scholarly journals ATP Depletion via Mitochondrial F1F0 Complex by Lethal Factor is an Early Event in B. Anthracis-Induced Sudden Cell Death

2009 ◽  
Vol 2 ◽  
pp. JCD.S2811 ◽  
Author(s):  
Mitchell W. Woodberry ◽  
Leopoldo Aguilera-Aguirre ◽  
Attila Bacsi ◽  
Ashok K. Chopra ◽  
Alexander Kurosky ◽  
...  
Keyword(s):  
Cell Death ◽  
Atpase Activity ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Underlying Mechanism ◽  
Atp Depletion ◽  
Mitogen Activated Protein Kinase ◽  
Early Event ◽  
Edema Factor ◽  
Mitogen Activated Protein

Bacillus anthracis' primary virulence factor is a tripartite anthrax toxin consisting of edema factor (EF), lethal factor (LF) and protective antigen (PA). In complex with PA, EF and LF are internalized via receptor-mediated endocytosis. EF is a calmodulin-dependent adenylate cyclase that induces tissue edema. LF is a zinc-metalloprotease that cleaves members of mitogen-activated protein kinase kinases. Lethal toxin (LT: PA plus LF)-induced death of macrophages is primarily attributed to expression of the sensitive Nalp1b allele, inflammasome formation and activation of caspase-1, but early events that initiate these processes are unknown. Here we provide evidence that an early essential event in pyroptosis of alveolar macrophages is LF-mediated depletion of cellular ATP. The underlying mechanism involves interaction of LF with F1F0-complex gamma and beta subunits leading to increased ATPase activity in mitochondria. In support, mitochondrial DNA-depleted MH-S cells have decreased F1F0 ATPase activity due to the lack of F06 and F08 polypeptides and show increased resistance to LT. We conclude that ATP depletion is an important early event in LT-induced sudden cell death and its prevention increases survival of toxin-sensitive cells.

scholarly journals Anthrax toxin lethal factor domain 3 is highly mobile and responsive to ligand binding

2014 ◽  
Vol 70 (11) ◽  
pp. 2813-2822 ◽  
Author(s):  
Kimberly M. Maize ◽  
Elbek K. Kurbanov ◽  
Teresa De La Mora-Rey ◽  
Todd W. Geders ◽  
Dong-Jin Hwang ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Mitogen Activated Protein Kinase ◽  
Host Immune System ◽  
Peptide Substrates ◽  
Edema Factor ◽  
Mitogen Activated Protein ◽  
Domain 3

The secreted anthrax toxin consists of three components: the protective antigen (PA), edema factor (EF) and lethal factor (LF). LF, a zinc metalloproteinase, compromises the host immune system primarily by targeting mitogen-activated protein kinase kinases in macrophages. Peptide substrates and small-molecule inhibitors bind LF in the space between domains 3 and 4 of the hydrolase. Domain 3 is attached on a hinge to domain 2viaresidues Ile300 and Pro385, and can move through an angular arc of greater than 35° in response to the binding of different ligands. Here, multiple LF structures including five new complexes with co-crystallized inhibitors are compared and three frequently populated LF conformational states termed `bioactive', `open' and `tight' are identified. The bioactive position is observed with large substrate peptides and leaves all peptide-recognition subsites open and accessible. The tight state is seen in unliganded and small-molecule complex structures. In this state, domain 3 is clamped over certain substrate subsites, blocking access. The open position appears to be an intermediate state between these extremes and is observed owing to steric constraints imposed by specific bound ligands. The tight conformation may be the lowest-energy conformation among the reported structures, as it is the position observed with no bound ligand, while the open and bioactive conformations are likely to be ligand-induced.

Manipulation of host signalling pathways by anthrax toxins

2007 ◽  
Vol 402 (3) ◽  
pp. 405-417 ◽  
Author(s):  
Benjamin E. Turk
Keyword(s):  
Protein Kinase ◽  
Pathogenic Bacteria ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Cell Types ◽  
Host Cells ◽  
Mitogen Activated Protein Kinase ◽  
Signalling Pathways ◽  
Common Strategy ◽  
Mitogen Activated Protein

Infectious microbes face an unwelcoming environment in their mammalian hosts, which have evolved elaborate multicelluar systems for recognition and elimination of invading pathogens. A common strategy used by pathogenic bacteria to establish infection is to secrete protein factors that block intracellular signalling pathways essential for host defence. Some of these proteins also act as toxins, directly causing pathology associated with disease. Bacillus anthracis, the bacterium that causes anthrax, secretes two plasmid-encoded enzymes, LF (lethal factor) and EF (oedema factor), that are delivered into host cells by a third bacterial protein, PA (protective antigen). The two toxins act on a variety of cell types, disabling the immune system and inevitably killing the host. LF is an extraordinarily selective metalloproteinase that site-specifically cleaves MKKs (mitogen-activated protein kinase kinases). Cleavage of MKKs by LF prevents them from activating their downstream MAPK (mitogen-activated protein kinase) substrates by disrupting a critical docking interaction. Blockade of MAPK signalling functionally impairs cells of both the innate and adaptive immune systems and induces cell death in macrophages. EF is an adenylate cyclase that is activated by calmodulin through a non-canonical mechanism. EF causes sustained and potent activation of host cAMP-dependent signalling pathways, which disables phagocytes. Here I review recent progress in elucidating the mechanisms by which LF and EF influence host signalling and thereby contribute to disease.

scholarly journals Involvement of Domain II in Toxicity of Anthrax Lethal Factor

2004 ◽  
Vol 279 (50) ◽  
pp. 52473-52478 ◽  
Author(s):  
Xudong Liang ◽  
John J. Young ◽  
Sherrie A. Boone ◽  
David S. Waugh ◽  
Nicholas S. Duesbery
Keyword(s):  
Proteolytic Activity ◽  
Active Site ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Site Directed Mutagenesis ◽  
Mitogen Activated Protein Kinase ◽  
Anthrax Lethal Factor ◽  
Fold Reduction ◽  
Mitogen Activated Protein

Anthrax lethal factor (LF) is a Zn2+-metalloprotease that cleaves and inactivates mitogen-activated protein kinase kinases (MEKs). We have used site-directed mutagenesis to identify a cluster of residues in domain II of LF that lie outside the active site and are required for cellular proteolytic activity toward MEKs. Alanine substituted for Leu293, Lys294, Leu514, Asn516, or Arg491caused a 10–50-fold reduction in LF toxicity. Further, whereas pairwise substitution of alanine for Leu514and either Leu293, Lys294, or Arg491completely abrogated LF toxicity, pairwise mutation of Leu514and Asn516resulted in toxicity comparable with N516A alone. The introduction of these mutations reduced LF-mediated cleavage of MEK2 in cell-based assays but altered neither the ability of LF to bind protective antigen nor its ability to translocate across a membrane. Interestingly, directin vitromeasurement of LF activity indicated that decreased toxicity was not always accompanied by reduced proteolytic activity. However, mutations in this region significantly reduced the ability of LF to competitively inhibit B-Raf phosphorylation of MEK. These results provide evidence that elements of domain II are involved in the association of LF into productive complex with MEKs.

scholarly journals Proteasome Activity Is Required for Anthrax Lethal Toxin To Kill Macrophages

1999 ◽  
Vol 67 (6) ◽  
pp. 3055-3060 ◽  
Author(s):  
Guangqing Tang ◽  
Stephen H. Leppla
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Proteasome Inhibitors ◽  
Lethal Toxin ◽  
Atp Depletion ◽  
Mitogen Activated Protein Kinase ◽  
Target Cells ◽  
Anthrax Lethal Toxin ◽  
Early Effect ◽  
Primary Mouse

ABSTRACT 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.

scholarly journals Anthrax lethal factor-cleavage products of MAPK (mitogen-activated protein kinase) kinases exhibit reduced binding to their cognate MAPKs

2004 ◽  
Vol 378 (2) ◽  
pp. 569-577 ◽  
Author(s):  
A. Jane BARDWELL ◽  
Mahsa ABDOLLAHI ◽  
Lee BARDWELL
Keyword(s):  
Protein Kinase ◽  
Protective Antigen ◽  
Signal Transmission ◽  
Lethal Factor ◽  
Lethal Toxin ◽  
Mitogen Activated Protein Kinase ◽  
Cell Surface Receptor ◽  
Common Mechanism ◽  
Anthrax Lethal Toxin ◽  
Mitogen Activated Protein

Anthrax lethal toxin is the major cause of death in systemic anthrax. Lethal toxin consists of two proteins: protective antigen and LF (lethal factor). Protective antigen binds to a cell-surface receptor and transports LF into the cytosol. LF is a metalloprotease that targets MKKs [MAPK (mitogen-activated protein kinase) kinases]/MEKs [MAPK/ERK (extracellular-signal-regulated kinase) kinases], cleaving them to remove a small N-terminal stretch but leaving the bulk of the protein, including the protein kinase domain, intact. LF-mediated cleavage of MEK1 and MKK6 has been shown to inhibit signalling through their cognate MAPK pathways. However, the precise mechanism by which this proteolytic cleavage inhibits signal transmission has been unclear. Here we show that the C-terminal LF-cleavage products of MEK1, MEK2, MKK3, MKK4, MKK6 and MKK7 are impaired in their ability to bind to their MAPK substrates, suggesting a common mechanism for the LF-induced inhibition of signalling.

scholarly journals A Chimeric Protein That Functions as both an Anthrax Dual-Target Antitoxin and a Trivalent Vaccine

2010 ◽  
Vol 54 (11) ◽  
pp. 4750-4757 ◽  
Author(s):  
Gaobing Wu ◽  
Yuzhi Hong ◽  
Aizhen Guo ◽  
Chunfang Feng ◽  
Sha Cao ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Vaccine Candidate ◽  
Lethal Factor ◽  
Lethal Dose ◽  
Chimeric Protein ◽  
Dominant Negative ◽  
Lethal Challenge ◽  
Edema Factor ◽  
Trivalent Vaccine

ABSTRACT Effective measures for the prophylaxis and treatment of anthrax are still required for counteracting the threat posed by inhalation anthrax. In this study, we first demonstrated that the chimeric protein LFn-PA, created by fusing the protective antigen (PA)-binding domain of lethal factor (LFn) to PA, retained the functions of the respective molecules. On the basis of this observation, we attempted to develop an antitoxin that targets the binding of lethal factor (LF) and/or edema factor (EF) to PA and the transportation of LF/EF. Therefore, we replaced PA in LFn-PA with a dominant-negative inhibitory PA (DPA), i.e., PAF427D. In in vitro models of anthrax intoxication, the LFn-DPA chimera showed 3-fold and 2-fold higher potencies than DPA in protecting sensitive cells against anthrax lethal toxin (LeTx) and edema toxin (EdTx), respectively. In animal models, LFn-DPA exhibited strong potency in rescuing mice from lethal challenge with LeTx. We also evaluated the immunogenicity and immunoprotective efficacy of LFn-DPA as an anthrax vaccine candidate. In comparison with recombinant PA, LFn-DPA induced significantly higher levels of the anti-PA immune response. Moreover, LFn-DPA elicited an anti-LF antibody response that could cross-react with EF. Mice immunized with LFn-DPA tolerated a LeTx challenge that was 5 times its 50% lethal dose. Thus, LFn-DPA represents a highly effective trivalent vaccine candidate for both preexposure and postexposure vaccination. Overall, we have developed a novel and dually functional reagent for the prophylaxis and treatment of anthrax.

scholarly journals Injection of Staphylococcus aureus EDIN by the Bacillus anthracis Protective Antigen Machinery Induces Vascular Permeability

2009 ◽  
Vol 77 (9) ◽  
pp. 3596-3601 ◽  
Author(s):  
Monica Rolando ◽  
Patrick Munro ◽  
Caroline Stefani ◽  
Patrick Auberger ◽  
Gilles Flatau ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Vascular Permeability ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Vascular Leakage ◽  
Host Cells ◽  
Mitogen Activated Protein Kinase ◽  
Systemic Injection ◽  
Pulmonary Endothelium ◽  
Endothelium Permeability

ABSTRACT Systemic injection of Bacillus anthracis lethal toxin (LT) produces vascular leakage and animal death. Recent studies suggest that LT triggers direct endothelial cell cytotoxicity that is responsible for the vascular leakage. LT is composed of heptamers of protective antigen (PA), which drives the endocytosis and translocation into host cells of the lethal factor (LF), a mitogen-activated protein kinase kinase protease. Here we investigated the consequences of injection of an endothelium-permeabilizing factor using LT as a “molecular syringe.” To this end, we generated the chimeric factor LE, corresponding to the PA-binding domain of LF (LF1-254) fused to EDIN exoenzyme. EDIN ADP ribosylates RhoA, leading to actin cable disruption and formation of transcellular tunnels in endothelial cells. We report that systemic injection of LET (LE plus PA) triggers a PA-dependent increase in the pulmonary endothelium permeability. We also report that native LT induces a progressive loss of endothelium barrier function. We established that there is a direct correlation between the extent of endothelium permeability induced by LT and the cytotoxic activity of LT. This suggests new ways to design therapeutic drugs against anthrax directed toward vascular permeability.

scholarly journals Inactivation of Rho GTPases by Statins Attenuates Anthrax Lethal Toxin Activity

2008 ◽  
Vol 77 (1) ◽  
pp. 348-359 ◽  
Author(s):  
Aimee M. deCathelineau ◽  
Gary M. Bokoch
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Protective Antigen ◽  
Biological Effects ◽  
Lethal Factor ◽  
Lethal Toxin ◽  
Mitogen Activated Protein Kinase ◽  
Gtpase Activity ◽  
Anthrax Lethal Toxin ◽  
Reductase Inhibitors

ABSTRACT 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.

Sign in / Sign up

Export Citation Format

Share Document