Anthrax Lethal Factor (LF) Mediated Block of the Anthrax Protective Antigen (PA) Ion Channel:  Effect of Ionic Strength and Voltage†

Biochemistry ◽  
2006 ◽  
Vol 45 (9) ◽  
pp. 3060-3068 ◽  
Author(s):  
Tobias Neumeyer ◽  
Fiorella Tonello ◽  
Federica Dal Molin ◽  
Bettina Schiffler ◽  
Frank Orlik ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Ion Channel ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Channel Effect ◽  
Anthrax Lethal Factor ◽  
Anthrax Protective Antigen

scholarly journals Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

2013 ◽  
Vol 20 (7) ◽  
pp. 986-997 ◽  
Author(s):  
Hang Lu ◽  
Jason Catania ◽  
Katalin Baranji ◽  
Jie Feng ◽  
Mili Gu ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Neutralization Assay ◽  
Dominant Factor ◽  
Anthrax Lethal Toxin ◽  
Serum Samples ◽  
Native Form ◽  
Anthrax Lethal Factor ◽  
Methionine Residues ◽  
Anthrax Vaccines

ABSTRACTThe cell-based anthrax toxin neutralization assay (TNA) is used to determine functional antibody titers of sera from animals and humans immunized with anthrax vaccines. The anthrax lethal toxin is a critical reagent of the TNA composed of protective antigen (PA) and lethal factor (LF), which are neutralization targets of serum antibodies. Cytotoxic potency of recombinant LF (rLF) lots can vary substantially, causing a challenge in producing a renewable supply of this reagent for validated TNAs. To address this issue, we characterized a more potent rLF variant (rLF-A) with the exact native LF amino acid sequence that lacks the additional N-terminal histidine and methionine residues present on the commonly used form of rLF (rLF-HMA) as a consequence of the expression vector. rLF-A can be used at 4 to 6 ng/ml (in contrast to 40 ng/ml rLF-HMA) with 50 ng/ml recombinant PA (rPA) to achieve 95 to 99% cytotoxicity. In the presence of 50 ng/ml rPA, both rLF-A and rLF-HMA allowed for similar potencies (50% effective dilution) among immune sera in the TNA. rPA, but not rLF, was the dominant factor in determining potency of serum samples containing anti-PA antibodies only or an excess of anti-PA relative to anti-rLF antibodies. Such anti-PA content is reflected in immune sera derived from most anthrax vaccines in development. These results support that 7- to 10-fold less rLF-A can be used in place of rLF-HMA without changing TNA serum dilution curve parameters, thus extending the use of a single rLF lot and a consistent, renewable supply.

scholarly journals Detection of Anthrax Toxin by an Ultrasensitive Immunoassay Using Europium Nanoparticles

2009 ◽  
Vol 16 (3) ◽  
pp. 408-413 ◽  
Author(s):  
Shixing Tang ◽  
Mahtab Moayeri ◽  
Zhaochun Chen ◽  
Harri Harma ◽  
Jiangqin Zhao ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Laboratory Research ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Serum Samples ◽  
Detection Range ◽  
Edema Factor ◽  
Anthrax Lethal Factor ◽  
Europium Nanoparticles

ABSTRACT We developed a europium nanoparticle-based immunoassay (ENIA) for the sensitive detection of anthrax protective antigen (PA). The ENIA exhibited a linear dose-dependent pattern within the detection range of 0.01 to 100 ng/ml and was approximately 100-fold more sensitive than enzyme-linked immunosorbent assay (ELISA). False-positive results were not observed with serum samples from healthy adults, mouse plasma without PA, or plasma samples collected from mice injected with anthrax lethal factor or edema factor alone. For the detection of plasma samples spiked with PA, the detection sensitivities for ENIA and ELISA were 100% (11/11 samples) and 36.4% (4/11 samples), respectively. The assay exhibited a linear but qualitative correlation between the PA injected and the PA detected in murine blood (r = 0.97731; P < 0.0001). Anthrax PA was also detected in the circulation of mice infected with spores from a toxigenic Sterne-like strain of Bacillus anthracis, but only in the later stages of infection. These results indicate that the universal labeling technology based on europium nanoparticles and its application may provide a rapid and sensitive testing platform for clinical diagnosis and laboratory research.

scholarly journals A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

2003 ◽  
Vol 100 (11) ◽  
pp. 6652-6657 ◽  
Author(s):  
N. Kushner ◽  
D. Zhang ◽  
N. Touzjian ◽  
M. Essex ◽  
J. Lieberman ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Lethal Factor

scholarly journals Development of Enzyme Linked Immunosorbent Assay for humoral immuneresponse and infection monitoring of anthrax

2020 ◽  
Vol 71 (1) ◽  
pp. 1935
Author(s):  
H. R. NOURI ◽  
H. RAZZAZ ◽  
M. TAGHDIRI ◽  
K. TADAYON ◽  
S. R. BANIHASHEMI
Keyword(s):  
Protective Antigen ◽  
Critical Role ◽  
Lethal Factor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibody ◽  
Elisa Kit ◽  
Gram Positive Bacteria ◽  
Anthrax Lethal Factor ◽  
Antibody Titers ◽  
Immune Assays

Immune assays were taken into consideration to diagnose and quantify metabolites such as antigen and antibody. Enzyme-Linked Immunosorbent Assays (ELISAs), which are used to detect antigens and antibodies, generated several periods of infectious and vaccination conditions. There is an extensive range of commercial infectious disease ELISA kits useful for the detection of human and animal IgG, IgA, IgM antibodies and microorganism antigens. Anthrax is one of the serious infectious diseases caused by rod-shaped, gram-positive bacteria known as Bacillus anthracis. Subunit or attenuated vaccines applied against anthrax disease increase the antibody against the Protective Antigen (PA) which has a critical role as a toxin of B. anthracis. Herein, the ELISA was developed using PA domain 4 and anthrax Lethal Factor to detect IgG antibody in serum. Besides, the level of anti-LF antibodies were determined as a complementary test to measure variance in antibody titers associated with vaccination or infection that leads to detection of anthrax in livestock. The results show that we developed high-quality ELISA kit that can be used to test immunogenicity of vaccines and infections in mice. We tried to develop the Anti- PA4 ELISA kit and conduct the validation studies to evaluate the fluctuation level of the antibody in the anthrax vaccine and distinction between disease and vaccination in mice.

scholarly journals Anthrax Protective Antigen Cleavage and Clearance from the Blood of Mice and Rats

2007 ◽  
Vol 75 (11) ◽  
pp. 5175-5184 ◽  
Author(s):  
Mahtab Moayeri ◽  
Jason F. Wiggins ◽  
Stephen H. Leppla
Keyword(s):  
Receptor Binding ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Dominant Negative ◽  
Furin Cleavage ◽  
Defective Mutant ◽  
Tissue Surfaces ◽  
Anthrax Protective Antigen

ABSTRACT Bacillus anthracis protective antigen (PA) is an 83-kDa (PA83) protein that is cleaved to the 63-kDa protein (PA63) as an essential step in binding and internalizing lethal factor (LF). To assess in vivo receptor saturating PA concentrations, we injected mice with PA variants and measured the PA remaining in the blood at various times using PA83- and PA63-specific enzyme-linked immunosorbent assays. We found that both wild-type PA (WT-PA) and a receptor-binding-defective mutant (Ub-PA) were cleaved to PA63 independent of their ability to bind cells. This suggested a PA-acting protease activity in the blood. The protease cleaved PA at the furin cleavage sequence because furin site-modified PA mutants were not cleaved. Cleavage measured in vitro was leupeptin sensitive and dependent on calcium. Cell surface cleavage was important for toxin clearance, however, as Ub-PA and uncleavable PA mutants were cleared at slower rates than WT-PA. The cell binding-independent cleavage of PA was also verified by using Ub-PA (which is still cleaved) to rescue mice from toxin challenge by competitively binding circulating LF. This mutant was able to rescue mice even when given 12 h before toxin challenge. Its therapeutic ability was comparable to that of dominant-negative PA, which binds cells but does not allow LF translocation, and to the protection afforded through receptor clearance by WT-PA and uncleavable receptor binding-competent mutants. The PA cleavage and clearance observed in mice did not appear to have a role in the differential mouse susceptibility as it occurred similarly in lethal toxin (LT)-resistant DBA/2J and LT-sensitive BALB/cJ mice. Interestingly, PA63 was not found in LT-resistant or -sensitive rats and PA83 clearance was slower in rats than in mice. Finally, to determine the minimum amount of PA required in circulation for LT toxicity in mice, we administered time-separated injections of PA and LF and showed that lethality of LF for mice after PA was no longer measurable in circulation, suggesting active PA sequestration at tissue surfaces.

scholarly journals Structural Determinants for the Binding of Anthrax Lethal Factor to Oligomeric Protective Antigen

2005 ◽  
Vol 281 (3) ◽  
pp. 1630-1635 ◽  
Author(s):  
Roman A. Melnyk ◽  
Krissi M. Hewitt ◽  
D. Borden Lacy ◽  
Henry C. Lin ◽  
Chris R. Gessner ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Structural Determinants ◽  
Anthrax Lethal Factor

scholarly journals Membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway

2004 ◽  
Vol 166 (5) ◽  
pp. 645-651 ◽  
Author(s):  
Laurence Abrami ◽  
Margaret Lindsay ◽  
Robert G. Parton ◽  
Stephen H. Leppla ◽  
F. Gisou van der Goot
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Endocytic Pathway ◽  
Cell Surface Receptor ◽  
Membrane Insertion ◽  
Edema Factor ◽  
Early Endosomes ◽  
Surface Receptor ◽  
A Cell ◽  
Anthrax Protective Antigen

The protective antigen (PA) of anthrax toxin binds to a cell surface receptor, undergoes heptamerization, and binds the enzymatic subunits, the lethal factor (LF) and the edema factor (EF). The resulting complex is then endocytosed. Via mechanisms that depend on the vacuolar ATPase and require membrane insertion of PA, LF and EF are ultimately delivered to the cytoplasm where their targets reside. Here, we show that membrane insertion of PA already occurs in early endosomes, possibly only in the multivesicular regions, but that subsequent delivery of LF to the cytoplasm occurs preferentially later in the endocytic pathway and relies on the dynamics of internal vesicles of multivesicular late endosomes.

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