Anthrax Lethal Toxin Activity Research Articles

Activation of the Nlrp1b inflammasome by reduction of cytosolic ATP.

The efficacy of the innate immune system depends on its ability to mount an appropriate response to diverse infections and damaging agents. Key components of this system are pattern recognition receptors that detect pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs). Nlrp1b is a pattern recognition receptor that forms a caspase-1 activation platform, known as an inflammasome, upon sensing the proteolytic activity of anthrax lethal toxin. The activation of caspase-1 leads to the release of proinflammatory cytokines that aid in the clearance of the anthrax infection. Here, we demonstrate that Nlrp1b also becomes activated in cells that are subjected to energy stress caused by metabolic inhibitors or by nutrient deprivation. Glucose starvation and hypoxia were used to correlate the level of cytosolic ATP to the degree of inflammasome activation. Because lowering the ratio of cytosolic ATP to AMP activates the main cellular energy sensor, AMP-activated protein kinase (AMPK), we assessed whether AMPK promoted inflammasome activity by using a combination of small interfering RNA (siRNA) and transfection of a dominant negative AMPK subunit. We found that AMPK promoted inflammasome activity, but activation of AMPK in the absence of ATP depletion was not sufficient for caspase-1-mediated pro-interleukin 1β (pro-IL-1β) processing. Finally, we found that mutation of the ATP-binding motif of Nlrp1b caused constitutive activation, suggesting that ATP might inhibit the Nlrp1b inflammasome instead of being required for its assembly.

Proteolytic Processing of Nlrp1b Is Required for Inflammasome Activity

Nlrp1b is a NOD-like receptor that detects the catalytic activity of anthrax lethal toxin and subsequently co-oligomerizes into a pro-caspase-1 activation platform known as an inflammasome. Nlrp1b has two domains that promote oligomerization: a NACHT domain, which is a member of the AAA+ ATPase family, and a poorly characterized Function to Find Domain (FIIND). Here we demonstrate that proteolytic processing within the FIIND generates N-terminal and C-terminal cleavage products of Nlrp1b that remain associated in both the auto-inhibited state and in the activated state after cells have been treated with lethal toxin. Functional significance of cleavage was suggested by the finding that mutations that block processing of Nlrp1b also prevent the ability of Nlrp1b to activate pro-caspase-1. By using an uncleaved mutant of Nlrp1b, we established the importance of cleavage by inserting a heterologous TEV protease site into the FIIND and demonstrating that TEV protease processed this site and induced inflammasome activity. Proteolysis of Nlrp1b was shown to be required for the assembly of a functional inflammasome: a mutation within the FIIND that abolished cleavage had no effect on self-association of a FIIND-CARD fragment, but did reduce the recruitment of pro-caspase-1. Our work indicates that a post-translational modification enables Nlrp1b to function.

Abstract 2334: Anti-angiogenic activity of anthrax lethal toxin using the mouse retina as a model system

Abstract Our laboratory has previously shown that inhibition of MKK pathways by anthrax lethal toxin (LeTx) inhibits tumor growth and angiogenesis, reduces VEGF secretion, and results in rapid cessation of blood flow to tumors in melanoma and sarcoma xenograft model systems. To gain insight into the anti-angiogenic nature of LeTx, we utilized both developmental and pathological models of vascularization in the murine retina. 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 in retinal vascular development followed by the formation of abnormal vascular tufts that co-stained with phosphorylated MAPK in the outer retinal region. Using a model of oxygen-induced retinopathy, western analysis revealed that MAPK activation preceded the onset of hypoxia and development of neovascularization in this model system. Intravitreal injection of LeTx during hypoxia resulted in a persistent lack of development and progression of all pathologic aspects of neovascularization, while having minimal effect on macrophage recruitment. VEGF levels remained at an elevated level even with the persistent lack of neovascular growth and continued hypoxic environment, implying a VEGF-independent mechanism for the effects of MKK inhibition. Additionally, immature retinal vasculature during normal development was persistently destroyed following MAPK inhibition, while mature vessels from adult animals were unaffected. Our studies not only reveal that MKK signaling plays a key role in retinal angiogenesis but also that perturbation of MKK signaling by LeTx can profoundly alter vascular morphogenesis. The persistent destruction of immature vessels during normal vascular development in the retina and in the model of oxygen-induced retinopathy suggests an extreme sensitivity of immature vessels to MKK signaling inhibition. Since MKK inhibition prevents oxygen-induced retinopathy by a mechanism independent of the VEGF-targeting strategies, this approach may be used in conjunction with existing strategies utilizing VEGF inhibitors to prevent pathologic angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2334. doi:1538-7445.AM2012-2334

Consequences and Utility of the Zinc-Dependent Metalloprotease Activity of Anthrax Lethal Toxin

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.

Inactivation of Rho GTPases by Statins Attenuates Anthrax Lethal Toxin Activity

Anthrax lethal factor (LF), secreted by Bacillus anthracis, interacts with protective antigen to form a bipartite toxin (lethal toxin [LT]) that exerts pleiotropic biological effects resulting in subversion of the innate immune response. Although the mitogen-activated protein kinase kinases (MKKs) are the major intracellular protein targets of LF, the pathology induced by LT is not well understood. The statin family of HMG-coenzyme A reductase inhibitors have potent anti-inflammatory effects independent of their cholesterol-lowering properties, which have been attributed to modulation of Rho family GTPase activity. The Rho GTPases regulate vesicular trafficking, cytoskeletal dynamics, and cell survival and proliferation. We hypothesized that disruption of Rho GTPase function by statins might alter LT action. We show here that statins delay LT-induced death and MKK cleavage in RAW macrophages and that statin-mediated effects on LT action are attributable to disruption of Rho GTPases. The Rho GTPase-inactivating toxin, toxin B, did not significantly affect LT binding or internalization, suggesting that the Rho GTPases regulate trafficking and/or localization of LT once internalized. The use of drugs capable of inhibiting Rho GTPase activity, such as statins, may provide a means to attenuate intoxication during B. anthracis infection.

Dendritic Cells Endocytose Bacillus anthracis Spores: Implications for Anthrax Pathogenesis

Phagocytosis of inhaled Bacillus anthracis spores and subsequent trafficking to lymph nodes are decisive events in the progression of inhalational anthrax because they initiate germination and dissemination of spores. Found in high frequency throughout the respiratory track, dendritic cells (DCs) routinely take up foreign particles and migrate to lymph nodes. However, the participation of DCs in phagocytosis and dissemination of spores has not been investigated previously. We found that human DCs readily engulfed fully pathogenic Ames and attenuated B. anthracis spores predominately by coiling phagocytosis. Spores provoked a loss of tissue-retaining chemokine receptors (CCR2, CCR5) with a concurrent increase in lymph node homing receptors (CCR7, CD11c) on the membrane of DCs. After spore infection, immature DCs displayed a mature phenotype (CD83(bright), HLA-DR(bright), CD80(bright), CD86(bright), CD40(bright)) and enhanced costimulatory activity. Surprisingly, spores activated the MAPK cascade (ERK, p38) within 30 min and stimulated expression of several inflammatory response genes by 2 h. MAPK signaling was extinguished by 6 h infection, and there was a dramatic reduction of secreted TNF-alpha, IL-6, and IL-8 in the absence of DC death. This corresponded temporally with enzymatic cleavage of proximal MAPK signaling proteins (MEK-1, MEK-3, and MAP kinase kinase-4) and may indicate activity of anthrax lethal toxin. Taken together, these results suggest that B. anthracis may exploit DCs to facilitate infection.

Anthrax Lethal Toxin Blocks MAPK Kinase-Dependent IL-2 Production in CD4+ T Cells

Anthrax lethal toxin (LT) is a critical virulence factor that cleaves and inactivates MAPK kinases (MAPKKs) in host cells and has been proposed as a therapeutic target in the treatment of human anthrax infections. Despite the potential use of anti-toxin agents in humans, the standard activity assays for anthrax LT are currently based on cytotoxic actions of anthrax LT that are cell-, strain-, and species-specific, which have not been demonstrated to occur in human cells. We now report that T cell proliferation and IL-2 production inversely correlate with anthrax LT levels in human cell assays. The model CD4+ T cell tumor line, Jurkat, is a susceptible target for the specific protease action of anthrax LT. Anthrax LT cleaves and inactivates MAPKKs in Jurkat cells, whereas not affecting proximal or parallel TCR signal transduction pathways. Moreover, anthrax LT specifically inhibits PMA/ionomycin- and anti-CD3-induced IL-2 production in Jurkat cells. An inhibitor of the protease activity of anthrax LT completely restores IL-2 production by anthrax LT-treated Jurkat cells. Anthrax LT acts on primary CD4+ T cells as well, cleaving MAPKKs and leading to a 95% reduction in anti-CD3-induced proliferation and IL-2 production. These findings not only will be useful in the development of new human cell-based bioassays for the activity of anthrax LT, but they also suggest new mechanisms that facilitate immune evasion by Bacillus anthracis. Specifically, anthrax LT inhibits IL-2 production and proliferative responses in CD4+ T cells, thereby blocking functions that are pivotal in the regulation of immune responses.

Anthrax Lethal Toxin Rapidly Activates Caspase-1/ICE and Induces Extracellular Release of Interleukin (IL)-1β and IL-18

Anthrax lethal toxin (LT), a critical virulence factor for Bacillus anthracis, has been demonstrated to cleave and to inactivate mitogen-activated protein kinase kinases (MAPKKs) that propagate prosurvival signals in macrophages (1-5). Whether this action of anthrax LT leads to the production of proinflammatory cytokines by macrophages has been more controversial (6, 7). We now report that anthrax LT treatment leads to the specific extracellular release of interleukin (IL)-1beta and IL-18 by the murine macrophage cell lines, RAW264.7 and J774A.1. Studies of the processing of IL-1beta reveal that the levels of activated/cleaved IL-1beta in RAW264.7 and J774.A1 cells are increased following treatment with anthrax LT. Enhanced processing of IL-1beta directly correlates with increased levels in the activation of its upstream regulator, IL-1beta-converting enzyme/Caspase-1 (ICE). The extracellular release of IL-1beta and IL-18 in response to anthrax LT is ICE-dependent, as an ICE-specific inhibitor blocks this process. These data indicate that ICE, IL-1beta, and IL-18 are downstream effectors of anthrax LT in macrophages, providing the basis for new bioassays for anthrax LT activity and representing potential therapeutic targets.

Asp 187 and Phe 190 residues in lethal factor are required for the expression of anthrax lethal toxin activity.

Anthrax toxin consists of three proteins, protective antigen, lethal factor, and edema factor. Protective antigen translocates lethal factor and edema factor to the cytosol of mammalian cells. The amino-termini of lethal factor and edema factor have several homologous stretches. These regions are presumably involved in binding to protective antigen. In the present study we have determined the role of one such homologous stretch in lethal factor. Residues 187AspLeuLeuPhe190 were replaced by alanine. Asp187Ala and Phe190Ala were found to be non-toxic in combination with protective antigen. Their protective antigen-binding ability was drastically reduced. We propose that Asp187 and Phe190 are crucial for the expression of anthrax lethal toxin activity.

Asp 187 and Phe 190 residues in lethal factor are required for the expression of anthrax lethal toxin activity

Anthrax toxin consists of three proteins, protective antigen, lethal factor, and edema factor. Protective antigen translocates lethal factor and edema factor to the cytosol of mammalian cells. The amino-termini of lethal factor and edema factor have several homologous stretches. These regions are presumably involved in binding to protective antigen. In the present study we have determined the role of one such homologous stretch in lethal factor. Residues 187AspLeuLeuPhe190 were replaced by alanine. Asp187Ala and Phe190Ala were found to be non-toxic in combination with protective antigen. Their protective antigen-binding ability was drastically reduced. We propose that Asp187 and Phe190 are crucial for the expression of anthrax lethal toxin activity.

Trp 346 and Leu 352 Residues in Protective Antigen Are Required for the Expression of Anthrax Lethal Toxin Activity

The three separate proteins that make up anthrax toxin-protective antigen (PA), edema factor (EF) and lethal factor (LF) act in binary combinations to produce two distinct reactions in experimental animals: edema (PA+EF) and death (PA+LF). PA is believed to interact with a membrane receptor and, after proteolytic processing, to mediate endocytosis and subsequent translocation of EF or LF into the cytosol. Residues W346, M350, and L352 in loop 3 of domain 2 have been implicated to induce a conformational change when the pH is lowered from 7.4 to 6.5. Modification of the residues Trp (346), Met (350), and Leu (352) to alanine individually and all the three residues together to alanine residues resulted in the loss of cytotoxic activity in combination with LF. The mutant proteins were able to bind to the cell surface receptor, become cleaved by trypsin, bind LF, and oligomerize. These residues might play an important role in the membrane insertion of PA and/or translocation of LF/EF into the cytosol.

Participation of residue F552 in domain III of the protective antigen in the biological activity of anthrax lethal toxin.

The protective antigen (PA) component of anthrax toxin translocates the catalytic moieties lethal factor (LF) and edema factor (EF) into the cytosol. The proteolytically activated 63 kDa form of PA (PA63) has the ability to oligomerize and bind LF/EF. PA has four distinct domains performing specialized functions; whereas the function of domains I, II and IV has been well characterized, domain III has no known role in the biological activity of PA. Here we report the role of amino acid residues lining an exposed hydrophobic patch of domain III in the biological activity of PA. The residues Phe552, Phe554, lIe562, Leu566 and lle574 were individually substituted with alanine and the effect was studied. All mutant PA proteins except Phe552Ala were equally active as wild-type PA in exhibiting a toxic phenotype to J774A.1 cells in the presence of LF. Substitution of Ala for Phe552 reduced the ability of PA to intoxicate cells by more than 250-fold. However, Phe552Ala was equally active in receptor binding and susceptibility to trypsin and chymotrypsin as wild-type PA, the activities that have been shown to be essential for the biological activity of PA. This mutated PA protein had a decreased ability to bind LF, oligomerize on cells and to induce release of 86Rb+ from Chinese hamster ovary cells. These results suggest that the residue Phe552 in PA plays an important role in LF binding and oligomerization. Our study provides a basis for further exploration of the biological significance of domain III of PA.

Influence of body weight on response of Fischer 344 rats to anthrax lethal toxin

Groups of Fischer 344 rats were injected intravenously with Bacillus anthracis culture supernatant containing crude anthrax toxin. Times to death of rats given identical toxin preparations varied directly with the weights of the rats (P = 0.0001). In contrast to previous reports, the data indicate that rat weight must be taken into account during in vivo assays of anthrax lethal toxin activity.

Calcium is required for the expression of anthrax lethal toxin activity in the macrophagelike cell line J774A.1

Anthrax lethal toxin, which consists of two separate proteins, protective antigen (Mr, 82,700) and lethal factor (Mr, approximately 83,000), is cytotoxic to the macrophagelike cell line J774A.1. Removal of calcium from the culture medium protected cells against the action of lethal toxin. Calcium depletion during the binding phase of intoxication afforded only partial protection. Further analysis showed that calcium removal caused some inhibition of protective antigen binding but that it had minimal effect on proteolytic conversion of protective antigen to the active 63-kilodalton fragment and that it had no effect on lethal factor binding. Cells to which lethal toxin had bound in the presence of calcium were protected when transferred to calcium-depleted culture medium, indicating a role for calcium at a postbinding stage. When ammonium chloride is present with lethal toxin, toxin accumulates in intracellular vesicles. Calcium-free medium protected these cells upon removal of the amine block, suggesting that calcium is also required at a step after internalization of lethal toxin. Calcium channel blockers inhibited 45Ca2+ uptake and protected cells against cytotoxicity. Calmodulin inhibitors also protected against the action of lethal toxin, suggesting involvement of calmodulin at a step during intoxication. We conclude that calcium is required at several steps in the intoxication of cells by anthrax lethal toxin.