A major virulence factor of Bacillus anthracis is the anthrax Lethal Toxin (LeTx), a bipartite toxin composed of Protective Antigen and Lethal Factor. Systemic administration of LeTx to laboratory animals leads to death associated with vascular leakage and pulmonary edema. In this study, we investigated whether systemic exposure of mice to LeTx would induce gene expression changes associated with vascular/capillary leakage in lung tissue. We observed enhanced susceptibility of A/J mice to death by systemic LeTx administration compared to the C57BL/6 strain. LeTx-induced groups of both up- and down-regulated genes were observed in mouse lungs 6 h after systemic administration of wild type toxin compared to lungs of mice exposed to an inactive mutant form of the toxin. Lungs of the less susceptible C57BL/6 strain showed 80% fewer differentially expressed genes compared to lungs of the more sensitive A/J strain. Expression of genes known to regulate vascular permeability was modulated by LeTx in the lungs of the more susceptible A/J strain. Unexpectedly, the largest set of genes with altered expression was immune specific, characterized by the up-regulation of lymphoid genes and the down-regulation of myeloid genes. Transcripts encoding neutrophil chemoattractants, modulators of tumor regulation and angiogenesis were also differentially expressed in both mouse strains. These studies provide new directions for the investigation of vascular leakage and pulmonary edema induced by anthrax LeTx.
Lethal Toxin; lung; gene expression
The bipartite anthrax lethal toxin (LeTx) consisting of protective antigen (PA) and lethal factor (LF) is a major virulence factor contributing to death from systemic Bacillus anthracis infection. The current vaccine elicits antibodies directed primarily to PA; however, in experimental settings serologic responses to LF can neutralize LeTx and contribute to protection against infection. The goals of the present study were to identify sequential B-cell epitopes of LF and to determine the capacity of these determinants to bind neutralizing antibodies. Sera of recombinant LF-immunized A/J mice exhibited high titers of immunoglobulin G anti-LF reactivity that neutralized LeTx in vitro 78 days after the final booster immunization and protected the mice from in vivo challenge with 3 50% lethal doses of LeTx. These sera bound multiple discontinuous epitopes, and there were major clusters of reactivity on native LF. Strikingly, all three neutralizing, LF-specific monoclonal antibodies tested bound specific peptide sequences that coincided with sequential epitopes identified in polyclonal antisera from recombinant LF-immunized mice. This study confirms that LF induces high-titer protective antibodies in vitro and in vivo. Moreover, the binding of short LF peptides by LF-specific neutralizing monoclonal antibodies suggests that generation of protective antibodies by peptide vaccination may be feasible for this antigen. This study paves the way for a more effective anthrax vaccine by identifying discontinuous peptide epitopes of LF.
We have recently shown that the anthrax lethal toxin (LeTx) selectively represses nuclear hormone receptors. In the current studies, we found that LeTx repressed activation of the MMTV promoter related to over-expression of the transcription factors, HNF3, Oct1 and c-Jun. LeTx transcriptional repression was associated with a decrease in protein levels of these transcription factors in a lethal factor (LF) protease activity dependent manner. Early administration of LeTx antagonists partially or completely abolished the repressive effects of LeTx. In contrast to the rapid cleavage of MAPKKs by LeTx, the degradation of these transcription factors occurred at a relatively late stage after LeTx treatment. In addition, LeTx repressed phorbol 12-myristate 13-acetate (PMA) -induced MMTV promoter activity and PMA-induction of endogenous c-Jun protein. Collectively, these findings suggest that transcription factors are intracellular targets of LeTx and expand our understanding of the molecular action of LeTx at a later stage of low-dose exposure.
anthrax lethal toxin (LeTx); MMTV promoter; transcription factors; over-expression; repression
Lethal factor, the enzymatic moiety of anthrax lethal toxin (LeTx) is a protease that inactivates mitogen activated protein kinase kinases (MEK or MKK). In vitro and in vivo studies demonstrate LeTx targets endothelial cells. However, the effects of LeTx on endothelial cells are incompletely characterized. To gain insight into this process we used a developmental model of vascularization in the murine retina. We hypothesized that application of LeTx would disrupt normal retinal vascularization, specifically during the angiogenic phase of vascular development. By immunoblotting and immunofluorescence microscopy we observed that MAPK activation occurs in a spatially and temporally regulated manner during retinal vascular development. Intravitreal administration of LeTx caused an early delay (4 d post injection) in retinal vascular development that was marked by reduced penetration of vessels into distal regions of the retina as well as failure of sprouting vessels to form the deep and intermediate plexuses within the inner retina. In contrast, later stages (8 d post injection) were characterized by the formation of abnormal vascular tufts that co-stained with phosphorylated MAPK in the outer retinal region. We also observed a significant increase in the levels of secreted VEGF in the vitreous 4 d and 8 d after LeTx injection. In contrast, the levels of over 50 cytokines other cytokines, including bFGF, EGF, MCP-1, and MMP-9, remained unchanged. Finally, co-injection of VEGF-neutralizing antibodies significantly decreased LeTx-induced neovascular growth. Our studies not only reveal that MAPK signaling plays a key role in retinal angiogenesis but also that perturbation of MAPK signaling by LeTx can profoundly alter vascular morphogenesis.
High mortality in the 2001 US and recent European anthrax outbreaks suggests that better understanding of the effects of this bacteria’s toxins is needed to improve treatment.
Methods and results
Here, 24h edema (ETx) and lethal (LeTx) toxin infusions were investigated for 96h in sedated and mechanically ventilated canines. Initial study compared similarly lethal doses of ETx (n=8) or LeTx (n=15) alone. ETx was 24 times less lethal than LeTx, while median time to death in non-survivors (n=6 and 9 respectively) was shorter with ETx (42 vs. 67h, p=0.04). Compared to controls (n=9), both toxins decreased arterial and central venous pressures (CVP) and systemic vascular resistance (SVRI) and increased heart rate (HR), cardiac index, blood urea nitrogen (BUN), creatinine (Cr), BUN:Cr ratio, and hepatic transaminases (p≤0.05, toxin effect or time interaction). However, ETx stimulated early diuresis, reduced serum sodium and had more pronounced vasodilatory effects than LeTx as reflected by greater or earlier CVP, SVRI, and BUN:Cr changes (p≤0.01). LeTx progressively decreased left ventricular ejection fraction (p≤0.002). In subsequent study, lethal dose LeTx with an equimolar nonlethal ETx dose (n=8) increased mortality versus LeTx alone (n=8) (p=0.05).
Shock with ETx or LeTx may require differing supportive therapies while toxin antagonists should likely target both toxins.
Anthrax; edema and lethal toxins; shock; organ injury
Background. High mortality in the 2001 US and recent European anthrax outbreaks suggests that better understanding of the effects of the toxins produced by this bacterium is needed to improve treatment.
Methods and results. Here, 24-h edema (ETx) and lethal (LeTx) toxin infusions were investigated for 96 h in sedated canines receiving mechanical ventilation. The initial study compared similarly lethal doses of ETx (n=8) or LeTx (n=15) alone. ETx was 24 times less lethal than LeTx, and the median time to death in nonsurvivors (n=6 and n=9, respectively) was shorter with ETx (42 vs 67 h; P=.04). Compared with controls (n=9), both toxins decreased arterial and central venous pressures and systemic vascular resistance and increased heart rate, cardiac index, blood urea nitrogen (BUN) level, creatinine (Cr) concentration, BUN:Cr ratio, and hepatic transaminase levels (P ⩽ .05 for toxin effect or time interaction). However, ETx stimulated early diuresis, reduced serum sodium levels, and had more pronounced vasodilatory effects, compared with LeTx, as reflected by greater or earlier central venous pressures, systemic vascular resistance, and changes in the BUN:Cr ratio (P ⩽ .01). LeTx progressively decreased the left ventricular ejection fraction (P ⩽ .002). In a subsequent study, a lethal dose of LeTx with an equimolar nonlethal ETx dose (n=8) increased mortality, compared with LeTx alone (n=8;P=.05).
Conclusion. Shock with ETx or LeTx may require differing supportive therapies, whereas toxin antagonists should likely target both toxins.
Passive transfer of antibody may be useful for preexposure prophylaxis against biological agents used as weapons of terror, such as Bacillus anthracis. Studies were performed to evaluate the ability of anthrax antiprotective antigen (anti-PA) and antilethal factor (anti-LF) neutralizing monoclonal antibodies (mAbs) to protect against an anthrax lethal toxin (LeTx) challenge in a mouse model and to identify correlates of immunity to LeTx challenge. Despite having similar affinities for their respective antigens, anti-PA (3F11) and anti-LF (9A11), passive transfer of up to 1.5 mg of anti-PA 3F11 mAb did not provide significant protection when transferred to mice 24 h before LeTx challenge, while passive transfer of as low as 0.375 mg of anti-LF 9A11 did provide significant protection. Serum collected 24 h after passive transfer had LeTx-neutralizing activity when tested using a standard LeTx neutralization assay, but neutralization titers measured using this assay did not correlate with protection against LeTx challenge. However, measurement of LeTx-neutralizing serum responses with an LeTx neutralization assay in vitro employing the addition of LeTx to J774A.1 cells 15 min before the addition of the serum did result in neutralization titers that correlated with protection against LeTx challenge. Our results demonstrate that only the LeTx neutralization titers measured utilizing the addition of LeTx to J774A.1 cells 15 min before the addition of sample correlated with protection in vivo. Thus, this LeTx neutralization assay may be a more biologically relevant neutralization assay to predict the in vivo protective capacity of LeTx-neutralizing antibodies.
Bacillus anthracis lethal toxin (LT) is a bipartite toxin composed of protective antigen (PA) and lethal factor (LF). Injection of LT produces clinical signs characteristic of anthrax infection, including pleural edema and vascular collapse in various animal models. We utilized the classic Miles leakage assay to quantify vascular leakage in mice. LT injected intradermally induced leakage as early as 15 to 25 min in some inbred mouse strains, but not in others, whereas PA or LF individually did not induce leakage. A third component of anthrax toxin, edema factor, did not induce leakage alone or with PA. Leakage was quantified in eight mouse strains, and no correlation was found between sensitivity to intradermal leakage and sensitivity to the lethality of systemically administered LT. The leakage could be inhibited by ketotifen, an inhibitor of mast cell degranulation, but not by azelastine, a histamine receptor 1 antagonist, or by ketanserin, a serotonin 5-HT2A receptor antagonist. LT was cytotoxic to MC/9 mast cells (in vitro) by 7 h after toxin treatment but did not induce histamine release from these cells. Mast cell-deficient mice exhibited the leakage event and had no increased resistance to systemic LT. Human umbilical vein endothelial cells were resistant to LT over 12 h, with only 20% of cells succumbing by 24 h, suggesting that endothelial cell killing is not the cause of the rapid LT-mediated leakage event. We describe here a ketotifen-sensitive vascular leakage event induced by LT which is the most rapid in vivo or in vitro LT-mediated effect reported to date.
Anthrax lethal toxin (LeTx) is a virulence factor of Bacilillus anthracis that is a bivalent toxin, containing lethal factor (LF) and protective Ag proteins, which causes cytotoxicity and altered macrophage function. LeTx exposure results in early K+ efflux from macrophages associated with caspase-1 activation and increased IL-1β release. The mechanism of this toxin-induced K+ efflux is unknown. The goals of the current study were to determine whether LeTx-induced K+ efflux from macrophages is mediated by toxin effects on specific K+ channels and whether altered K+-channel activity is involved in LeTx-induced IL-1β release. Exposure of macrophages to LeTx induced a significant increase in the activities of two types of K+ channels that have been identified in mouse macrophages: Ba2+-sensitive inwardly rectifying K+ (Kir) channels and 4-aminopyridine–sensitive outwardly rectifying voltage-gated K+ (Kv) channels. LeTx enhancement of both Kir and Kv required the proteolytic activity of LF, because exposure of macrophages to a mutant LF-protein (LFE687C) combined with protective Ag protein had no effect on the currents. Furthermore, blocking Kir and Kv channels significantly decreased LeTx-induced release of IL-1β. In addition, retroviral transduction of macrophages with wild-type Kir enhanced LeTx-induced release of IL-1β, whereas transduction of dominant-negative Kir blocked LeTx-induced release of IL-1β. Activation of caspase-1 was not required for LeTx-induced activation of either of the K+ channels. These data indicate that a major mechanism through which LeTx stimulates macrophages to release IL-1β involves an LF-protease effect that enhances Kir and Kv channel function during toxin stimulation.
We investigated the ability of using monoclonal antibodies (MAbs) against anthrax protective antigen (PA), an anthrax exotoxin component, to modulate exotoxin cytotoxic activity on target macrophage cell lines. Anthrax PA plays a critical role in the pathogenesis of Bacillus anthracis infection. PA is the cell-binding component of the two anthrax exotoxins: lethal toxin (LeTx) and edema toxin. Several MAbs that bind the PA component of LeTx are known to neutralize LeTx-mediated killing of target macrophages. Here we describe for the first time an overlooked population of anti-PA MAbs that, in contrast, function to increase the potency of LeTx against murine macrophage cell lines. The results support a possible mechanism of enhancement: binding of MAb to PA on the macrophage cell surface stabilizes the PA by interaction of MAb with macrophage Fcγ receptors. This results in an increase in the amount of PA bound to the cell surface, which in turn leads to an enhancement in cell killing, most likely due to increased internalization of LF. Blocking of PA-receptor binding eliminates enhancement by MAb, demonstrating the importance of this step for the observed enhancement. The additional significance of these results is that, at least in mice, immunization with PA appears to elicit a poly-clonal response that has a significant prevalence of MAbs that enhance LeTx-mediated killing in macrophages.
Recent research regarding the structure and function of Bacillus anthracis lethal (LeTx) and edema (ETx) toxins provides growing insights into the pathophysiology and treatment of shock with this lethal bacteria. These are both binary-type toxins composed of protective antigen necessary for their cellular uptake and either lethal or edema factors, the toxigenic moieties. The primary cellular receptors for protective antigen have been identified and constructed and key steps in the extracellular processing and internalization of the toxins clarified. Consistent with the lethal factor's primary action as an intracellular endopeptidase targeting mitogen-activated protein kinase kinases, growing evidence indicates that shock with this toxin does not result from an excessive inflammatory response. In fact, the potent immunosuppressive effects of LeTx may actually contribute to the establishment and persistence of infection. Instead, shock with LeTx may be related to the direct injurious effects of lethal factor on endothelial cell function. Despite the importance of LeTx, very recent studies show that edema factor, a potent adenyl cyclase, has the ability to make a substantial contribution to shock caused by B. anthracis and works additively with LeTx. Furthermore, ETx may contribute to the immunosuppressive effects of LeTx. Therapies under development that target several different steps in the cellular uptake and function of these two toxins have been effective in in vitro and in vivo systems. Understanding how best to apply these agents clinically and how they interact with conventional treatments should be goals for future research.
anthrax; toxin; shock; treatment
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.
anthrax; lethal toxin; TTP; IL-8; P-body
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.
anthrax; lethal factor; mitogen-activated protein kinase kinase; pathogenesis; metalloprotease; tumorigenesis; retinal neovascularization
The lethal toxin (LeTx) of Bacillus anthracis plays a key role in the pathogenesis of anthrax. The protective antigen (PA) is a primary part of the anthrax toxin and forms LeTx by combination with lethal factor (LF). Phenylalanine-427 (F427) is crucial for PA function. This study was designed to discover potential novel therapeutic agents and vaccines for anthrax. This was done by screening PA mutants that were mutated at the F427 residue for a dominant-negative inhibitory (DNI) phenotype which was nontoxic but inhibited the toxicity of the wild-type LeTx. For this, PA residue F427 was first mutated to each of the other 19 naturally occurring amino acids. The cytotoxicity and DNI phenotypes of the mutated PA proteins were tested in the presence of 1 μg/ml LF in RAW264.7 cells and were shown to be dependent on the individual amino acid replacements. A total of 16 nontoxic mutants with various levels of DNI activity were identified in vitro. Among them, F427D and F427N mutants had the highest DNI activities in RAW264.7 cells. Both mutants inhibited LeTx intoxication in mice in a dose-dependent way. Furthermore, they induced a Th2-predominant immune response and protected mice against a challenge with five 50% lethal doses of LeTx. The protection was correlated mainly with a low level of interleukin-1β (IL-1β) and with high levels of PA-specific immunoglobulin G1, IL-6, and tumor necrosis factor alpha. Thus, PA DNI mutants, such as F427D and F427N mutants, may serve in the development of novel therapeutic agents and vaccines to fight B. anthracis infections.
Anthrax lethal and edema toxins (LeTx and EdTx, respectively) form by binding of lethal factor (LF) or edema factor (EF) to the pore-forming moiety protective antigen (PA). Immunity to LF and EF protects animals from anthrax spore challenge and neutralizes anthrax toxins. The goal of the present study is to identify linear B-cell epitopes of EF and to determine the relative contributions of cross-reactive antibodies of EF and LF to LeTx and EdTx neutralization. A/J mice were immunized with recombinant LF (rLF) or rEF. Pools of LF or EF immune sera were tested for reactivity to rLF or rEF by enzyme-linked immunosorbent assays, in vitro neutralization of LeTx and EdTx, and binding to solid-phase LF and EF decapeptides. Cross-reactive antibodies were isolated by column absorption of EF-binding antibodies from LF immune sera and by column absorption of LF-binding antibodies from EF immune sera. The resulting fractions were subjected to the same assays. Major cross-reactive epitopes were identified as EF amino acids (aa) 257 to 268 and LF aa 265 to 274. Whole LF and EF immune sera neutralized LeTx and EdTx, respectively. However, LF sera did not neutralize EdTx, nor did EF sera neutralize LeTx. Purified cross-reactive immunoglobulin G also failed to cross-neutralize. Cross-reactive B-cell epitopes in the PA-binding domains of whole rLF and rEF occur and have been identified; however, the major anthrax toxin-neutralizing humoral responses to these antigens are constituted by non-cross-reactive epitopes. This work increases understanding of the immunogenicity of EF and LF and offers perspective for the development of new strategies for vaccination against anthrax.
Anthrax infections are frequently associated with severe and often irreversible hypotensive shock. The isolated toxic proteins of Bacillus anthracis produce a non-cytokine-mediated hypotension in rats by unknown mechanisms. These observations suggest the anthrax toxins have direct cardiovascular effects. Here, we characterize these effects. As a first step, we administered systemically anthrax lethal toxin (LeTx) and edema toxin (EdTx) to cohorts of three to twelve rats at different doses and determined the time of onset, degree of hypotension and mortality. We measured serum concentrations of the protective antigen (PA) toxin component at various time points after infusion. Peak serum levels of PA were in the µg/mL range with half-lives of 10–20 minutes. With doses that produced hypotension with delayed lethality, we then gave bolus intravenous infusions of toxins to groups of four to six instrumented rats and continuously monitored blood pressure by telemetry. Finally, the same doses used in the telemetry experiments were given to additional groups of four rats, and echocardiography was performed pretreatment and one, two, three and twenty-four hours post-treatment. LeTx and EdTx each produced hypotension. We observed a doubling of the velocity of propagation and 20% increases in left ventricular diastolic and systolic areas in LeTx-treated rats, but not in EdTx-treated rats. EdTx-but not LeTx-treated rats showed a significant increase in heart rate. These results indicate that LeTx reduced left ventricular systolic function and EdTx reduced preload. Uptake of toxins occurs readily into tissues with biological effects occurring within minutes to hours of serum toxin concentrations in the µg/mL range. LeTx and EdTx yield an irreversible shock with subsequent death. These findings should provide a basis for the rational design of drug interventions to reduce the dismal prognosis of systemic anthrax infections.
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.
Anthrax lethal toxin (LeTx) and edema toxin (EdTx) have been shown to alter hemodynamics in the rodent model, while LeTx primarily is reported to induce extensive tissue pathology. However, the rodent model has limitations when used for comparison to higher organisms such as humans. The rabbit model, on the other hand, has gained recognition as a useful model for studying anthrax infection and its pathophysiological effects. In this study, we assessed the hemodynamic effects of lethal toxin (LeTx) and edema toxin (EdTx) in the rabbit model using physiologically relevant amounts of the toxins. Moreover, we further examine the pathological effects of LeTx on cardiac tissue. We intravenously injected Dutch-belted rabbits with either low-dose and high-dose recombinant LeTx or a single dose of EdTx. The animals’ heart rate and mean arterial pressure were continuously monitored via telemetry until either 48 or 72 h post-challenge. Additional animals challenged with LeTx were used for cardiac troponin I (cTnI) quantitation, cardiac histopathology, and echocardiography. LeTx depressed heart rate at the lower dose and mean arterial pressure (MAP) at the higher dose. EdTx, on the other hand, temporarily intensified heart rate while lowering MAP. Both doses of LeTx caused cardiac pathology with the higher dose having a more profound effect. Lastly, left-ventricular dilation due to LeTx was not apparent at the given time-points. Our study demonstrates the hemodynamic effects of anthrax toxins, as well as the pathological effects of LeTx on the heart in the rabbit model, and it provides further evidence for the toxins’ direct impact on the heart.
anthrax; lethal toxin; rabbit model; cardiac
Solid tumor growth is dependent on angiogenesis, the formation of neovasculature from existing vessels. Endothelial activation of the ERK1/2, JNK, and p38 mitogen activated protein kinase (MAPK) pathways is central to this process, and thus presents an attractive target for the development of angiogenesis inhibitors. Anthrax lethal toxin (LeTx) has potent catalytic MAPK inhibition activity. Preclinical studies showed LeTx induced potent tumor growth inhibition via the inhibition of xenograft vascularization. However, LeTx receptors and the essential furin-like activating proteases are expressed in many normal tissues, potentially limiting the specificity of LeTx as an anti-tumor agent. To circumvent nonspecific LeTx activation and simultaneously enhance tumor vascular targeting, a substrate preferably cleaved by the gelatinases class of matrix metalloproteinases (MMPs) was substituted for the furin LeTx activation site. In vivo efficacy studies demonstrated that this MMP-activated LeTx inhibited tumor xenografts growth via the reduced migration of endothelial cells into the tumor parenchyma. Here we have expanded on these initial findings by demonstrating that this MMP-activated LeTx reduces endothelial proangiogenic MMP expression, thus causing a diminished proteolytic capacity for extracellular matrix remodeling and endothelial differentiation into capillary networks. Additionally, our data suggests that inhibition of the JNK and p38, but not ERK1/2 pathways is significant in the anti-angiogenic activity of the MMP-activated LeTx. Collectively, these results support the clinical development of the MMP-activated LeTx for the treatment of solid tumors.
anthrax lethal toxin; lethal factor; matrix metalloproteinase; protective antigen; tumor angiogenesis; vascular endothelial growth factor
Antibody (Ab) responses to Bacillus anthracis toxins are protective, but relatively few protective monoclonal antibodies (MAbs) have been reported. Protective antigen (PA) is essential for the action of B. anthracis lethal toxin (LeTx) and edema toxin. In this study, we generated two MAbs to PA, MAbs 7.5G and 10F4. These MAbs did not compete for binding to PA, consistent with specificities for different epitopes. The MAbs were tested for their ability to protect a monolayer of cultured macrophages against toxin-mediated cytotoxicity. MAb 7.5G, the most-neutralizing MAb, bound to domain 1 of PA and reduced LeTx toxicity in BALB/c mice. Remarkably, MAb 7.5G provided protection without blocking the binding of PA or lethal factor or the formation of the PA heptamer complex. However, MAb 7.5G slowed the proteolytic digestion of PA by furin in vitro, suggesting a potential mechanism for Ab-mediated protection. These observations indicate that some Abs to domain 1 can contribute to host protection.
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.
Cellular adaptation to different stresses related to survival and function has been demonstrated in several cell types. Anthrax lethal toxin (LeTx) induces rapid cell death, termed “pyroptosis,” by activating NLRP1b/caspase-1 in murine macrophages. We and others (S. D. Ha et al., J. Biol. Chem. 282:26275-26283, 2007; I. I. Salles et al., Proc. Natl. Acad. Sci. U. S. A. 100:12426 –12431, 2003) have shown that RAW264.7 cells preexposed to sublethal doses of LeTx become resistant to subsequent high cytolytic doses of LeTx, termed toxin-induced resistance (TIR). To date, the cellular mechanisms of pyroptosis and TIR are largely unknown. We found that LeTx caused NLRP1b/caspase-1-dependent mitochondrial dysfunction, including hyperpolarization and generation of reactive oxygen species, which was distinct from that induced by stimuli such as NLRP3-activating ATP. In TIR cells, these mitochondrial events were not detected, although caspase-1 was activated, in response to LeTx. We identified that downregulation of the late endosomal cholesterol-transferring protein MLN64 in TIR cells was involved in TIR. The downregulation of MLN64 in TIR cells was at least in part due to DNA methyltransferase 1-mediated DNA methylation. In wild-type RAW264.7 cells and primary bone marrow-derived macrophages, LeTx caused NLRP1b/caspase-1-dependent mitochondrial translocation of MLN64, resulting in cholesterol enrichment, membrane hyperpolarization, reactive oxygen species (ROS) generation, and depletion of free glutathione (GSH). This study demonstrates for the first time that MLN64 plays a key role in LeTx/caspase-1-induced mitochondrial dysfunction.
We performed GeneChip analyses on RNA from Bacillus anthracis lethal toxin (LeTx)-treated RAW 264.7 murine macrophages to investigate global effects of anthrax toxin on host cell gene expression. Stringent analysis of data revealed that the expression of several mitogen-activated protein kinase kinase-regulatory genes was affected within 1.5 h post-exposure to LeTx. By 3.0 h, the expression of 103 genes was altered, including those involved in intracellular signaling, energy production, and protein metabolism.
Bacillus anthracis secretes several virulence factors targeting different host organs and cell types during inhalational anthrax infection. The bacterial expression of a key virulence factor, lethal toxin (LeTx) is closely tied to another factor, edema toxin (EdTx). Both are transcribed on the same virulence plasmid (pXO1) and both have been the subject of much individual study. Their combined effect during virulent anthrax likely modulates both the global transcriptional and the phenotypic response of macrophages and phagocytes. In fact, responses brought about by the toxins may be different than each of their individual effects.
Here we report the transcriptional and apoptotic responses of the macrophage-like phagocytic cell line THP-1 exposed to B. anthracis Sterne (pXO1+) spores, and B. anthracis Δ Sterne (pXO1-) spores. These cells are resistant to LeTx-induced cytolysis, a phenotype seen in macrophages from several mouse strains which are sensitive to toxigenic anthrax infection. Our results indicate that the pXO1-containing strain induces higher pro-inflammatory transcriptional responses during the first 4 hours of interaction with bacterium, evident in the upregulation of several genes relevant to Nf-κB, phosphatases, prostaglandins, and TNF-α, along with decreases in expression levels of genes for mitochondrial components. Both bacterial strains induce apoptosis, but in the toxigenic strain-challenged cells, apoptosis is delayed.
This delay in apoptosis occurs despite the much higher level of TNF-α secretion induced by the toxigenic-strain challenge. Interestingly, CFLAR, an important apoptotic inhibitor which blocks apoptosis induced by large amounts of extracellular TNF-α, is upregulated significantly during toxigenic-strain infection, but not at all during non-toxigenic-strain infection, indicating that it may play a role in blocking or delaying TNF-α-mediated apoptosis. The suppression of apoptosis by the toxigenic anthrax strain is consistent with the notion that apoptosis itself may represent a protective host cell response.
Anthrax Lethal Toxin (LeTx) demonstrates potent MAPK pathway inhibition and apoptosis in melanoma cells that harbor the activating V600E B-RAF mutation. LeTx is composed of two proteins, PA and LF. Uptake of the toxin into cells is dependent upon proteolytic activation of PA by the ubiquitously expressed furin or furin-like proteases. In order to circumvent nonspecific LeTx activation, a substrate preferably cleaved by gelatinases was substituted for the furin LeTx activation site. Here we have shown the toxicity of this MMP-activated LeTx is dependent on host cell surface MMP-2 and −9 activity as well as the presence of the activating V600E B-RAF mutation, making this toxin dual specific. This additional layer of tumor cell specificity would potentially decrease systemic toxicity from the reduction of nonspecific toxin activation while retaining anti-tumor efficacy in patients with V600E B-RAF melanomas. Moreover, our results indicate that cell surface-associated gelatinase expression can be used to predict sensitivity among V600E B-RAF melanomas. This finding will aid in the better selection of patients that will potentially respond to MMP-activated LeTx therapy.
anthrax lethal toxin; B-RAF; lethal factor; matrix metalloproteinase; protective antigen