The Anthrax Protective Antigen (PA63) Bound Conformation of a Peptide Inhibitor of the Binding of Lethal Factor to PA63:  As Determined by trNOESY NMR and Molecular Modeling†

2004 ◽  
Vol 47 (22) ◽  
pp. 5347-5355 ◽  
Author(s):  
Rickey P. Hicks ◽  
Apurba K. Bhattacharjee ◽  
Brandon W. Koser ◽  
Daniel D. Traficante
Keyword(s):  
Molecular Modeling ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Peptide Inhibitor ◽  
Anthrax Protective Antigen

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 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.

scholarly journals Structure of a dominant negative mutant reveals a stalled intermediate state of anthrax protective antigen pore maturation

2020 ◽  
Author(s):  
Harry Scott ◽  
Wei Huang ◽  
Srinivas Gonti ◽  
Kaiming Zhang ◽  
Nurjahan Mehzabeen ◽  
...  
Keyword(s):  
Pore Formation ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Edema Factor ◽  
Beta Barrel ◽  
Anthrax Protective Antigen ◽  
Maturation State ◽  
Membrane Spanning

AbstractAnthrax is a severe bacterial infection caused by Bacillus anthracis, which produces a tripartite toxin that includes protective antigen (PA), lethal factor (LF) and edema factor (EF). A series of dominant-negative mutations have been previously identified that prevent the heptameric PA prepore from forming the pH-induced, membrane spanning beta-barrel pore that is required for translocation of EF and LF to the cytoplasm of the infected cell. Here we show that the dominant negative D425A mutation stalls the formation of the pore at a reversible intermediate maturation state, which exhibits many of the structural aspects of the pore but fails to form the phi(ϕ)-clamp and beta-barrel structure needed for full pore maturation. Overall, this structure reveals that ϕ-clamp and beta-barrel pore formation are later steps in the pathway to pore formation, thereby providing a regulatory mechanism to prevent premature translocation of EF and LF.

scholarly journals Purification of Anthrax Edema Factor fromEscherichia coli and Identification of Residues Required for Binding to Anthrax Protective Antigen

2001 ◽  
Vol 69 (10) ◽  
pp. 6532-6536 ◽  
Author(s):  
Praveen Kumar ◽  
Nidhi Ahuja ◽  
Rakesh Bhatnagar
Keyword(s):  
Recombinant Protein ◽  
Protective Antigen ◽  
Metal Chelate ◽  
Lethal Factor ◽  
Exchange Chromatography ◽  
Biologically Active ◽  
E Coli ◽  
Edema Factor ◽  
Step Procedure ◽  
Anthrax Protective Antigen

ABSTRACT The structural gene for anthrax edema factor (EF) was expressed in Escherichia coli under the control of a powerful T5 promoter to yield the 89-kDa recombinant protein that reacted with anti-EF antibodies. Recombinant EF was purified to homogeneity by a two-step procedure involving metal chelate affinity chromatography and cation-exchange chromatography. From 1 liter of culture, 2.5 mg of biologically active EF was easily purified. This is the first report of purification of anthrax EF from E. coli. EF purified from E. coli was biologically and functionally as active as its Bacillus anthracis counterpart. The recombinant protein could compete with lethal factor for binding to protective antigen. Sequence analysis revealed a stretch of seven amino acids, Val Tyr Tyr Glu Ile Gly Lys, present both in EF (residues 136 to 142) and lethal factor (residues 147 to 153). To investigate the role of these seven residues in binding to protective antigen, the residues were individually mutated to alanine in EF. Mutations in residues Tyr137, Tyr138, Ile140, and Lys142 of EF specifically blocked its interaction with anthrax protective antigen. The adenylate cyclase activity of the mutants remained unaffected. The results suggested that residues Tyr137, Tyr138, Ile140, and Lys142 are required for binding of EF to anthrax protective antigen, which facilitates its entry into susceptible cells.

scholarly journals Protection from Anthrax Toxin-Mediated Killing of Macrophages by the Combined Effects of Furin Inhibitors and Chloroquine

2005 ◽  
Vol 49 (9) ◽  
pp. 3875-3882 ◽  
Author(s):  
Tomoko Komiyama ◽  
Joel A. Swanson ◽  
Robert S. Fuller
Keyword(s):  
Antigen Processing ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Proteolytic Processing ◽  
Anthrax Toxin ◽  
Acid Inhibitor ◽  
Therapeutic Benefits ◽  
Anthrax Protective Antigen ◽  
Apoptotic Pathways ◽  
And Function

ABSTRACT Cell surface proteolytic processing of anthrax protective antigen by furin or other furin-related proteases is required for its oligomerization, endocytosis, and function as a translocon for anthrax lethal and edema factors. Countering toxin lethality is essential to developing effective chemotherapies for anthrax infections that have proceeded beyond the stage at which antibiotics are effective. The primary target for toxin is the macrophage, which can be killed by lethal factor via both necrotic and apoptotic pathways. Here we show that three high-affinity inhibitors of furin efficiently blocked killing of murine J774A.1 macrophages by recombinant protective antigen plus lethal factor: RRD-eglin and RRDG-eglin, developed by engineering the protein protease inhibitor eglin c, and the peptide boronic acid inhibitor acetyl-Arg-Glu-Lys-boroArg pinanediol. Inhibition of killing was dose dependent and correlated with prevention of protective antigen processing. Previous studies have shown that weak bases, such as chloroquine, which neutralize acidic compartments, also interfere with toxin-dependent killing. Here we show that combining furin inhibitors and chloroquine strongly augments the inhibition of toxin-dependent killing, suggesting that combined use of antifurin drugs and chloroquine might provide enhanced therapeutic benefits. Reversible furin inhibitors protected against anthrax toxin killing for at least 5 h, but by 8 h, toxin-dependent killing resumed even though furin inhibitors were still active. An irreversible chloromethylketone inhibitor did not exhibit this loss of protection.

scholarly journals Large-Scale Structural Changes Accompany Binding of Lethal Factor to Anthrax Protective Antigen

Structure ◽  
2004 ◽  
Vol 12 (11) ◽  
pp. 2059-2066 ◽  
Author(s):  
Gang Ren ◽  
Joel Quispe ◽  
Stephen H. Leppla ◽  
Alok K. Mitra
Keyword(s):  
Large Scale ◽  
Structural Changes ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Protective Antigen

scholarly journals Investigation of New Dominant-Negative Inhibitors of Anthrax Protective Antigen Mutants for Use in Therapy and Vaccination

2009 ◽  
Vol 77 (10) ◽  
pp. 4679-4687 ◽  
Author(s):  
Sha Cao ◽  
Aizhen Guo ◽  
Ziduo Liu ◽  
Yadi Tan ◽  
Gaobing Wu ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Therapeutic Agents ◽  
Dominant Negative ◽  
Raw264.7 Cells ◽  
Necrosis Factor Alpha ◽  
Anthrax Protective Antigen ◽  
The Individual ◽  
Novel Therapeutic

ABSTRACT The lethal toxin (LeTx) of Bacillus anthracis plays a key role in the pathogenesis of anthrax. The protective antigen (PA) is a primary part of the anthrax toxin and forms LeTx by combination with lethal factor (LF). Phenylalanine-427 (F427) is crucial for PA function. This study was designed to discover potential novel therapeutic agents and vaccines for anthrax. This was done by screening PA mutants that were mutated at the F427 residue for a dominant-negative inhibitory (DNI) phenotype which was nontoxic but inhibited the toxicity of the wild-type LeTx. For this, PA residue F427 was first mutated to each of the other 19 naturally occurring amino acids. The cytotoxicity and DNI phenotypes of the mutated PA proteins were tested in the presence of 1 μg/ml LF in RAW264.7 cells and were shown to be dependent on the individual amino acid replacements. A total of 16 nontoxic mutants with various levels of DNI activity were identified in vitro. Among them, F427D and F427N mutants had the highest DNI activities in RAW264.7 cells. Both mutants inhibited LeTx intoxication in mice in a dose-dependent way. Furthermore, they induced a Th2-predominant immune response and protected mice against a challenge with five 50% lethal doses of LeTx. The protection was correlated mainly with a low level of interleukin-1β (IL-1β) and with high levels of PA-specific immunoglobulin G1, IL-6, and tumor necrosis factor alpha. Thus, PA DNI mutants, such as F427D and F427N mutants, may serve in the development of novel therapeutic agents and vaccines to fight B. anthracis infections.

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
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