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1.  Bacillus anthracis Lethal Toxin Disrupts TCR Signaling in CD1d-Restricted NKT Cells Leading to Functional Anergy 
PLoS Pathogens  2009;5(9):e1000588.
Exogenous CD1d-binding glycolipid (α-Galactosylceramide, α-GC) stimulates TCR signaling and activation of type-1 natural killer–like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis–derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.
Author Summary
The bacterium Bacillus anthracis is the causative agent of anthrax infection. Anthrax is a life-threatening disease caused by inhalation or ingestion of spores, or transmission through wounds and abrasions. The bacterium secretes toxins, proteins that enter numerous cell types in an infected individual altering their function and contributing to the disease process. Consequently, we and other researchers are dedicated to identifying cells affected by anthrax toxins and to understanding the implications for the function of those cells. We have observed that anthrax toxins adversely affect a type of cell in the immune system known as a natural killer–like T (NKT) cell. NKT cells are found in the bone marrow, blood, spleen, lymph nodes and liver and are required for optimal immune responses. We report that anthrax toxins effectively shut down NKT cells, preventing them from functioning normally. We propose that this has serious consequences because anyone infected with Bacillus anthracis will be less able to mount an immune response against it.
PMCID: PMC2742733  PMID: 19779559
2.  Anthrax Lethal Toxin-Mediated Killing of Human and Murine Dendritic Cells Impairs the Adaptive Immune Response 
PLoS Pathogens  2005;1(2):e19.
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.
Dendritic cells (DCs) are specialized white blood cells that identify and present antigens to immune cells, T cells, in order to mount an immune response targeted against specific pathogens. DCs are critical to a host's defense against infection. Previous work has shown that the anthrax bacterium disables many immune cells, including DCs, through the action of a released toxin, lethal toxin. Here the authors show that lethal toxin efficiently kills both human and murine DCs. The means by which DCs were killed by the anthrax toxin were notably distinct and dependent on their genetic background. Human DCs, as well as those derived from the murine strain C57BL/6, died over the course of 72 h via activation of apoptosis, or programmed cell death. DCs from BALB/c mice, however, died rapidly of a necrotic cell death following toxin exposure. As human and C57BL/6 DCs share an identical response to anthrax toxin, C57BL/6 mice appear to provide an excellent model for human anthrax. The study's findings suggest that specific targeting of DCs by the anthrax toxin impairs the immune response of the infected host, and the authors believe that this strategy promotes spread of the bacterium and disease progression.
PMCID: PMC1266308  PMID: 16254597
3.  Edema Toxin Impairs Anthracidal Phospholipase A2 Expression by Alveolar Macrophages 
PLoS Pathogens  2007;3(12):e187.
Bacillus anthracis, the etiological agent of anthrax, is a spore-forming Gram-positive bacterium. Infection with this pathogen results in multisystem dysfunction and death. The pathogenicity of B. anthracis is due to the production of virulence factors, including edema toxin (ET). Recently, we established the protective role of type-IIA secreted phospholipase A2 (sPLA2-IIA) against B. anthracis. A component of innate immunity produced by alveolar macrophages (AMs), sPLA2-IIA is found in human and animal bronchoalveolar lavages at sufficient levels to kill B. anthracis. However, pulmonary anthrax is almost always fatal, suggesting the potential impairment of sPLA2-IIA synthesis and/or action by B. anthracis factors. We investigated the effect of purified ET and ET-deficient B. anthracis strains on sPLA2-IIA expression in primary guinea pig AMs. We report that ET inhibits sPLA2-IIA expression in AMs at the transcriptional level via a cAMP/protein kinase A–dependent process. Moreover, we show that live B. anthracis strains expressing functional ET inhibit sPLA2-IIA expression, whereas ET-deficient strains induced this expression. This stimulatory effect, mediated partly by the cell wall peptidoglycan, can be counterbalanced by ET. We conclude that B. anthracis down-regulates sPLA2-IIA expression in AMs through a process involving ET. Our study, therefore, describes a new molecular mechanism implemented by B. anthracis to escape innate host defense. These pioneering data will provide new molecular targets for future intervention against this deathly pathogen.
Author Summary
All mammals are susceptible to infection by Bacillus anthracis, the etiological agent of anthrax. Infection can occur either accidentally or as a potential consequence of a terrorism threat. Pulmonary infection is the most life-threatening form of the disease, causing a near 100% mortality. Despite appropriate therapy, all forms of infection may progress to fatal systemic anthrax, characterized by sepsis and respiratory failure. Thus, it is important to understand the mechanisms of host defense against B. anthracis. We have previously shown that alveolar macrophages produce an enzyme involved in innate defense that can kill B. anthracis: the enzyme is known as secreted phospholipase A2-IIA (sPLA2-IIA). The alveolar macrophage is one of the first cell types to come in contact with B. anthracis. In this study, we show that live B. anthracis spores stimulate the synthesis of sPLA2-IIA, this stimulation being counterbalanced by the inhibitory effect of the edema toxin produced by germinated spores and bacilli. Our study suggests that inhibition of sPLA2-IIA synthesis by edema toxin is a mechanism by which B. anthracis can escape innate host defense. These pioneering data provide new molecular targets for future intervention against this deadly pathogen.
PMCID: PMC2134952  PMID: 18069891
4.  Lethality in a Murine Model of Pulmonary Anthrax is Reduced by Combining Nuclear Transport Modifier with Antimicrobial Therapy 
PLoS ONE  2012;7(1):e30527.
In the last ten years, bioterrorism has become a serious threat and challenge to public health worldwide. Pulmonary anthrax caused by airborne Bacillus anthracis spores is a life- threatening disease often refractory to antimicrobial therapy. Inhaled spores germinate into vegetative forms that elaborate an anti-phagocytic capsule along with potent exotoxins which disrupt the signaling pathways governing the innate and adaptive immune responses and cause endothelial cell dysfunction leading to vascular injury in the lung, hypoxia, hemorrhage, and death.
Methods/Principal Findings
Using a murine model of pulmonary anthrax disease, we showed that a nuclear transport modifier restored markers of the innate immune response in spore-infected animals. An 8-day protocol of single-dose ciprofloxacin had no significant effect on mortality (4% survival) of A/J mice lethally infected with B. anthracis Sterne. Strikingly, mice were much more likely to survive infection (52% survival) when treated with ciprofloxacin and a cell-penetrating peptide modifier of host nuclear transport, termed cSN50. In B. anthracis-infected animals treated with antibiotic alone, we detected a muted innate immune response manifested by cytokines, tumor necrosis factor alpha (TNFα), interleukin (IL)-6, and chemokine monocyte chemoattractant protein-1 (MCP-1), while the hypoxia biomarker, erythropoietin (EPO), was greatly elevated. In contrast, cSN50-treated mice receiving ciprofloxacin demonstrated a restored innate immune responsiveness and reduced EPO level. Consistent with this improvement of innate immunity response and suppression of hypoxia biomarker, surviving mice in the combination treatment group displayed minimal histopathologic signs of vascular injury and a marked reduction of anthrax bacilli in the lungs.
We demonstrate, for the first time, that regulating nuclear transport with a cell-penetrating modifier provides a cytoprotective effect, which enables the host's immune system to reduce its susceptibility to lethal B. anthracis infection. Thus, by combining a nuclear transport modifier with antimicrobial therapy we offer a novel adjunctive measure to control florid pulmonary anthrax disease.
PMCID: PMC3266913  PMID: 22291977
5.  Mechanisms of NK Cell-Macrophage Bacillus anthracis Crosstalk: A Balance between Stimulation by Spores and Differential Disruption by Toxins 
PLoS Pathogens  2012;8(1):e1002481.
NK cells are important immune effectors for preventing microbial invasion and dissemination, through natural cytotoxicity and cytokine secretion. Bacillus anthracis spores can efficiently drive IFN-γ production by NK cells. The present study provides insights into the mechanisms of cytokine and cellular signaling that underlie the process of NK-cell activation by B. anthracis and the bacterial strategies to subvert and evade this response. Infection with non-toxigenic encapsulated B. anthracis induced recruitment of NK cells and macrophages into the mouse draining lymph node. Production of edema (ET) or lethal (LT) toxin during infection impaired this cellular recruitment. NK cell depletion led to accelerated systemic bacterial dissemination. IFN-γ production by NK cells in response to B. anthracis spores was: i) contact-dependent through RAE-1-NKG2D interaction with macrophages; ii) IL-12, IL-18, and IL-15-dependent, where IL-12 played a key role and regulated both NK cell and macrophage activation; and iii) required IL-18 for only an initial short time window. B. anthracis toxins subverted both NK cell essential functions. ET and LT disrupted IFN-γ production through different mechanisms. LT acted both on macrophages and NK cells, whereas ET mainly affected macrophages and did not alter NK cell capacity of IFN-γ secretion. In contrast, ET and LT inhibited the natural cytotoxicity function of NK cells, both in vitro and in vivo. The subverting action of ET thus led to dissociation in NK cell function and blocked natural cytotoxicity without affecting IFN-γ secretion. The high efficiency of this process stresses the impact that this toxin may exert in anthrax pathogenesis, and highlights a potential usefulness for controlling excessive cytotoxic responses in immunopathological diseases. Our findings therefore exemplify the delicate balance between bacterial stimulation and evasion strategies. This highlights the potential implication of the crosstalk between host innate defences and B. anthracis in initial anthrax control mechanisms.
Author Summary
NK cells are important immune effectors that perform a surveillance task and react to transformed, stressed, and virally infected cells. They represent a first-line defence against cancer and pathogen invasion. Different pathogens trigger distinct NK-cell activation pathways. The Bacillus anthracis spore is the highly resistant form that enters the host and provokes anthrax. This microbe kills through a combination of acute bacterial infection and devastating toxemia. In the present study, we characterise the crosstalk between NK cells and spores, as well as the strategies used by B. anthracis to evade initial control mechanisms and impact anthrax pathogenesis. Our findings exemplify the spores' property to efficiently drive a high production of IFN-γ by NK cells, as well as the complex pathways used for activation which require both cytokine and cellular signaling. B. anthracis subverts this response through its toxins by paralysing essential NK cell functions. Furthermore, edema toxin from B. anthracis blocks natural cytotoxicity without affecting IFN-γ secretion. The CyaA toxin of Bordetella pertussis possesses the same enzymatic activity and has a similar effect. The high efficiency of these toxins in blocking cytotoxicity in vivo implies possible exploitation of their subverting activity to modulate excessive cytotoxic responses in immunopathological diseases.
PMCID: PMC3257302  PMID: 22253596
6.  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.
PMCID: PMC3747219  PMID: 23977125
7.  Anthrax Lethal Toxin Impairs IL-8 Expression in Epithelial Cells through Inhibition of Histone H3 Modification 
PLoS Pathogens  2009;5(4):e1000359.
Lethal toxin (LT) is a critical virulence factor of Bacillus anthracis, the etiological agent of anthrax, whose pulmonary form is fatal in the absence of treatment. Inflammatory response is a key process of host defense against invading pathogens. We report here that intranasal instillation of a B. anthracis strain bearing inactive LT stimulates cytokine production and polymorphonuclear (PMN) neutrophils recruitment in lungs. These responses are repressed by a prior instillation of an LT preparation. In contrast, instillation of a B. anthracis strain expressing active LT represses lung inflammation. The inhibitory effects of LT on cytokine production are also observed in vitro using mouse and human pulmonary epithelial cells. These effects are associated with an alteration of ERK and p38-MAPK phosphorylation, but not JNK phosphorylation. We demonstrate that although NF-κB is essential for IL-8 expression, LT downregulates this expression without interfering with NF-κB activation in epithelial cells. Histone modifications are known to induce chromatin remodelling, thereby enhancing NF-κB binding on promoters of a subset of genes involved in immune response. We show that LT selectively prevents histone H3 phosphorylation at Ser 10 and recruitment of the p65 subunit of NF-κB at the IL-8 and KC promoters. Our results suggest that B. anthracis represses the immune response, in part by altering chromatin accessibility of IL-8 promoter to NF-κB in epithelial cells. This epigenetic reprogramming, in addition to previously reported effects of LT, may represent an efficient strategy used by B. anthracis for invading the host.
Author Summary
Bacillus anthracis, the etiological agent of anthrax, can infect mammals either accidentally or as a potential consequence of a terrorism threat. Pulmonary infection is a life-threatening form of the disease, causing a near 100% mortality rate in the absence of appropriate therapy. Thus, it is important to understand the mechanisms of host defense against B. anthracis. We examined the effects of various B. anthracis strains on lung inflammation in a mouse model of pulmonary anthrax and on human lung epithelial cells, the first barrier of lung against invading pathogens. We showed that a B. anthracis strain expressing lethal toxin inhibits inflammation. In contrast, a strain in which this toxin has been inactivated induces lung inflammation. We next examined the mechanisms involved in the inhibitory effect of lethal toxin. We showed that B. anthracis injects lethal toxin into epithelial cells, blocks the molecules associated on the chromosome, and thus represses production of mediators involved in inflammation. As the latter is a key process in host defense, its alteration by lethal toxin predisposes the host to infection by B. anthracis. This effect on the chromosomal machinery may represent an efficient strategy used by B. anthracis for invading the host.
PMCID: PMC2655721  PMID: 19343203
8.  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.
PMCID: PMC1273843  PMID: 16239558
9.  An anthrax lethal factor mutant that is defective at causing pyroptosis retains pro-apoptotic activity 
The FEBS journal  2009;277(1):119-127.
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.
PMCID: PMC2805769  PMID: 19922472
lethal toxin; anthrax; MAPKK; Nlrp1b
10.  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.
PMCID: PMC1307061  PMID: 16299324
11.  Capsules, Toxins and AtxA as Virulence Factors of Emerging Bacillus cereus Biovar anthracis 
PLoS Neglected Tropical Diseases  2015;9(4):e0003455.
Emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon (CA strain) and Côte d’Ivoire (CI strain). These strains are unusual, because their genomic characterisation shows that they belong to the B. cereus species, although they harbour two plasmids, pBCXO1 and pBCXO2, that are highly similar to the pXO1 and pXO2 plasmids of B. anthracis that encode the toxins and the polyglutamate capsule respectively. The virulence factors implicated in the pathogenicity of these B. cereus bv anthracis strains remain to be characterised. We tested their virulence by cutaneous and intranasal delivery in mice and guinea pigs; they were as virulent as wild-type B. anthracis. Unlike as described for pXO2-cured B. anthracis, the CA strain cured of the pBCXO2 plasmid was still highly virulent, showing the existence of other virulence factors. Indeed, these strains concomitantly expressed a hyaluronic acid (HA) capsule and the B. anthracis polyglutamate (PDGA) capsule. The HA capsule was encoded by the hasACB operon on pBCXO1, and its expression was regulated by the global transcription regulator AtxA, which controls anthrax toxins and PDGA capsule in B. anthracis. Thus, the HA and PDGA capsules and toxins were co-regulated by AtxA. We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants. Expression of the HA capsule by itself led to local multiplication and, during intranasal infection, to local dissemination to the adjacent brain tissue. Co-expression of either toxins or PDGA capsule with HA capsule enabled systemic dissemination, thus providing a clear evolutionary advantage. Protection against infection by B. cereus bv anthracis required the same vaccination formulation as that used against B. anthracis. Thus, these strains, at the frontier between B. anthracis and B. cereus, provide insight into how the monomorphic B. anthracis may have emerged.
Author Summary
Anthrax is caused by the bacterium Bacillus anthracis that affects all mammals worldwide. It emerged more than 10,000 years ago from a Bacillus cereus precursor. In the past decade, B. cereus bacteria were isolated in the USA from anthrax-like pneumonia cases. They harbour one virulence plasmid very similar to the toxin–encoding plasmid of B. anthracis. Recently, an anthrax-like disease in great apes in Africa was caused by emerging B. cereus strains, named B. cereus biovar anthracis. These strains are atypical as they possess both plasmids coding for toxin and capsule similar to those so far found only in B. anthracis. These unusual pathogenic B. cereus are currently neglected. We explored the virulence of these pathogens and their colonisation and dissemination capacity within the murine host. We found that these toxinogenic strains harbour two capsules, the classical B. anthracis capsule and an additional polysaccharidic capsule. This latter capsule confers virulence alone or in combination with toxins. Both capsules are concomitantly expressed, under the control of a common global regulator and host signals. Our results show that acquisition of new genetic information by these B. cereus clearly gives them a selective advantage, favouring their dissemination within infected hosts and the environment.
PMCID: PMC4382292  PMID: 25830379
12.  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.
PMCID: PMC2981334  PMID: 20855511
13.  Contribution of Lethal Toxin and Edema Toxin to the Pathogenesis of Anthrax Meningitis ▿ 
Infection and Immunity  2011;79(7):2510-2518.
Bacillus anthracis is a Gram-positive spore-forming bacterium that causes anthrax disease in humans and animals. Systemic infection is characterized by septicemia, toxemia, and meningitis, the main neurological complication associated with high mortality. We have shown previously that B. anthracis Sterne is capable of blood-brain barrier (BBB) penetration, establishing the classic signs of meningitis, and that infection is dependent on the expression of both major anthrax toxins, lethal toxin (LT) and edema toxin (ET). Here we further investigate the contribution of the individual toxins to BBB disruption using isogenic toxin mutants deficient in lethal factor, ΔLF, and edema factor, ΔEF. Acute infection with B. anthracis Sterne and the ΔLF mutant resulted in disruption of human brain microvascular endothelial cell (hBMEC) monolayer integrity and tight junction protein zona occludens-1, while the result for cells infected with the ΔEF mutant was similar to that for the noninfected control. A significant decrease in bacterial invasion of BBB endothelium in vitro was observed during infection with the ΔLF strain, suggesting a prominent role for LT in promoting BBB interaction. Further, treatment of hBMECs with purified LT or chemicals that mimic LT action on host signaling pathways rescued the hypoinvasive phenotype of the ΔLF mutant and resulted in increased bacterial uptake. We also observed that toxin expression reduced bacterial intracellular survival by inducing the bulk degradative autophagy pathway in host cells. Finally, in a murine model of anthrax meningitis, mice infected with the ΔLF mutant exhibited no mortality, brain bacterial load, or evidence of meningitis compared to mice infected with the parental or ΔEF strains.
PMCID: PMC3191953  PMID: 21518787
14.  Anthrax Toxins Inhibit Neutrophil Signaling Pathways in Brain Endothelium and Contribute to the Pathogenesis of Meningitis 
PLoS ONE  2008;3(8):e2964.
Anthrax meningitis is the main neurological complication of systemic infection with Bacillus anthracis approaching 100% mortality. The presence of bacilli in brain autopsies indicates that vegetative bacteria are able to breach the blood-brain barrier (BBB). The BBB represents not only a physical barrier but has been shown to play an active role in initiating a specific innate immune response that recruits neutrophils to the site of infection. Currently, the basic pathogenic mechanisms by which B. anthracis penetrates the BBB and causes anthrax meningitis are poorly understood.
Methodology/Principal Findings
Using an in vitro BBB model, we show for the first time that B. anthracis efficiently invades human brain microvascular endothelial cells (hBMEC), the single cell layer that comprises the BBB. Furthermore, transcriptional profiling of hBMEC during infection with B. anthracis revealed downregulation of 270 (87%) genes, specifically key neutrophil chemoattractants IL-8, CXCL1 (Groα) and CXCL2 (Groβ), thereby strongly contrasting hBMEC responses observed with other meningeal pathogens. Further studies using specific anthrax toxin-mutants, quantitative RT-PCR, ELISA and in vivo assays indicated that anthrax toxins actively suppress chemokine production and neutrophil recruitment during infection, allowing unrestricted proliferation and dissemination of the bacteria. Finally, mice challenged with B. anthracis Sterne, but not the toxin-deficient strain, developed meningitis.
These results suggest a significant role for anthrax toxins in thwarting the BBB innate defense response promoting penetration of bacteria into the central nervous system. Furthermore, establishment of a mouse model for anthrax meningitis will aid in our understanding of disease pathogenesis and development of more effective treatment strategies.
PMCID: PMC2493037  PMID: 18698416
15.  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.
PMCID: PMC3497415  PMID: 23027535
16.  Murine Macrophage Transcriptional Responses to Bacillus anthracis Infection and Intoxication  
Infection and Immunity  2005;73(2):1069-1080.
Interactions between Bacillus anthracis and host macrophages represent critical early events in anthrax pathogenesis, but their details are not clearly understood. Here we report the first genomewide characterization of the transcriptional changes within macrophages infected with B. anthracis and the identification of several hundred host genes that were differentially expressed during this intracellular stage of infection. These loci included both genes that are known to be regulated differentially in response to many other bacterial pathogens and those that appear to be differentially regulated in response to B. anthracis but not other bacterial species that have been tested. These data provide a transcriptional basis for a variety of physiological changes observed during infection, including the induction of apoptosis caused by the infecting bacteria. The expression patterns underlying B. anthracis-induced apoptosis led us to test further the importance of one very highly induced macrophage gene, that for ornithine decarboxylase. Our data show that this enzyme plays an important and previously unrecognized role in suppressing apoptosis in B. anthracis-infected cells. We have also characterized the transcriptional response to anthrax lethal toxin in activated macrophages and found that, following toxin treatment, many of the host inflammatory response pathways are dampened. These data provide insights into B. anthracis pathogenesis as well as potential leads for the development of new diagnostic and therapeutic options.
PMCID: PMC547061  PMID: 15664951
17.  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.
PMCID: PMC356021  PMID: 14977930
18.  Bacillus anthracis Peptidoglycan Stimulates an Inflammatory Response in Monocytes through the p38 Mitogen-Activated Protein Kinase Pathway 
PLoS ONE  2008;3(11):e3706.
We hypothesized that the peptidoglycan component of B. anthracis may play a critical role in morbidity and mortality associated with inhalation anthrax. To explore this issue, we purified the peptidoglycan component of the bacterial cell wall and studied the response of human peripheral blood cells. The purified B. anthracis peptidoglycan was free of non-covalently bound protein but contained a complex set of amino acids probably arising from the stem peptide. The peptidoglycan contained a polysaccharide that was removed by mild acid treatment, and the biological activity remained with the peptidoglycan and not the polysaccharide. The biological activity of the peptidoglycan was sensitive to lysozyme but not other hydrolytic enzymes, showing that the activity resides in the peptidoglycan component and not bacterial DNA, RNA or protein. B. anthracis peptidoglycan stimulated monocytes to produce primarily TNFα; neutrophils and lymphocytes did not respond. Peptidoglycan stimulated monocyte p38 mitogen-activated protein kinase and p38 activity was required for TNFα production by the cells. We conclude that peptidoglycan in B. anthracis is biologically active, that it stimulates a proinflammatory response in monocytes, and uses the p38 kinase signal transduction pathway to do so. Given the high bacterial burden in pulmonary anthrax, these findings suggest that the inflammatory events associated with peptidoglycan may play an important role in anthrax pathogenesis.
PMCID: PMC2577892  PMID: 19002259
19.  The Role of NF-κB and H3K27me3 Demethylase, Jmjd3, on the Anthrax Lethal Toxin Tolerance of RAW 264.7 Cells 
PLoS ONE  2010;5(3):e9913.
In Bacillus anthracis, lethal toxin (LeTx) is a critical virulence factor that causes immune suppression and toxic shock in the infected host. NF-κB is a key mediator of the inflammatory response and is crucial for the plasticity of first level immune cells such as macrophages, monocytes and neutrophils. In macrophages, this inflammatory response, mediated by NF-κB, can regulate host defense against invading pathogens. A Jumonji C family histone 3 lysine-27 (H3K27) demethylase, Jmjd3, plays a crucial role in macrophage plasticity and inflammation. Here we report that NF-κB and Jmjd3 can modulate the LeTx intoxication resistance of RAW 264.7 cells.
Principal Findings
This study showed that a 2 h exposure of macrophages to LeTx caused substantial cell death with a survival rate of around 40%. The expression of the Jmjd3 gene was induced 8-fold in intoxication-resistant cells generated by treatment with lipopolysaccharides of RAW 264.7 cells. These intoxication-resistant cell lines (PLx intox and PLxL intox) were maintained for 8 passages and had a survival rate of around 100% on secondary exposure to LeTx and lipopolysaccharides. Analysis of NF-κB gene expression showed that the expression of p100, p50 and p65 was induced around 20, 7 and 4 fold, respectively, in both of the intoxication-resistant cell lines following a 2 h treatment with PLxL (0.1+0.1+1 µg/ml). In contrast, these NF-κB genes were not induced following treatment with PLx treatment at the same concentrations.
Although LeTx influences macrophage physiology and causes defects of some key signaling pathways such as GSK3β which contributes to cytotoxicity, these results indicate that modulation of NF-κB by p50, p100 and Jmjd3 could be vital for the recovery of murine macrophages from exposure to the anthrax lethal toxin.
PMCID: PMC2848010  PMID: 20360974
20.  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.
PMCID: PMC343952  PMID: 14688124
21.  Human Monoclonal Antibodies against Anthrax Lethal Factor and Protective Antigen Act Independently To Protect against Bacillus anthracis Infection and Enhance Endogenous Immunity to Anthrax▿  
Infection and Immunity  2007;75(11):5425-5433.
The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-μg dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] of B. anthracis Sterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected with B. anthracis Sterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.
PMCID: PMC2168292  PMID: 17646360
22.  Effective antiprotease-antibiotic treatment of experimental anthrax 
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.
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.
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.
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.
PMCID: PMC1090577  PMID: 15819985
23.  Bacillus anthracis Capsule Activates Caspase-1 and Induces Interleukin-1β Release from Differentiated THP-1 and Human Monocyte-Derived Dendritic Cells▿ †  
Infection and Immunity  2009;78(1):387-392.
The poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infection. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Recently, efforts have been made to include PGA as a component of anthrax vaccine; however, the innate immune response of PGA itself has been poorly investigated. In this study, we characterized the innate immune response elicited by PGA in the human monocytic cell line THP-1, which was differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA) and human monocyte-derived dendritic cells (hMoDCs). PGA capsules were isolated from the culture supernatant of either the pXO1-cured strain of B. anthracis H9401 or B. licheniformis ATCC 9945a. PGA treatment of differentiated THP-1 cells and hMoDCs led to the specific extracellular release of interleukin-1β (IL-1β) in a dose-dependent manner. Evaluation of IL-1β processing by Western blotting revealed that cleaved IL-1β increased in THP-1 cells and hMoDCs after PGA treatment. Enhanced processing of IL-1β directly correlated with increased activation of its upstream regulator, caspase-1, also known as IL-1β-converting enzyme (ICE). The extracellular release of IL-1β in response to PGA was ICE dependent, since the administration of an ICE inhibitor prior to PGA treatment blocked induction of IL-1β. These results demonstrate that B. anthracis PGA elicits IL-1β production through activation of ICE in PMA-differentiated THP-1 cells and hMoDCs, suggesting the potential for PGA as a therapeutic target for anthrax.
PMCID: PMC2798191  PMID: 19737897
24.  Anthrax edema toxin differentially regulates lipopolysaccharide-induced monocyte production of tumor necrosis factor alpha and interleukin-6 by increasing intracellular cyclic AMP. 
Infection and Immunity  1994;62(10):4432-4439.
Bacillus anthracis exotoxins mediate most of the symptomatology of severe anthrax. In addition to a clinical syndrome reminiscent of septic shock, which may be mediated by cytokines produced by macrophages stimulated with lethal toxin, infected patients show profound edema at sites of infection. Edema is mediated by edema toxin (ET), which comprises of a binding molecule, protective antigen, and an active moiety, edema factor, which possesses intrinsic adenylyl cyclase activity. Intracellular cyclic AMP (cAMP) regulates the production of several cytokines that modulate edema formation and play important roles in host defense against invading bacteria. To determine whether ET enhanced the accumulation of cAMP in monocytes and thereby influenced cytokine production, we cultured human monocytes with endotoxin (lipopolysaccharide [LPS]) and dilutions of ET and determined the levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) in culture supernatant fluids. We further estimated cytokine-specific mRNA accumulation in monocytes by reverse transcription PCR and examined intracellular cAMP concentrations following treatment with ET. ET and LPS each induced monocytes to secrete comparable amounts of IL-6. ET did not inhibit and in most experiments modestly enhanced LPS-induced IL-6 production. In contrast to this stimulatory effect on IL-6 production, ET induced little or no TNF-alpha production. Moreover, ET profoundly inhibited LPS-induced TNF-alpha synthesis. These regulatory phenomena were also observed at the mRNA level in association with dose-related enhancement of intracellular cAMP in ET-treated monocytes. Monocytes treated with dibutyryl cAMP, an active analog of cAMP, produced cytokines in a pattern identical to that of cells treated with ET. The disruption of cytokine networks as a consequence of unregulated, ET-induced cAMP accumulation in human monocytes may impair cellular antimicrobial responses and contribute to clinical signs and symptoms.
PMCID: PMC303127  PMID: 7927706
25.  Human anti-anthrax protective antigen neutralizing monoclonal antibodies derived from donors vaccinated with anthrax vaccine adsorbed 
Potent anthrax toxin neutralizing human monoclonal antibodies were generated from peripheral blood lymphocytes obtained from Anthrax Vaccine Adsorbed (AVA) immune donors. The anti-anthrax toxin human monoclonal antibodies were evaluated for neutralization of anthrax lethal toxin in vivo in the Fisher 344 rat bolus toxin challenge model.
Human peripheral blood lymphocytes from AVA immunized donors were engrafted into severe combined immunodeficient (SCID) mice. Vaccination with anthrax protective antigen and lethal factor produced a significant increase in antigen specific human IgG in the mouse serum. The antibody producing lymphocytes were immortalized by hybridoma formation. The genes encoding the protective antibodies were rescued and stable cell lines expressing full-length human immunoglobulin were established. The antibodies were characterized by; (1) surface plasmon resonance; (2) inhibition of toxin in an in vitro mouse macrophage cell line protection assay and (3) in vivo in a Fischer 344 bolus lethal toxin challenge model.
The range of antibodies generated were diverse with evidence of extensive hyper mutation, and all were of very high affinity for PA83~1 × 10-10-11M. Moreover all the antibodies were potent inhibitors of anthrax lethal toxin in vitro. A single IV dose of AVP-21D9 or AVP-22G12 was found to confer full protection with as little as 0.5× (AVP-21D9) and 1× (AVP-22G12) molar equivalence relative to the anthrax toxin in the rat challenge prophylaxis model.
Here we describe a powerful technology to capture the recall antibody response to AVA vaccination and provide detailed molecular characterization of the protective human monoclonal antibodies. AVP-21D9, AVP-22G12 and AVP-1C6 protect rats from anthrax lethal toxin at low dose. Aglycosylated versions of the most potent antibodies are also protective in vivo, suggesting that lethal toxin neutralization is not Fc effector mediated. The protective effect of AVP-21D9 persists for at least one week in rats. These potent fully human anti-PA toxin-neutralizing antibodies are attractive candidates for prophylaxis and/or treatment against Anthrax Class A bioterrorism toxins.
PMCID: PMC420254  PMID: 15140257

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