scholarly journals Metalloproteinase Inhibitors, Nonantimicrobial Chemically Modified Tetracyclines, and Ilomastat Block Bacillus anthracis Lethal Factor Activity in Viable Cells

2005 ◽  
Vol 73 (11) ◽  
pp. 7548-7557 ◽  
Author(s):  
Salih S. Kocer ◽  
Stephen G. Walker ◽  
Brad Zerler ◽  
Lorne M. Golub ◽  
Sanford R. Simon
Keyword(s):  
Bacillus Anthracis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Lethal Factor ◽  
Sodium Dodecyl ◽  
Viable Cell ◽  
Lethal Toxin ◽  
Mitogen Activated Protein Kinase ◽  
Metalloproteinase Inhibitors ◽  
Mitogen Activated Protein ◽  
Chemically Modified Tetracyclines

ABSTRACT 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.

scholarly journals Fc gamma R-dependent mitogen-activated protein kinase activation in leukocytes: a common signal transduction event necessary for expression of TNF-alpha and early activation genes.

1996 ◽  
Vol 184 (3) ◽  
pp. 1027-1035 ◽  
Author(s):  
R Trotta ◽  
P Kanakaraj ◽  
B Perussia
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Kinase Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mitogen Activated Protein Kinase ◽  
Monocytic Cell ◽  
Fast Kinetics ◽  
Mitogen Activated Protein ◽  
Stimulation Of

Cross-linking the receptors for the Fc domain of IgG (Fc gamma R) on leukocytes induces activation of protein tyrosine kinases. The intermediary molecules that transduce to the nucleus the signals leading to induction of the diverse biological responses mediated by these receptors are not clearly identified. We have investigated whether mitogen-activated protein kinases (MAPK) are involved in transmembrane signaling via the three Fc gamma R present on monocytic, polymorphonuclear, and natural killer (NK) cells. Our results indicate that occupancy of Fc gamma RI and Fc gamma RII on the monocytic cell line THP-I and on polymorphonuclear leukocytes (PMN) induces, transiently and with fast kinetics, MAPK phosphorylation, as indicated by decreased electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and increased amounts of the proteins in antiphosphotyrosine antibody immunoprecipitates. This, associated with increased enzymatic activity, also occurs upon stimulation of the transmembrane isoform of CD16 (Fc gamma RIIIA) in NK cells and in a T cell line expressing transfected Fc gamma RIIIA alpha ligand-binding chain in association with zeta, but not upon stimulation of the glycosil-phosphatidylinositol-anchored Fc gamma RIIIB on PMN. Using the specific MAP kinase kinase inhibitor-PD 098059, we show that activation of MAPK is necessary for the Fc gamma R-dependent induction of c-fos and tumor necrosis factor alpha mRNA expression in monocytes and NK cells. These results underscore the role of MAPK as signal-transducing molecules controlling the expression of different genes relevant to leukocyte biology upon Fc gamma R stimulation.

scholarly journals Glycerol Monolaurate Inhibits Virulence Factor Production in Bacillus anthracis

2005 ◽  
Vol 49 (4) ◽  
pp. 1302-1305 ◽  
Author(s):  
Sara M. Vetter ◽  
Patrick M. Schlievert
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Lethal Factor ◽  
Sodium Dodecyl ◽  
The United States ◽  
Human Populations ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Glycerol Monolaurate

ABSTRACT Anthrax, caused by Bacillus anthracis, has been brought to the public's attention because of the 2001 bioterrorism attacks. However, anthrax is a disease that poses agricultural threats in the United States as well as human populations in Europe, China, Africa, and Australia. Glycerol monolaurate (GML) is a compound that has been shown to inhibit exotoxin production by Staphylococcus aureus and other gram-positive bacteria. Here, we study the effects of GML on growth and toxin production in B. anthracis. The Sterne strain of B. anthracis was grown to post-exponential phase with 0-, 10-, 15-, or 20-μg/ml concentrations of GML and then assayed quantitatively for protective antigen (PA) and lethal factor (LF). After 8 h, GML at concentrations greater than 20 μg/ml was bacteriostatic to growth of the organism. However, a 10-μg/ml concentration of GML was not growth inhibitory, but amounts of PA and LF made were greatly reduced. This effect was not global for all proteins when total secreted protein from culture fluids was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Through quantitative reverse transcription-PCR assays, this toxin-inhibitory effect was shown to occur at the transcriptional level, since amounts of mRNA for pagA (PA), lef (LF), and cya (edema factor) were reduced. Surprisingly, mRNA levels of atxA, a regulator of exotoxin gene expression, rose in the presence of GML. These data will be useful in developing therapeutic tools to treat anthrax disease, whether in animals or humans. These results also suggest that mechanisms of virulence regulation exist independent of atxA.

scholarly journals Anthrax Lethal Toxin Impairs CD1d-Mediated Antigen Presentation by Targeting the Extracellular Signal-Related Kinase 1/2 Mitogen-Activated Protein Kinase Pathway

2010 ◽  
Vol 78 (5) ◽  
pp. 1859-1863 ◽  
Author(s):  
Masood A. Khan ◽  
Richard M. Gallo ◽  
Randy R. Brutkiewicz
Keyword(s):  
Immune System ◽  
Protein Kinase ◽  
Bacillus Anthracis ◽  
Antigen Presentation ◽  
Specific Inhibitor ◽  
Lethal Toxin ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Lethal toxin (LT) is a critical virulence factor of Bacillus anthracis and an important means by which this bacterium evades the host's immune system. In this study, we demonstrate that CD1d-expressing cells treated with LT have reduced CD1d-mediated antigen presentation. We earlier showed an important role for the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2) in the regulation of CD1d-mediated antigen presentation, and we report here that LT impairs antigen presentation by CD1d in an ERK1/2-dependent manner. Similarly, LT and the ERK1/2 pathway-specific inhibitor U0126 caused a decrease in major histocompatibility complex (MHC) class II-mediated antigen presentation. Confocal microscopy analyses revealed altered intracellular distribution of CD1d and LAMP-1 in LT-treated cells, similar to the case for ERK1/2-inhibited cells. These results suggest that Bacillus anthracis has the ability to evade the host's innate immune system by reducing CD1d-mediated antigen presentation through targeting the ERK1/2 pathway.

scholarly journals GeneChip Analyses of Global Transcriptional Responses of Murine Macrophages to the Lethal Toxin of Bacillus anthracis

2005 ◽  
Vol 73 (3) ◽  
pp. 1879-1885 ◽  
Author(s):  
Jason E. Comer ◽  
Cristi L. Galindo ◽  
Ashok K. Chopra ◽  
Johnny W. Peterson
Keyword(s):  
Bacillus Anthracis ◽  
Intracellular Signaling ◽  
Lethal Toxin ◽  
Mitogen Activated Protein Kinase ◽  
Transcriptional Responses ◽  
Murine Macrophages ◽  
Mitogen Activated Protein ◽  
Cell Gene Expression ◽  
Cell Gene ◽  
Protein Kinase Kinase

ABSTRACT 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.

scholarly journals The Poly-γ-d-Glutamic Acid Capsule of Bacillus anthracis Enhances Lethal Toxin Activity

2011 ◽  
Vol 79 (9) ◽  
pp. 3846-3854 ◽  
Author(s):  
Jeyoun Jang ◽  
Minhui Cho ◽  
Jeong-Hoon Chun ◽  
Min-Hee Cho ◽  
Jungchan Park ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Immune Surveillance ◽  
Lethal Toxin ◽  
Mitogen Activated Protein Kinase ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
Anthrax Infection ◽  
Processed Form

ABSTRACTThe poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors ofBacillus anthracis, which causes a highly lethal infectious disease. The PGA capsule disguisesB. anthracisfrom immune surveillance and allows its unimpeded growth in the host. The PGA capsule recently was reported to be associated with lethal toxin (LT) in the blood of experimentally infected animals (M. H. Cho, et al., Infect. Immun. 78:387-392, 2010). The effect of PGA, either alone or in combination with LT, on macrophages, which play an important role in the progression of anthrax disease, has not been thoroughly investigated. In this study, we investigated the effect of PGA on LT cytotoxicity using the mouse macrophage cell line J774A.1. PGA produced a concentration-dependent enhancement of the cytotoxicity of LT on J774A.1 cells through an enhancement in the binding and accumulation of protective antigen to its receptors. The increase of LT activity was confirmed using Western blot analysis, which showed that the combination of PGA and LT produced a greater degree of degradation of mitogen-activated protein kinase kinases and an increased level of the activation of the proform of caspase-1 to its processed form compared to the effects of LT alone. In addition, mice that received a tail vein injection of both PGA and LT had a significantly increased rate of death compared to that of mice injected with LT alone. PGA had no effect when added to cultures or administered to mice in the absence of LT. These results emphasize the importance of PGA in the pathogenesis of anthrax infection.

scholarly journals Expression and Purification of the Recombinant Lethal Factor of Bacillus anthracis

1998 ◽  
Vol 66 (2) ◽  
pp. 862-865 ◽  
Author(s):  
Pankaj Gupta ◽  
Smriti Batra ◽  
Arun P. Chopra ◽  
Yogendra Singh ◽  
Rakesh Bhatnagar
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Lethal Factor ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Nitrilotriacetic Acid ◽  
Biologically Active ◽  
Liquid Chromatograph ◽  
E Coli

ABSTRACT The structural gene for the 90-kDa lethal factor (LF) isolated fromBacillus anthracis was expressed as a fusion protein with six histidine residues in Escherichia coli. Expression of LF in E. coli under the transcriptional regulation of the T5 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 90 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant LF reacted with anti-LF antibodies. The protein was purified to homogeneity by nickel nitrilotriacetic acid affinity chromatography and gel filtration on a Sephacryl S-200 column followed by anion exchange on a fast-performance liquid chromatograph with a Resource-Q column. The yield of purified LF from this procedure was 1.5 mg/liter. In solution, trypsin cleaved protective antigen bound to native and recombinant LF with comparable affinities. In macrophage lysis assays, native and recombinant LF exhibited identical potencies. The results suggest that large amounts of biologically active LF can be purified by this procedure.

scholarly journals Interleukin 1 alpha activates two forms of p54 alpha mitogen-activated protein kinase in rabbit liver.

1994 ◽  
Vol 180 (6) ◽  
pp. 2017-2025 ◽  
Author(s):  
M Kracht ◽  
O Truong ◽  
N F Totty ◽  
M Shiroo ◽  
J Saklatvala
Keyword(s):  
Protein Kinase ◽  
Map Kinases ◽  
Interleukin 1 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Systemic Injection ◽  
Mitogen Activated Protein

We have identified in rabbits two hepatic forms of T669 peptide kinases that are very strongly activated after systemic injection with the inflammatory cytokine interleukin 1 (IL-1). The T669 peptide contains a major phosphorylation site of the epidermal growth factor receptor, threonine 699 and is a substrate for mitogen-activated protein (MAP) kinases. The kinases were purified to homogeneity and corresponded to 50- and 55-kD proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequencing of 12 tryptic peptides of both kinases identified them as p54 MAP kinase alpha. This kinase belongs to the novel family of stress-activated protein kinases. This is the first evidence of IL-1 activating a specific protein kinase in vivo.

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 Oxidized ATP Protection against Anthrax Lethal Toxin

2006 ◽  
Vol 74 (7) ◽  
pp. 3707-3714 ◽  
Author(s):  
Mahtab Moayeri ◽  
Katherine E. Wickliffe ◽  
Jason F. Wiggins ◽  
Stephen H. Leppla
Keyword(s):  
Fluorescent Dyes ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Sodium Dodecyl ◽  
Inbred Strains ◽  
Lethal Toxin ◽  
Anthrax Lethal Toxin ◽  
P2x7 Receptors ◽  
Intracellular Location ◽  
Mitogen Activated Protein

ABSTRACT Bacillus anthracis lethal toxin (LT) induces rapid lysis (<90 min) of murine macrophages from certain inbred strains. The mechanism for LT-induced cytolysis is currently unknown. We hypothesized that the ATP-activated macrophage P2X7 receptors implicated in nucleotide-mediated macrophage lysis could play a role in LT-mediated cytolysis and discovered that a potent P2X7 antagonist, oxidized ATP (o-ATP), protects macrophages against LT. Other P2X7 receptor antagonists, however, had no effect on LT function, while oxidized nucleotides, o-ADP, o-GTP, and o-ITP, which did not act as receptor ligands, provided protection. Cleavage of the LT substrates, the mitogen-activated protein kinases, was inhibited by o-ATP in RAW274.6 macrophages and CHO cells. We investigated the various steps in the intoxication pathway and found that binding of the protective-antigen (PA) component of LT to cells and the enzymatic proteolytic ability of the lethal factor (LF) component of LT were unaffected by o-ATP. Instead, the drug inhibited formation of the sodium dodecyl sulfate-resistant PA oligomer, which occurs in acidified endosomes, but did not prevent cell surface PA oligomerization, as evidenced by binding and translocation of LF to a protease-resistant intracellular location. We found that o-ATP also protected cells from anthrax edema toxin and diphtheria toxin, which also require an acidic environment for escape from endosomes. Confocal microscopy using pH-sensitive fluorescent dyes showed that o-ATP increased endosomal pH. Finally, BALB/cJ mice injected with o-ATP and LT were completely protected against lethality.

Sign in / Sign up

Export Citation Format

Share Document