Anthrax Lethal Factor Cleaves the N-Terminus of MAPKKs and Induces Tyrosine/Threonine Phosphorylation of MAPKs in Cultured Macrophages

Vitale, Gaetano; Pellizzari, Rossella; Recchi, Chiara; Napolitani, Giorgio; Mock, Michèle; Montecucco, Cesare

doi:10.1006/bbrc.1998.9040

Cited by 398 publications

(253 citation statements)

References 29 publications

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“…With other multiplex microsphere sets, experiments with up to 100 substrates could be attained. Additionally, this system is surface based and has the option of using full length protein substrates, which will be used in extending our studies to full length substrates to evaluate the contributions of MAPKK distal element (3,4). Inclusion of substrates with the distal element could be greatly beneficial in the creation of a screening assay to detect LF inhibitors.…”

Section: Discussionmentioning

confidence: 99%

“…Lethal factor inhibitor (In-2-LF, suspended in 5% acetic acid) was added to wells 2, 4,6,8,10,14,16,18,20, and 22 to a final concentration of 1.5 lM. Factor Xa inhibitor, Antithrombin III, was added to a final concentration of 1 lM to wells 3,4,7,8,11,15,16,19,20,23. LF was added to wells 1-11 and 13-23 at a final concentration of 525 nM and Xa was added to wells 25-35 and 37-47 at a concentration of 575 nM.…”

Section: Hypercyt Analysismentioning

confidence: 99%

“…These toxins use a cellular receptor binding protein to deliver a zinc dependent metalloprotease into the cell, where it cleaves key host proteins to exert toxic effects on the host organism (1). The metalloprotease for lethal toxin is the lethal factor protease (LF), which specifically cleaves the Map-kinasekinase (MAPKK) family of proteins at an N-terminal cleavage site (2)(3)(4). Subsequent studies have demonstrated the presence of a C-terminal distal binding element on the MAPKK family of proteins that is required for efficient cleavage by LF (4,5).…”

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confidence: 99%

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Microsphere‐based protease assays and screening application for lethal factor and factor Xa

Saunders¹,

Kim²,

Woods³

et al. 2006

Cytometry Pt A

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Background: Proteases regulate many biological pathways in humans and are components of several bacterial toxins. Protease studies and development of protease inhibitors do not follow a single established methodology and are mostly protease specific. Methods: We have created recombinant fusion proteins consisting of a biotinylated attachment sequence linked to a GFP via a protease cleavage site to develop a multiplexable microsphere-based protease assay system. Using the proteases lethal factor and factor Xa, we performed kinetic experiments to determine optimal conditions for inhibitor screens and detect known inhibitors using the HyperCytÒ flow cytometry system. Results: We have demonstrated specific cleavage of lethal factor and factor Xa substrates, optimized screening conditions for these substrates, shown specific inhibition

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Section: Discussionmentioning

confidence: 99%

Section: Hypercyt Analysismentioning

confidence: 99%

mentioning

confidence: 99%

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Microsphere‐based protease assays and screening application for lethal factor and factor Xa

Saunders¹,

Kim²,

Woods³

et al. 2006

Cytometry Pt A

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“…Lethal toxin (LeTx) consists of PA, the moiety that is responsible for cellular binding and entry, and LF, which is a zinc metalloprotease. LeTx is implicated in both macrophage (Mf) and host death (Klimpel et al, 1994;Duesbery et al, 1998;Vitale et al, 1998). Oedema toxin (EdTx) consists of PA and EF, a calmodulin-dependent adenylate cyclase.…”

Section: Introductionmentioning

confidence: 99%

Early Bacillus anthracis-macrophage interactions: intracellular survival and escape

et al. 2000

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This study describes early intracellular events occurring during the establishment phase of Bacillus anthracis infections. Anthrax infections are initiated by dormant endospores gaining access to the mammalian host and becoming engulfed by regional macrophages (Mφ). During systemic anthrax, late stage events include vegetative growth in the blood to very high titres and the synthesis of the anthrax exotoxin complex, which causes disease symptoms and death. Experiments focus on the early events occurring during the first few hours of the B. anthracis infectious cycle, from endospore germination up to and including release of the vegetative cell from phagocytes. We found that newly vegetative bacilli escape from the phagocytic vesicles of cultured Mφ and replicate within the cytoplasm of these cells. Release from the Mφ occurs 4–6 h after endospore phagocytosis, timing that correlates with anthrax infection of test animals. Genetic analysis from this study indicates that the toxin plasmid pXO1 is required for release from the Mφ, whereas the capsule plasmid pXO2 is not. The transactivator atxA, located on pXO1, is also found to be essential for release, but the toxin genes themselves are not required. This suggests that Mφ release of anthrax bacilli is atxA regulated. The putative ‘escape’ genes may be located on the chromosome and/or on pXO1.

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“…Anthrax PA is essential for pore formation, allowing intracellular transport of the active toxin enzymes, EF, and LF. EF is an adenylate cyclase, whereas LF is a metalloprotease that inactivates MAPK kinases (MAPKKs) through cleavage at specific recognition sites (5)(6)(7)(8).…”

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confidence: 99%

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

Fang

Cordoba-Rodriguez

Lankford

et al. 2005

The Journal of Immunology

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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.

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Anthrax Lethal Factor Cleaves the N-Terminus of MAPKKs and Induces Tyrosine/Threonine Phosphorylation of MAPKs in Cultured Macrophages

Cited by 398 publications

References 29 publications

Microsphere‐based protease assays and screening application for lethal factor and factor Xa

Microsphere‐based protease assays and screening application for lethal factor and factor Xa

Early Bacillus anthracis-macrophage interactions: intracellular survival and escape

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

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