scholarly journals Cytosolic Delivery and Characterization of the TcdB Glucosylating Domain by Using a Heterologous Protein Fusion

2001 ◽  
Vol 69 (1) ◽  
pp. 599-601 ◽  
Author(s):  
Lea M. Spyres ◽  
Maen Qa'Dan ◽  
Amy Meader ◽  
James J. Tomasek ◽  
Eric W. Howard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Small Gtpases ◽  
Anthrax Toxin ◽  
Coding Region ◽  
Protein Fusion ◽  
Actin Reorganization ◽  
Amino Terminal ◽  
Cell Extracts

ABSTRACT TcdB from Clostridium difficile glucosylates small GTPases (Rho, Rac, and Cdc42) and is an important virulence factor in the human disease pseudomembranous colitis. In these experiments, in-frame genetic fusions between the genes for the 255 amino-terminal residues of anthrax toxin lethal factor (LFn) and the TcdB1-556 coding region were constructed, expressed, and purified from Escherichia coli. LFnTcdB1-556was enzymatically active and glucosylated recombinant RhoA, Rac, Cdc42, and substrates from cell extracts. LFnTcdB1-556 plus anthrax toxin protective antigen intoxicated cultured mammalian cells and caused actin reorganization and mouse lethality, all similar to those caused by wild-type TcdB.

scholarly journals Anthrax Toxin as a Molecular Tool for Stimulation of Cytotoxic T Lymphocytes: Disulfide-Linked Epitopes, Multiple Injections, and Role of CD4+ Cells

1998 ◽  
Vol 66 (10) ◽  
pp. 4696-4699 ◽  
Author(s):  
Jimmy D. Ballard ◽  
R. John Collier ◽  
Michael N. Starnbach
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Mammalian Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Ctl Response ◽  
Amino Terminal ◽  
Carboxy Terminal

ABSTRACT We have previously demonstrated that anthrax toxin-derived proteins, protective antigen (PA) and the amino-terminal portion of lethal factor (LFn), can be used in combination to deliver heterologous molecules to the cytosol of mammalian cells. In this study we examined the ability of an LFn-peptide disulfide-linked heterodimer to prime cytotoxic T lymphocytes (CTL) in the presence of PA. A mutant of LFn that contains a carboxy-terminal reactive cysteine was generated. This form of LFn could be oxidized with a synthetic cysteine containing peptide to form a heterodimer of the protein and peptide. Mice injected with the heterodimer plus PA mounted a peptide-specific CTL response, indicating that this molecule functioned similarly to the genetically fused forms used previously. We also report the results of an analysis of two aspects of this system important for the development of experimental vaccines. First, CD4 knockout mice were unable to generate a CTL response when treated with PA plus an LFn-epitope fusion protein, suggesting that CD4+ helper responses are essential for stimulating specific CTL with the PA-LFn system. Second, we now show that primary injection with this system does not generate any detectable antibody response to the vaccine components and that prior immunization has no effect on priming a CTL response to an unrelated epitope upon subsequent injection.

Oligomerization of Anthrax Toxin Protective Antigen and Binding of Lethal Factor during Endocytic Uptake into Mammalian Cells

1999 ◽  
Vol 67 (4) ◽  
pp. 1853-1859
Author(s):  
Yogendra Singh ◽  
Kurt R. Klimpel ◽  
Seema Goel ◽  
Prabodha K. Swain ◽  
Stephen H. Leppla
Keyword(s):  
Mammalian Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin

scholarly journals Cytotoxic T-Lymphocyte Epitopes Fused to Anthrax Toxin Induce Protective Antiviral Immunity

1999 ◽  
Vol 67 (7) ◽  
pp. 3290-3296 ◽  
Author(s):  
Amy M. Doling ◽  
Jimmy D. Ballard ◽  
Hao Shen ◽  
Kaja Murali Krishna ◽  
Rafi Ahmed ◽  
...  
Keyword(s):  
Fusion Protein ◽  
T Lymphocyte ◽  
Mammalian Cells ◽  
Protective Antigen ◽  
Cytotoxic T Lymphocyte ◽  
Lethal Factor ◽  
Antiviral Immunity ◽  
Anthrax Toxin ◽  
Listeriolysin O

ABSTRACT We have investigated the use of the protective antigen (PA) and lethal factor (LF) components of anthrax toxin as a system for in vivo delivery of cytotoxic T-lymphocyte (CTL) epitopes. During intoxication, PA directs the translocation of LF into the cytoplasm of mammalian cells. Here we demonstrate that antiviral immunity can be induced in BALB/c mice immunized with PA plus a fusion protein containing the N-terminal 255 amino acids of LF (LFn) and an epitope from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. We also demonstrate that BALB/c mice immunized with a single LFn fusion protein containing NP and listeriolysin O protein epitopes in tandem mount a CTL response against both pathogens. Furthermore, we show that NP-specific CTL are primed in both BALB/c and C57BL/6 mice when the mice are immunized with a single fusion containing two epitopes, one presented by Ld and one presented by Db. The data presented here demonstrate the versatility of the anthrax toxin delivery system and indicate that this system may be used as a general approach to vaccinate outbred populations against a variety of pathogens.

scholarly journals Cross-Linked Forms of the Isolated N-Terminal Domain of the Lethal Factor Are Potent Inhibitors of Anthrax Toxin

2007 ◽  
Vol 75 (10) ◽  
pp. 5052-5058 ◽  
Author(s):  
Stephen J. Juris ◽  
Roman A. Melnyk ◽  
Robert E. Bolcome ◽  
Joanne Chan ◽  
R. John Collier
Keyword(s):  
Mammalian Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Zebrafish Model ◽  
Ion Conductance ◽  
New Approach ◽  
Terminal Domain ◽  
Edema Factor ◽  
C Terminus

ABSTRACT The proteins that comprise anthrax toxin self-assemble at the mammalian cell surface into a series of toxic complexes, each containing a heptameric form of protective antigen (PA) plus up to a total of three molecules of the enzymatic moieties of the toxin (lethal factor [LF] and edema factor [EF]). These complexes are trafficked to the endosome, where the PA heptamer forms a pore in the membrane under the influence of low pH, and bound LF and EF unfold and translocate through the pore to the cytosol. To explore the hypothesis that the PA pore can translocate multiple, cross-linked polypeptides simultaneously, we cross-linked LFN, the N-terminal domain of LF, via an introduced cysteine at its N or C terminus and characterized the products. Both dimers and trimers of LFN retained the ability to bind to PA pores and block ion conductance, but they were unable to translocate across the membrane, even at high voltages or with a transmembrane pH gradient. The multimers were remarkably potent inhibitors of toxin action in mammalian cells (20- to 50-fold more potent than monomeric LFN) and in a zebrafish model system. These findings show that the PA pore cannot translocate multimeric, cross-linked polypeptides and demonstrate a new approach to generating potent inhibitors of anthrax toxin.

scholarly journals Oligomerization of Anthrax Toxin Protective Antigen and Binding of Lethal Factor during Endocytic Uptake into Mammalian Cells

1999 ◽  
Vol 67 (4) ◽  
pp. 1853-1859 ◽  
Author(s):  
Yogendra Singh ◽  
Kurt R. Klimpel ◽  
Seema Goel ◽  
Prabodha K. Swain ◽  
Stephen H. Leppla
Keyword(s):  
Gel Electrophoresis ◽  
Mammalian Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Proteolytic Activation ◽  
Edema Factor ◽  
Plastic Surface ◽  
Kinetics Of

ABSTRACT The protective antigen (PA) protein of anthrax toxin binds to a cellular receptor and is cleaved by cell surface furin to produce a 63-kDa fragment (PA63). The receptor-bound PA63 oligomerizes to a heptamer and acts to translocate the catalytic moieties of the toxin, lethal factor (LF) and edema factor (EF), from endosomes to the cytosol. In this report, we used nondenaturing gel electrophoresis to show that each PA63 subunit in the heptamer can bind one LF molecule. Studies using PA immobilized on a plastic surface showed that monomeric PA63 is also able to bind LF. The internalization of PA and LF by cells was studied with radiolabeled and biotinylated proteins. Uptake was relatively slow, with a half-time of 30 min. The number of moles of LF internalized was nearly equal to the number of moles of PA subunit internalized. The essential role of PA oligomerization in LF translocation was shown with PA protein cleaved at residues 313-314. The oligomers formed by these proteins during uptake into cells were not as stable when subjected to heat and detergent as were those formed by native PA. The results show that the structure of the toxin proteins and the kinetics of proteolytic activation, LF binding, and internalization are balanced in a way that allows each PA63 subunit to internalize an LF molecule. This set of proteins has evolved to achieve highly efficient internalization and membrane translocation of the catalytic components, LF and EF.

scholarly journals The chymotrypsin-sensitive site, FFD315, in anthrax toxin protective antigen is required for translocation of lethal factor.

1994 ◽  
Vol 269 (46) ◽  
pp. 29039-29046
Author(s):  
Y Singh ◽  
K R Klimpel ◽  
N Arora ◽  
M Sharma ◽  
S H Leppla
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Sensitive Site

scholarly journals Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

2006 ◽  
Vol 13 (6) ◽  
pp. 671-677 ◽  
Author(s):  
Robert Mabry ◽  
Kathleen Brasky ◽  
Robert Geiger ◽  
Ricardo Carrion ◽  
Gene B. Hubbard ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Systemic Circulation ◽  
Rapid Identification ◽  
Anthrax Toxin ◽  
Soluble Form ◽  
Before And After

ABSTRACT Several strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. The assays utilize as capture agents an engineered high-affinity antibody to PA, a soluble form of the extracellular domain of the anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwich immunoassays were used to detect and quantify PA and LF in animals infected with the Ames or Vollum strains of anthrax spores. PA and LF were detected before and after signs of toxemia were observed, with increasing levels reported in the late stages of the infection. These results represent the detection of free PA and LF by ELISA in the systemic circulation of two animal models exposed to either of the two fully virulent strains of anthrax. Simple anthrax toxin detection ELISAs could prove useful in the evaluation of potential therapies and possibly as a clinical diagnostic to complement other strategies for the rapid identification of B. anthracis infection.

scholarly journals Anthrax Toxin Characterization

2002 ◽  
Vol 45 (1) ◽  
pp. 3-5 ◽  
Author(s):  
Jiří Patočka ◽  
Miroslav Špliňo
Keyword(s):  
Blood Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
White Blood Cells ◽  
The Body ◽  
Anthrax Toxin ◽  
Signaling System ◽  
Animal Cells ◽  
The Third ◽  
Edema Factor

The anthrax toxin comprises three proteins. When they work together, they can kill humans, especially after spores of the bacteria have been inhaled. One anthrax protein, called protective antigen (PA), chaperones the two other toxins into human or animal cells and shields them from the body’s immune system. The second, lethal factor (LF), destroys the white blood cells that hosts send in defence. The third toxin molecule, edema factor (EF), hijacks the signaling system in the body. This disrupts the energy balance of cells and leads to them accumulating fluid and complete destroy of cells.

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