430-439.
Because of its ease of dispersal and high lethality, Bacillus anthracis is one of the most feared biowarfare agents. A better understanding of anthrax pathogenesis is urgently needed to develop new therapies for systemic disease that is relatively unresponsive to antibiotics. Although experimental evidence has implicated a role for macrophages in anthrax pathogenesis, clinical and pathological observations suggest that a direct insult to the host vasculature may also be important. Two bacterial toxins, lethal toxin and edema toxin, are believed to mediate the clinical sequelae of anthrax. Here, I examined whether these toxins are directly toxic to endothelial cells, the cell type that lines the interior of blood vessels. I show for the first time that lethal toxin but not edema toxin reduces the viability of cultured human endothelial cells and induces caspase-dependent endothelial apoptosis. In addition, this toxicity affects both microvascular and large vessel endothelial cells as well as endothelial cells that have differentiated into tubules within a type I collagen extracellular matrix. Finally, lethal toxin induces cleavage of mitogen-activated protein kinase kinases in endothelial cells and inhibits phosphorylation of ERK, p38, and JNK p46. Based on the contributions of these pathways to endothelial survival, I propose that lethal toxin-mediated cytotoxicity/apoptosis results primarily through inhibition of the ERK pathway. I also hypothesize that the observed endothelial toxicity contributes to vascular pathology and hemorrhage during systemic anthrax.
doi:10.1128/IAI.72.1.430-439.2004
PMCID: PMC343952  PMID: 14688124
19.  Anthrax toxins suppress T lymphocyte activation by disrupting antigen receptor signaling 
Anthrax is an infection caused by pathogenic strains of Bacillus anthracis, which secretes a three-component toxic complex consisting of protective antigen (PA), edema factor (EF), and lethal factor (LF). PA forms binary complexes with either LF or EF and mediates their entry into host cells. Although the initial phases of bacterial growth occur in the lymph node, the host fails to mount an effective immune response. Here, we show that LT and ET are potent suppressors of human T cell activation and proliferation triggered through the antigen receptor. Both LT and ET inhibit the mitogen-activated protein and stress kinase pathways, and both toxins inhibit activation of NFAT and AP-1, two transcription factors essential for cytokine gene expression. These data identify a novel strategy of immune evasion by B. anthracis, based on both effector subunits of the toxic complex, and targeted to a key cellular component of adaptive immunity.
doi:10.1084/jem.20041557
PMCID: PMC2213032  PMID: 15699068
20.  Constitutive MEK1 Activation Rescues Anthrax Lethal Toxin-Induced Vascular Effects In Vivo▿  
Infection and Immunity  2010;78(12):5043-5053.
Anthrax lethal toxin (LT) increases vascular leakage in a number of mammalian models and in human anthrax disease. Using a zebrafish model, we determined that vascular delivery of LT increased permeability, which was phenocopied by treatment with a selective chemical inhibitor of MEK1 and MEK2 (also known as mitogen-activated protein kinase [MAPK] kinase, MEK, or MKK). Here we investigate further the role of MEK1/phospho-ERK (pERK) in the action of LT. Overexpression of wild-type zebrafish MEK1 at high levels did not induce detrimental effects. However, a constitutively activated version, MEK1S219D,S223D (MEK1DD), induced early defects in embryonic development that correlated with increased ERK/MAPK phosphorylation. To bypass these early developmental defects and to provide a genetic tool for examining the action of lethal factor (LF), we generated inducible transgenic zebrafish lines expressing either wild-type or activated MEK1 under the control of a heat shock promoter. Remarkably, induction of MEK1DD transgene expression prior to LT delivery prevented vascular damage, while the wild-type MEK1 line did not. In the presence of both LT and MEK1DD transgene expression, cardiovascular development and function proceeded normally in most embryos. The resistance to microsphere leakage in transgenic animals demonstrated a protective role against LT-induced vascular permeability. A consistent increase in ERK phosphorylation among LT-resistant MEK1DD transgenic animals provided additional confirmation of transgene activation. These findings provide a novel genetic approach to examine mechanism of action of LT in vivo through one of its known targets. This approach may be generally applied to investigate additional pathogen-host interactions and to provide mechanistic insights into host signaling pathways affected by pathogen entry.
doi:10.1128/IAI.00604-10
PMCID: PMC2981334  PMID: 20855511
21.  Recombinant Anthrax Toxin Receptor-Fc Fusion Proteins Produced in Plants Protect Rabbits against Inhalational Anthrax ▿ †  
Inhalational anthrax, a zoonotic disease caused by the inhalation of Bacillus anthracis spores, has a ∼50% fatality rate even when treated with antibiotics. Pathogenesis is dependent on the activity of two toxic noncovalent complexes: edema toxin (EdTx) and lethal toxin (LeTx). Protective antigen (PA), an essential component of both complexes, binds with high affinity to the major receptor mediating the lethality of anthrax toxin in vivo, capillary morphogenesis protein 2 (CMG2). Certain antibodies against PA have been shown to protect against anthrax in vivo. As an alternative to anti-PA antibodies, we produced a fusion of the extracellular domain of human CMG2 and human IgG Fc, using both transient and stable tobacco plant expression systems. Optimized expression led to the CMG2-Fc fusion protein being produced at high levels: 730 mg/kg fresh leaf weight in Nicotiana benthamiana and 65 mg/kg in N. tabacum. CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with B. anthracis spores at a dose of 2 mg/kg body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later. The glycosylation of the Fc (or lack thereof) had no significant effect on the protective potency of CMG2-Fc in rabbits or on its serum half-life, which was about 5 days. Significantly, CMG2-Fc effectively neutralized, in vitro, LeTx-containing mutant forms of PA that were not neutralized by anti-PA monoclonal antibodies.
doi:10.1128/AAC.00592-10
PMCID: PMC3019684  PMID: 20956592
22.  Both Lethal and Edema Toxins of Bacillus anthracis Disrupt the Human Dendritic Cell Chemokine Network 
PLoS ONE  2012;7(8):e43266.
Bacillus anthracis, the agent of anthrax, produces two main virulence factors: a capsule and two toxins. Both lethal toxin (LT) and edema toxin (ET) paralyze the immune defense system. Here, we analyze the effects of LT and ET on the capacity of human monocyte-derived dendritic cells (MoDC) to produce proinflammatory chemokines. We show that both toxins disrupt proinflammatory chemokine production. LT has more pronounced effects than ET on CXCL8 production, which is correlated with impaired recruitment of neutrophils in vitro. Finally, we show that both toxins also differentially disrupt IL-12p70, IL-10, and TNF-α production. Taken together, these results demonstrate that both B. anthracis toxins alter MoDC functions and the activation of the innate immune system.
doi:10.1371/journal.pone.0043266
PMCID: PMC3427382  PMID: 22937027
23.  Anthrax Edema Toxin Induces Maturation of Dendritic Cells and Enhances Chemotaxis towards Macrophage Inflammatory Protein 3β▿  
Infection and Immunity  2009;77(5):2036-2042.
Bacillus anthracis secretes two bipartite toxins, edema toxin (ET) and lethal toxin (LT), which impair immune responses and contribute directly to the pathology associated with the disease anthrax. Edema factor, the catalytic subunit of ET, is an adenylate cyclase that impairs host defenses by raising cellular cyclic AMP (cAMP) levels. Synthetic cAMP analogues and compounds that raise intracellular cAMP levels lead to phenotypic and functional changes in dendritic cells (DCs). Here, we demonstrate that ET induces a maturation state in human monocyte-derived DCs (MDDCs) similar to that induced by lipopolysaccharide (LPS). ET treatment results in downregulation of DC-SIGN, a marker of immature DCs, and upregulation of DC maturation markers CD83 and CD86. Maturation of DCs by ET is accompanied by an increased ability to migrate toward the lymph node-homing chemokine macrophage inflammatory protein 3β, like LPS-matured DCs. Interestingly, cotreating with LT differentially affects the ET-induced maturation of MDDCs while not inhibiting ET-induced migration. These findings reveal a mechanism by which ET impairs normal innate immune function and may explain the reported adjuvant effect of ET.
doi:10.1128/IAI.01329-08
PMCID: PMC2681763  PMID: 19273556
24.  MEK2 Is Sufficient but Not Necessary for Proliferation and Anchorage-Independent Growth of SK-MEL-28 Melanoma Cells 
PLoS ONE  2011;6(2):e17165.
Mitogen-activated protein kinase kinases (MKK or MEK) 1 and 2 are usually treated as redundant kinases. However, in assessing their relative contribution towards ERK-mediated biologic response investigators have relied on tests of necessity, not sufficiency. In response we developed a novel experimental model using lethal toxin (LeTx), an anthrax toxin-derived pan-MKK protease, and genetically engineered protease resistant MKK mutants (MKKcr) to test the sufficiency of MEK signaling in melanoma SK-MEL-28 cells. Surprisingly, ERK activity persisted in LeTx-treated cells expressing MEK2cr but not MEK1cr. Microarray analysis revealed non-overlapping downstream transcriptional targets of MEK1 and MEK2, and indicated a substantial rescue effect of MEK2cr on proliferation pathways. Furthermore, LeTx efficiently inhibited the cell proliferation and anchorage-independent growth of SK-MEL-28 cells expressing MKK1cr but not MEK2cr. These results indicate in SK-MEL-28 cells MEK1 and MEK2 signaling pathways are not redundant and interchangeable for cell proliferation. We conclude that in the absence of other MKK, MEK2 is sufficient for SK-MEL-28 cell proliferation. MEK1 conditionally compensates for loss of MEK2 only in the presence of other MKK.
doi:10.1371/journal.pone.0017165
PMCID: PMC3041822  PMID: 21365009
25.  Exogenous Gamma and Alpha/Beta Interferon Rescues Human Macrophages from Cell Death Induced by Bacillus anthracis  
Infection and Immunity  2004;72(3):1291-1297.
During the recent bioterrorism-related outbreaks, inhalational anthrax had a 45% mortality in spite of appropriate antimicrobial therapy, underscoring the need for better adjuvant therapies. The variable latency between exposure and development of disease suggests an important role for the host's innate immune response. Alveolar macrophages are likely the first immune cells exposed to inhalational anthrax, and the interferon (IFN) response of these cells comprises an important arm of the host innate immune response to intracellular infection with Bacillus anthracis. Furthermore, IFNs have been used as immunoadjuvants for treatment of another intracellular pathogen, Mycobacterium tuberculosis. We established a model of B. anthracis infection with the Sterne strain (34F2) which contains lethal toxin (LeTx). 34F2 was lethal to murine and human macrophages. Treatment with IFNs significantly improved cell viability and reduced the number of germinated intracellular spores. Infection with 34F2 failed to induce the latent transcription factors signal transducer and activators of transcription 1 (STAT1) and ISGF-3, which are central to the IFN response. Furthermore, 34F2 reduced STAT1 activation in response to exogenous alpha/beta IFN, suggesting direct inhibition of IFN signaling. Even though 34F2 has LeTx, there was no mitogen-activated protein kinase kinase 3 cleavage and p38 was normally induced, suggesting that these early effects of B. anthracis infection in macrophages are independent of LeTx. These data suggest an important role for both IFNs in the control of B. anthracis and the potential benefit of using exogenous IFN as an immunoadjuvant therapy.
doi:10.1128/IAI.72.3.1291-1297.2004
PMCID: PMC356021  PMID: 14977930

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